> Chemicals & Drugs > Amino Acid > Activating transcription factor 6, human

← Activated-Leukocyte Cell Adhesion Molecule

Activating transcription factor 6, human

Q: How can PubCompare.ai help in research related to Activating transcription factor 6 (ATF6)?

PubCompare.ai allows you to screen protocol literature more efficietly and leverage AI to pinpoint critical insights. The platform can help researchers identify the most effective protocols related to Activating transcription factor 6 (ATF6) for their specific research goals. PubCompare.ai's AI-driven analysis can highlight key differences in protocol effectiveness, enabling you to choose the best option for reproducibility and accuracy.

Q: What are some common applications of Activating transcription factor 6 (ATF6) in human research?

Activating transcription factor 6 (ATF6) is widely studied for its role in maintaining cellular proteostasis and modulating key processes like metabolism, inflammation, and apoptosis. Researchers often investigate how ATF6 dysregulation contributes to the pathogenesis of various human diseases, such as neurodegenerative disorders, cardiovascular disease, and metabolic syndromes. Understanding the power of ATF6 activation can provide valuable insights into disease mechanisms and identify potential therapeutic targets.

Q: Are there different variants or types of Activating transcription factor 6 (ATF6) in humans?

Yes, there are two main isoforms of Activating transcription factor 6 (ATF6) found in humans: ATF6α and ATF6β. Both isoforms serve as sensors for endoplasmic reticulum stress and trigger adaptive signaling pathways, but they may have distinct roles and regulation in the unfolded protein response and other cellular processes. Researchers often study the individual and combined effects of these ATF6 isoforms in various human cell types and disease models.

Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response in humans.
It serves as a sensor for endoplasmic reticulum stress, triggering adaptive signaling pathways that promote cell survival and homeostasis.
ATF6 plays a central role in maintaining proteostasis and modulating various cellular processes, including metabolism, inflammation, and apoptosis.
Disregulation of ATF6 activity has been implicated in the pathogenesis of diverse human diseases, such as neurodegenerative disorders, cardiovascular disease, and metabolic syndromes.
Exploring the power of ATF6 activation in humans can provide valuable insights into disease mechanisms and potential therapeutic targets.
PubCompare.ai's AI-driven approach can help researchers discover protocols from literature, preprints, and patents, while providing AI-powered comparisons to enhance research reproducibility and accuracy.
This single, typo-free platform can uncover the best protocols and products for your ATF6-related studies.

Most cited protocols related to «Activating transcription factor 6, human»

Comprehensive Immunoprecipitation-HPLC Analysis

Cited 5 times

Protein extracts (100 μg) were subjected to immunoprecipitation using a protein A/G agarose column (Amicogen, Korea). Protein A/G agarose columns were separately pre-incubated with 1 μg of 216 different antisera for; growth and proliferation-related proteins (n = 10), cMyc/MAX/MAD signaling proteins (n = 3), p53/Rb/E2F signaling proteins (n = 5), epigenetic modification-related proteins (n = 6), protein translation-related proteins (n = 5), RAS signaling proteins (n = 17), growth factor-related proteins (n = 16), NFkB signaling proteins (n = 12), cellular protection-related proteins (n = 15), upregulated inflammatory proteins (n = 26), downregulated inflammatory proteins (n = 13), p53-mediated apoptosis-related proteins (n = 17), FAS-mediated apoptosis-related proteins (n = 8), angiogenesis-related proteins (n = 20), osteogenesis-related proteins (n = 12), antioxidant-related proteins (n = 8), oncogenic proteins (n = 15), and control housekeeping proteins (n = 3) (Table 2).Table 2

Antibodies used in the study.

Signaling proteins	No.	Antibodies
Cellular proliferation	10	Ki-67^, PCNA^, CDK4^, PLK4^, lamin A/C, MPM2^, p14^, p16^, p21^, p27^*
cMyc/MAX/MAD signaling	3	cMyc^, MAX^, MAD^*
p53/Rb/E2F signaling	5 (2)	p53, Rb-1^#, E2F-1^*, (p21, CDK4)
Wnt/β-catenin signaling	5	Wnt1^, β-catenin^, APC^, snail^, TCF-1^*
Epigenetic modification	6	DMAP1^, histone H1^, KDM4D^$, HDAC-10^$, MBD4^, DNMT1^
Protein translation signaling	5	DOHH^, DHS^, elF5A-1^$, elF5A-2^$, eIF2AK3^*
RAS signaling	17	NRAS^$, KRAS^$, STAT3^, SOS-1^, SOS-2^, RAF-B^, JAK2^$, JNK-1^, ERK1^, Rab^, p-ERK^$, AP-1^@, SP-1^@, SP-3^@, AMPK^@, (PKC^, p-PKC^@)
Growth factor signaling	16	FGF-1^, FGF-2^, HGF^, TGF-β1^#, TGF-β2, SMAD4^, PDGF-A^, IGF-1^, IGFIIR^, GH^, GHRH^, HER1^, HER2^, ERβ^, insulin^@, Met^*
NFkB signaling	12 (3)	NFkB^, IKK^, GADD45^, MDR, mTOR^@, p38^, p-p38^, NRF-2^, IL-1^, (ERK1^, p-ERK^*, AMPK)
Upregulated inflammatory proteins	26 (2)	IL-12^, CD3^, CD28^, CD31^, CD34^, CD40^, CD56^, CD68^, CD80^, CD99^, LL-37^, M-CSF^, MMP-1^$, -2^$, -3^$, -9^$, -10^$, -12^$, TIMP-1^&, TIMP-2^&, CXCR4^, COX1^, COX2^, integrin- α5^, (TGF- β1^#, TGF- β2^*)
Downregulated inflammatory proteins	13 (1)	TNFα^@, IL-1^, IL-6^, IL-8^, IL-10^, IL-28^, LTA4H^&, α1- antitrypsin ^&, lysozyme^, CD20^$, cathepsin C^, cathepsin G^, cathepsin K^*,
Cellular protection-related	15 (2)	LC3, PLC- β2, PI3K, PKC^, p-PKC^, FAK^, caveolin-1^, PGC-1α^, HSP-27^, HSP-70^, HSP-90^, TGase 2^$, p63^$, (pAKT1/2/3^*, HO-1)
Antioxidant-related	8 (3)	HO-1^, SOD-1^, GST-1^, SVCT2^&, NOS-1^$, (PGC-1α ^$, LC3^, NRF-2)
p53-mediated cellular apoptosis	17 (1)	(p53^), PUMA^, NOXA^, MDM2^, BCL2^, BAX^, BAD^, BAK^, BID^, AIF^, APAF-1^, caspase 9^, c-caspase 9^, caspase 3^, c-caspase 3^, PARP^, c-PARP^*
FAS-mediated cellular apoptosis	8 (3)	FASL^, FAS^, FADD^, FLIP^, caspase 8^, (BID^, caspase 3^, c-caspase 3^)
Oncogenic proteins	15 (2)	PTEN^&, MUC1, MUC4, maspin^, BRCA1^&, BRCA2^&, NF-1^, ATM^, CEA^$, 14-3-3^, survivin^@, DMBT1^, TERT^, (pAKT1/2/3^*, MBD4)
Angiogenesis-related proteins	20 (7)	HIF^&, VEGF-A^, VEGF-C^, angiogenin^$, LYVE-1^, CMG2^$, vWF^$, FLT-4^$, ET-1^, PAI-1^, VEGFR^00, plasminogen^, leptin^, (CD31, MMP-2, MMP-9, MMP-10, FGF-1, FGF-2, PDGF-A)
Osteogenesis-related proteins	12 (2)	OPG^, RANKL^, BMP-2^, BMP-4^, ALP^, osteocalcin^, osteopontin^, osteonectin^, RUNX2^, osterix^, (HSP-90, cathepsin K)
Control housekeeping proteins	3	α-tubulin^, β-actin^, GAPDH^*
Total	216 (28)

*Santa Cruz Biotechnology, USA; ^#DAKO, Denmark; ^$Neomarkers, CA, USA; ^@ZYMED, CA, USA; ^&Abcam, Cambridge, UK; the number of antibodies overlapped; ().

Abbreviations: AMPK; AMP-activated protein kinase, pAKT; v-akt murine thymoma viral oncogene homolog, p-Akt1/2/3 phosphorylated (p-Akt, Thr 308), APAF-1; apoptotic protease-activating factor 1, AP-1; activating protein-1, BAD; BCL2 associated death promoter, BAK; BCL2 antagonist/killer, BAX; BCL2 associated X, BCL-2; B-cell leukemia/lymphoma-2, BID; BH3 interacting-domain death agonist, c-caspase 3; cleaved-caspase 3, CD3; cluster of differentiation 3, CDK4; cyclin dependent kinase 4, CEA; carcinoembryonic antigen, CMG2: capillary morphogenesis protein 2, COX-1; cyclooxygenase-2, COX-2; cyclooxygenase-2, c-PARP; cleaved- PARP (poly-ADP ribose polymerase), DMAP1; DNA methyltransferase 1 associated protein, DMBT1; deleted in malignant brain tumors 1, DOHH; deoxyhypusine hydroxylase, DHS; deoxyhypusine synthase, E2F-1; transcription factor, eIF2AK3 (PERK); eukaryotic translation initiation factor 2 (protein kinase R (PKR)-like endoplasmic reticulum kinase), elF5A-1; eukaryotic translation initiation factor 5A-1, elF5A-2; eukaryotic translation initiation factor 5A-2, ERβ; estrogen receptor beta, ERK; extracellular signal-regulated protein kinases, ET-1: endothelin-1, FAS; CD95/Apo1, FASL; FAS ligand, FADD; FAS associated via death domain, FGF-1; fibroblast growth factor-1, FLIP; FLICE-like inhibitory protein, FLT-4; Fms-related tyrosine kinase 4, GADD45; growth arrest and DNA-damage-inducible 45, GAPDH; glyceraldehyde 3-phosphate dehydrogenase, GH; growth hormone, GHRH; growth hormone-releasing hormone, GST-1; glutathione S-transferase ω 1, HDAC-10; histone deacetylase 10, HIF-1α: hypoxia inducible factor-1α, HO-1; heme oxygenase 1, HER1; human epidermal growth factor receptor 1, HGFα; hepatocyte growth factor α, HSP-70; heat shock protein-70, IKK; ikappaB kinase, IGF-1; insulin-like growth factor 1, IGFIIR; insulin-like growth factor 2 receptor, IgK; immunoglobulin kappa (light chain), IL-1; interleukin-1, KDM4D; Lysine-specific demethylase 4D, JNK-1; Jun N-terminal protein kinase, KRAS; V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog, LC3; microtubule-associated protein 1 A/1B-light chain 3, LYVE-1: lymphatic vessel endothelial hyaluronan receptor 1, MAX; myc-associated factor X, MBD4; methyl-CpG-binding domain protein 4, M-CSF; macrophage colony-stimulating factor, MDM2; mouse double minute 2 homolog, MDR; multiple drug resistance, MMP-1; matrix metalloprotease-1, MPM2; mitotic protein monoclonal 2, mTOR; mammalian target of rapamycin, cMyc; V-myc myelocytomatosis viral oncogene homolog, NFkB; nuclear factor kappa-light-chain-enhancer of activated B cells, NOS-1; nitric oxide synthase 1, NRAS; neuroblastoma RAS Viral Oncogene homolog, NRF2; nuclear factor (erythroid-derived)-like 2, p14, p16, p21, p27, p38, PAI-1; plasminogen activator inhibitor-1, PARP; poly-ADP ribose polymerase, PCNA; proliferating cell nuclear antigen, PDGF-A: platelet-derived growth factor-A, PLC-β2; 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterse β-2, PI3K; phosphatidylinositol-3-kinase, PLK4; polo like kinase 4 or serine/threonine-protein kinase, PKC; protein kinase C, p-p38; phosphor-p38, PTEN; phosphatase and tensin homolog, RANKL; receptor activator of nuclear factor kappa-B ligand, Rb-1; retinoblastoma-1, RUNX2; Runt-related transcription factor-2, SMAD4; mothers against decapentaplegic, drosophila homolog 4, SOD-1; superoxide dismutase-1, SP-1; specificity protein 1, STAT3; signal transducer and activator of transcription-3, TGF-β1; transforming growth factor-β1, TERT; human telomerase reverse transcriptase, TNFα; tumor necrosis factor-α, β-actin, 14-3-3, VEGF vascular endothelial growth factor, VEGFR2: vascular endothelial growth factor receptor 2, p-VEGFR2: vascular endothelial growth factor receptor 2 (Y951), vWF: von Willebrand factor.

Briefly, protein samples were mixed with 5 mL of binding buffer (150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF and 0.5% NP-40) and incubated in protein A/G agarose (Amicogen, Korea) columns at 4 °C for 1 hour (columns were placed on a rotating stirrer during incubation). After washing each column with sufficient phosphate buffered saline solution, target proteins were eluted with 150 μL of IgG elution buffer (Pierce, USA). Immunoprecipitated proteins were analyzed using a HPLC unit (1100 series, Agilent, USA) equipped with a reverse phase column and a micro-analytical detector system (SG Highteco, Korea). Elution was performed using 0.15 M NaCl/20% acetonitrile solution at 0.4 mL/min for 30 min, and proteins were detected by UV spectroscopy at 280 nm. Control and experimental samples were run sequentially to allow comparisons^{12 (link),30 ,31}. For IP-HPLC, whole protein peak areas (mAU*s) were calculated after subtracting negative control antibody peak areas, and the square roots of protein peak areas were calculated to normalize concentrations (Supplementary Fig. 2). Protein percentages in total proteins in experimental and control groups were plotted. Analyses were repeated two to six times to achieve mean standard deviations of ≤±5%. Results were analyzed using the Chi-squared test.
The expressions of housekeeping proteins, that is, β-actin, α-tubulin, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal controls. Expressional changes of housekeeping proteins were adjusted to <±5% using a proportional basal line algorithm. To describe protein expressional changes, we tentatively defined a ≤±5% change as minimal, ±5–10% as slight, ±10–20% as meaningful, and a ≥±20% change as marked.

Kim M.K., Yoon C.S., Kim S.G., Park Y.W., Lee S.S, & Lee S.K. (2019). Effects of 4-Hexylresorcinol on Protein Expressions in RAW 264.7 Cells as Determined by Immunoprecipitation High Performance Liquid Chromatography. Scientific Reports, 9, 3379.

Full text: Click here

Publication 2019

Comprehensive Protein Profiling by Optimized Immunoprecipitation-HPLC

Cited 3 times

Protein extracts (~ 100 μg) were immunoprecipitated using protein A/G agarose columns (Amicogen, South Korea), which were separately pre-incubated with 1 μg of 197 different antisera, including proliferation-related proteins (n = 11), cMyc/MAX/MAD network proteins (n = 3), p53/Rb/E2F signaling proteins (n = 5 (1)), epigenetic modification proteins (n = 6), protein translation proteins (n = 5), growth factors (n = 16), RAS signaling proteins (n = 12), NFkB signaling proteins (n = 12 (2)), cellular stress and adaptation proteins (16 (8)), cellular differentiation proteins (n = 11), inflammatory proteins upregulated (n = 13 (2)), inflammatory proteins downregulated (n = 17), cell protection-related proteins (n = 19), antioxidant-related proteins (n = 6), p53-mediated apoptosis proteins (n = 13 (2)), FAS-mediated apoptosis proteins (n = 9 (1)), angiogenesis-related proteins (n = 17 (4)), antioxidant and protection-related (11 (2)), oncogenic proteins (n = 12 (4)), and cytoplasmic housekeeping proteins (n = 3) (Table 2). All antibodies were acquired commercially and were suitable for IP and specific for proteins of mouse origin.Table 2

Antibodies used in the study

Signaling proteins	No.	Antibodies
Proliferation-related proteins	11	Ki-67^*, PCNA^a, CDK4^a, PLK4^a, lamin A/C, MPM2^a, cyclin D2, p14, p16^a, p21^a, p27^a
cMyc/MAX/MAD network proteins	3	cMyc^a, MAX^a, MAD^a
p53/Rb/E2F signaling proteins	5 (1)	p53, Rb-1^#, E2F-1^a, MDM2^a, (CDK4)
Wnt/β-catenin signaling proteins	5	Wnt1^a, β-catenin^a, APC^a, snail^a, TCF-1^a
Epigenetic modification proteins	6	DMAP1^a, histone H1^a, KDM4D^$, HDAC-10^$, MBD4^a, DNMT1^a
Protein translation proteins	5	DOHH^a, DHS^a, elF5A-1^$, elF5A-2^$, eIF2AK3^a
Growth factor proteins	16	FGF-1^a, FGF-2^a, HGF-1^a, TGF-β1^#, TGF-β2, SMAD4^a, IGF-1^a, IGFIIR^a, GH^a, GHRH^a, HER1^a, HER2^a, Erβ^a, insulin^@, Met^a, CTGF^a
RAS signaling proteins	12	NRAS^$, KRAS^$, STAT3^a, SOS-1^a, SOS-2^a, RAF-B^a, JNK-1^a, ERK1^a, p-ERK^$, MEKK^a, pAKT1/2/3, PI3K^a
NFkB signaling proteins	12 (2)	NFkB^a, IKK^a, GADD45^a, GADD153, MDR^a, mTOR^@, p38^a, p-p38^a, AMPK, PGC-1α, (ERK1^a, p-ERK^a)
Cellular stress and adaptation proteins	16 (8)	LC3^a, PLC-β2^a, PKC^a, p-PKC^a, AKAP^a, NFAT5^a, leptin^a, HXK II^a, (pAKT1/2/3^a, p38^a, GADD45^a, PI3K^a, MDM2^a, mTOR^a, ERK-1^a, MDR^a)
Inflammatory proteins upregulated	13 (2)	IL-8, IL-12^a, CD31^a, COX1, α1-AT, LL-37, hepcidin^a, (TGF- β1^#, TGF- β2), MMP-1^a, MMP-3^a, MMP-9^a, MMP-10^a
Inflammation proteins downregulated	17	TNFα^@, IL-1^a, IL-6^a, IL-10^a, IL-28^a, M-CSF^a, lysozyme^a, COX-2^a, CD56^a, CD68^a, CD99^a, cathepsin C^a, cathepsin G^a, CRP-1^a, lactoferrin^a, MMP-2^a, MMP-12^a
Cellular differentiation proteins	11	TGase-2^$, p63^$, caveolin^a, Jagged2^a, Notch 1^a, GLI-1^a, Muc1^a, Muc4^a, AP-1^a, SP-1^a, SP-3^a
Antioxidant and protection-related	11 (2)	HO-1^a, SOD-1^a, GST-1^a, NOS-1^$, LC3^a, HSP-27^a, HSP-70^a, HSP-90^a, (leptin^a, hepcidin), NRF2^a
p53-mediated apoptosis proteins	13 (2)	BCL2^a, BAX^a, BAD^a, BAK^a, BID^a, AIF^a, APAF-1^a, caspase 9^a, c-caspase 9^a, PARP^a, c-PARP^a(p53^a, MDM2^a)
FAS-mediated apoptosis proteins	9 (1)	FASL^a, FAS^a, FADD^a, FLIP^a, caspase 8^a, caspase 3^a, c-caspase 3^a, (BID)
Oncogenic proteins	12 (4)	PTEN^&, DMBT-1^a, CEA^a, 14–3-3^a, survivin^@, YAP^a 1, TERT^a, (pAKT1/2/3^a, MBD4^a, Muc1^a, Muc4^a), ATM^a
Angiogenesis-related proteins	17 (4)	HIF^&, VEGF-A^a, VEGF-C^a, angiogenin^$, LYVE-1^a, CMG2^$, vWF^$, FLT-4^$, ET-1^a, PAI-1^a, plasminogen, PDGF-A^a, VCAM^a, (COX-1^a, leptin^a, CD31, FGF-2^a)
Control housekeeping proteins	3	α-tubulin^a, β-actin^a, GAPDH^a
Total	223 (26)	tTotal 197 antibodies

The number inside the parenthesis represents the antibodies overlapped

^aSanta Cruz Biotechnology, USA

^#DAKO, Denmark

^$Neomarkers, CA, USA

^@ZYMED, CA, USA

^&Abcam, Cambridge, UK

Abbreviations: α1-AT α-1 antitrypsin; AMPK AMP-activated protein kinase; p-AKT1/2/3 phosphorylated (p-Akt, Thr 308); APAF-1 apoptotic protease-activating factor 1; AP-1 activating protein-1; BAD BCL2 associated death promoter; BAK BCL2 antagonist/killer; BAX BCL2-associated X; BCL-2 B-cell leukemia/lymphoma-2; BID BH3 interacting-domain death agonist; c-caspase 3 cleaved-caspase 3, caveolin; CD31/56/68/99 cluster of differentiation 31/56/68/99; CDK4 cyclin-dependent kinase 4; CEA carcinoembryonic antigen; CMG2 capillary morphogenesis protein 2; COX-1 cyclooxygenase-2; COX-2 cyclooxygenase-2; c-PARP cleaved-PARP (poly-ADP ribose polymerase); CTGF connective tissue growth factor, cyclin D2; DMAP1 DNA methyltransferase 1-associated protein; DMBT1 deleted in malignant brain tumors 1; DOHH deoxyhypusine hydroxylase; DHS deoxyhypusine synthase; E2F-1 transcription factor; eIF2AK3 (PERK) eukaryotic translation initiation factor 2 (protein kinase R (PKR)-like endoplasmic reticulum kinase); elF5A-1 eukaryotic translation initiation factor 5A-1; elF5A-2 eukaryotic translation initiation factor 5A-2; ERβ estrogen receptor beta; ERK extracellular signal-regulated protein kinases; ET-1 endothelin-1; FAS; CD95/Apo1; FASL FAS ligand; FADD FAS associated via death domain; FGF-1 fibroblast growth factor-1; FLIP FLICE-like inhibitory protein; FLT-4 Fms-related tyrosine kinase 4; GADD45 growth arrest and DNA-damage-inducible 45; GAPDH glyceraldehyde 3-phosphate dehydrogenase; GH growth hormone; GHRH growth hormone-releasing hormone; GST-1 glutathione S-transferase-1; HDAC-10 hepcidin; HIF hypoxia inducible factor-1α; HO-1 heme oxygenase 1; HER2 human epidermal growth factor receptor 2; HGF hepatocyte growth factor; HSP-27/70/90 heat shock protein-27/70/90; HXK II hexokinase II; IKK IkappaB kinase, IGF-1, IGFIIR; IL-1/6/8/12/28 interleukin-1/6/8/12/28, Jagged2; JNK-1 Jun N-terminal protein kinase; KRAS V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; LC3 microtubule-associated protein 1A/1B-light chain 3, leptin; LYVE-1 lymphatic vessel endothelial hyaluronan receptor 1, MAX Myc-associated factor X; MBD4 methyl-CpG-binding domain protein 4; M-CSF macrophage colony-stimulating factor; MEKK MEK kinase; MDM2 mouse double minute 2 homolog; MDR multiple drug resistance; MPM2 mitotic protein monoclonal 2; mTOR mammalian target of rapamycin; cMyc V-myc myelocytomatosis viral oncogene homolog; NOS-1 nitric oxide synthase 1; NRAS neuroblastoma RAS viral oncogene homolog, Notch 1, p16, p21, p27, p38, p53, p63, PAI plasminogen activator inhibitor-1; PARP poly-ADP ribose polymerase; PCNA proliferating cell nuclear antigen, PDGF-A platelet-derived growth factor-A; PLC-β2 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterse β-2; PI3K phosphatidylinositol-3-kinases; PLK4 polo-like kinase 4 or serine/threonine-protein kinase; PKC protein kinase C, p-p38 phosphor-p38; PTEN phosphatase and tensin homolog; Rb-1 retinoblastoma-1; SMAD4 mothers against decapentaplegic; drosophila homolog 4; SOD-1 superoxide dismutase-1; SP-1 specificity protein 1; STAT3 signal transducer and activator of transcription-3; TGF-β1 transforming growth factor-β1; TERT human telomerase reverse transcriptase; TNF-α tumor necrosis factor-α, β-actin, 14-3-3; VEGF vascular endothelial growth factor; VCAM vascular cell adhesion, vWF Von Willebrand factor; YAP 1 yes-associated protein

Briefly, protein samples were mixed with 5 mL of binding buffer (150 mM NaCl, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5% Tergitol-type NP-40 (nonyl phenoxypolyethoxylethanol) and incubated in protein A/G agarose columns at 4 °C for 1 h (columns were placed on a rotating stirrer during the incubation). After washing each column with sufficient PBS (phosphate-buffered saline), target proteins were eluted using 150 μL of IgG elution buffer (Pierce, USA). Immunoprecipitated proteins were analyzed using a HPLC unit (1100 series, Agilent, USA) equipped with a reverse phase column and a micro-analytical detector system (SG Highteco, South Korea). Elution was performed using 0.15 M NaCl containing 20% acetonitrile at 0.4 mL/min for 30 min, and detection by UV spectroscopy at 280 nm. Control and experimental samples were run sequentially to allow comparisons. For IP-HPLC, whole protein peak areas (mAU*s) were calculated by subtracting the antibody peak areas of negative controls, and experimental protein peak area square roots were compared with control one (Additional file 2) [16 (link)].
When IP-HPLC results were compared with western blot data for cytoplasmic housekeeping protein (β-actin), IP-HPLC errors were < ± 5%, whereas western blot errors exceeded ± 20% and were not suitable for statistical analysis (Additional file 3) [16 (link)]. In particular, repeat IP-HPLC runs (4–10 runs) to determine errors associated with protein expression revealed errors were ± 5% (Additional file 4) [16 (link)]. Based on these findings, IP-HPLC was used rather than western blot to analyze protein expressional changes.

Yoon C.S., Kim M.K., Kim Y.S, & Lee S.K. (2018). In vivo protein expression changes in mouse livers treated with dialyzed coffee extract as determined by IP-HPLC. Maxillofacial Plastic and Reconstructive Surgery, 40(1), 44.

Full text: Click here

Publication 2018

Assessing UPR and Apoptosis in Tissue Sections

Cited 1 time

Human and rat tissues were sectioned at 5 μm for either hematoxylin and eosin staining or immunohistochemical staining. Activation of UPR was assessed by antibody reactivity to activating transcription factor 6 (ATF6, Imgenex-273 1:100) and C/EBP homologous protein (CHOP, Abcam Ab11419 1:100). Apoptotic cells were identified by immunoreactivity to cleaved caspase-3 antibody (Abcam Ab47131 1:200). The cleaved caspase-3-positive cells were counted and divided by the total number of cells in three randomly chosen high-power field (×400) for each section from each of three animals per group. The mean percentage of caspase-3+ cells for each animal was then obtained by averaging the results of the countings. Macrophages were identified by staining with antibodies against CD68 (AbBiotec 250594 1:200). Secondary antibodies conjugated to fluorescent dyes (Invitrogen), or biotin combined with immunoperoxidase/avidin-biotin, were as per the manufacturer protocol (Vectastain ABC kit, Vector Labs), and either hematoxylin or Nuclear Fast Red (Vector Labs) was used as a counterstain. Avidin/biotin blocking kits (Vector Labs) were used to block endogenous enzyme activity. Antibody isotype negative controls were included with each sample group. Images were acquired at room temperature using a Zeiss Axiovert S100 fitted with Zeiss 20×0.4 numerical aperture (n.a.) and 10×0.3 n.a. objectives and Axiocam camera. Acquisition of images was by Axiovision 4.6 software (Zeiss).

Yeager M.E., Reddy M.B., Nguyen C.M., Colvin K.L., Ivy D.D, & Stenmark K.R. (2012). Activation of the unfolded protein response is associated with pulmonary hypertension. Pulmonary Circulation, 2(2), 229-240.

Publication 2012

activating transcription factor 6, human Animals Antibodies Apoptosis Avidin Biotin Cardiac Arrest Caspase 3 Cloning Vectors DDIT3 protein, human enzyme activity Eosin Fever Fluorescent Dyes Hematoxylin Homo sapiens Immunoglobulin Isotypes Immunoglobulins Immunoperoxidase Techniques Macrophage S100 Proteins Tissues

Mapping MOR Interacting Proteins Using Y2H and MYTH Screens

Cited 1 time

The traditional Y2H screening method involves the reconstitution of the GAL4 transcription factor through the interaction of a bait protein fused to the GAL4 DNA binding domain and a prey protein (from a fetal brain cDNA library) fused to the transcriptional activating domain of GAL4 [21] (link). This method is biased for cytosolic and nuclear proteins, as the protein complex must be imported into the nucleus to activate transcription. Therefore, only the cytosolic portion of integral membrane proteins is usually employed in this type of screen. Traditional Y2H screens in this study were performed as previously described [22] , [23] using the second intracellular loop (IL2; amino acids 166–187) of the MOR as bait to screen a fetal human brain cDNA library. The MOR-IL2 was cloned into the yeast GAL4 DNA binding domain expression vector pAS2-1 (Clontech, Palo Alto, CA), while the human fetal brain cDNA library was provided in the GAL4 activation domain vector pACT2 (Clonetech). Bait and prey plasmids were successively transformed into yeast strain MaV103 [22] . Transformation of yeast with the fetal human brain library produced ∼2×10⁶ transformants/µg of DNA on quadruple dropout plates (-Leu/−Trp/−His/−Ura; Clonetech) containing 3-amino-1,2,4-triazol (3AT). Interactions were assayed for β-galactosidase (β-gal) activity via the nitrocellulose lift method [22] . cDNAs were extracted from yeast colonies, sequenced, and subjected to Basic Local Alignment Search Tool (BLAST) analysis to determine their identities.
To identify additional MOR interacting proteins (MORIPs), a modified split-ubiquitin membrane yeast two-hybrid (MYTH) screen was performed as previously described [24] (link). The MYTH system uses the split-ubiquitin method, in which the reconstitution of ubiquitin is mediated by a specific protein-protein interaction. Ubiquitin-specific proteases cleave at the C-terminus of ubiquitin, which releases a transcription factor that can translocate to the nucleus and activate transcription of a reporter gene [25] (link). The unique advantage of MYTH is that full-length integral membrane proteins can be used as bait and are amenable to protein-protein interaction analyses in their natural membrane environment [26] (link), [27] (link). For this study, full-length human MOR cDNA in the bait vector pCCW-STE (Dualsystems Biotech AG, Switzerland) and a human fetal brain library in the prey vector pPR3-N (Dualsystems) were sequentially transformed into S. cerevisiae reporter strain THY.AP4. Transformation of yeast with the human brain library yielded 6×10⁶ transformants/µg DNA on quadruple drop out plates (−Trp/−Leu/−His/−Ade; Clonetech) containing 3AT. Fifty transformants were positive for β-gal activity. These colonies were picked and their cDNAs extracted, sequenced and subjected to BLAST analysis. From this screen we identified four novel MORIPs (Table 1) that were subjected to further biochemical analysis.
To map sites of interaction between the MOR and the newly identified MORIPs, each MOR intracellular loop (IL) was tested for interaction with individual MORIPs using the traditional Y2H method. MOR-IL domains (IL1, amino acids 97–102; IL2, amino acids 166–187; IL3, amino acids 259–282; and C-tail residues 361–420) were separately ligated into pAS2-1 and assayed for interaction with candidate MORIP cDNA clones in pACT2. Bait and prey plasmids were simultaneously co-transformed into S. cerevisiae strain MaV103 and interactions assayed for β-gal activity as described above.

Petko J., Justice-Bitner S., Jin J., Wong V., Kittanakom S., Ferraro T.N., Stagljar I, & Levenson R. (2013). MOR Is Not Enough: Identification of Novel mu-Opioid Receptor Interacting Proteins Using Traditional and Modified Membrane Yeast Two-Hybrid Screens. PLoS ONE, 8(6), e67608.

Full text: Click here

Publication 2013

Uncovering TGFB Isoform Dysregulation in Pediatric Brain Tumors

Cited 1 time

We downloaded clinical metadata and RNA sequencing (RNAseq)-based mRNA expression data for 41 pediatric DMG patients (mean age at diagnosis in months = 7.02 ± 0.44; median = 6; range = 2–14) and 116 pediatric GBM patients (mean age at diagnosis in months = 60.01 ± 1.24; median = 60.4; range = 21–89.3) regarding TGFB isoforms TGFB1, TGFB2, and TGFB3 from the genomic data set repository stored in the cBioPortal for Cancer Genomics (https://pedcbioportal.kidsfirstdrc.org/ (accessed on 4 November 2022)) using the interactive web interface with full filtering functionality provided by the portal [41 (link),42 (link)]. By utilizing the cBioPortal database and an archived dataset that was compiled from harmonized and annotated across multiple data consortiums, such as the open Pediatric Brain Tumor Atlas (PBTA) Project (Project ID = openpbta) and the Pacific Pediatric Neuro-Oncology Consortium Clinical Genomics Atlas (Project ID = pbta_pnoc) [10 (link),43 (link),44 (link),45 (link)], we examined the effect of TGFB2 mRNA expression levels on PFS and OS outcomes. The general treatment strategies are outlined in the clinical trials associated with the treatment of DIPG patients (https://clinicaltrials.gov/ct2/show/NCT02274987 (accessed on 1 January 2023)) that included standard radiation therapy followed by biomarker-guided specialized therapy with FDA-approved targeted therapeutics drugs, which was guided by gene expression analysis, whole exome-sequencing (WES), and predictive modeling. As a limitation of this study, patient-specific treatment information was not available through the cBioPortal database.
The downloaded mRNA expression levels of TGFB1, TGFB2, TGFB3, Transforming Growth Factor Beta Receptor 1 (TGFBR1), Transforming Growth Factor Beta Receptor 2 (TGFBR2) and Transforming Growth Factor Beta Receptor 3 (TGFBR3) were reported using the RNAseq V2 values (datafiles for the expression profiles appended with “mRNA_expression_(RNA_Seq_V2_RSEM).txt”) normalized to “transcripts per million” (“TPM”) values calculated using RSEM, which is a software package for estimating gene and isoform expression levels from RNAseq data [46 (link)]. The RSEM-based process consists of two main steps: 1. A set of reference transcript sequences are generated for gene level mRNA abundance assessment; 2. A set of RNAseq reads are aligned to these reference transcripts for TPM abundance estimations. This process allows for a direct comparison of mRNA abundance and ranking across samples.
The Kaplan–Meier (KM) method, log-rank chi-square test, and the software packages survival_3.2-13, survminer_0.4.9 and survMisc_0.5.5 operated in the R environment were used to compare the PFS and OS outcomes of patient subsets. Graphical representations of the treatment outcomes were generated using graph drawing packages implemented in the R programming environment: dplyr_1.0.7, ggplot2_3.3.5, and ggthemes_4.2.4. The statistical significance of differences in the outcomes of the compared patient subsets was examined using the log-rank chi-square test and p-values less than 0.05 were deemed significant.
TBFB1, TGFB2, and TGFB3 mRNA expression values for normal pons specimens measured by RNAseq (rna_tissue_hpa.tsv.zip) were downloaded from https://www.proteinatlas.org/about/download accessed on 19 December 2022. We compiled the mRNA expression values (in TPM) for 29 distinct pons regions of the brain by filtering the “Tissue Group” annotations the accompanying description file (“rna_tissue_hpa_description.tsv.zip”). A two-way analysis of variance (ANOVA) model was used to compare the TGFB1/TGFB2/TGFB3 mRNA expression levels for the normal pons specimens with the TGFB1/TGFB2/TGFB3 mRNA expression levels in brain tumor specimens from 41 DIPG patients. Contrasts were performed between normal pons and DIPG samples for each of the transcripts and p-values were false discovery rate (FDR)-adjusted. Calculations were performed using multcomp_1.4-17 and emmeans_1.7.0 statistical packages in R version 4.1.2 (1 November 2021) with RStudio front end (RStudio 2021.09.0 + 351 “Ghost Orchid” Release)). Bar chart graphics were constructed using the ggplot2_3.3.5 R package.
mRNA expression levels of TGFB2 in log₂ TPM were correlated with the mRNA expression levels for 11 transcription factors known to augment TGFB2 expression, namely activating transcription factor 1 (ATF1), activating transcription factor 2 (ATF2), cyclic AMP-responsive element binding protein 1 (CREB1), E1A binding protein P300 (EP300), forkhead box protein O3 (FOXO3), polymerase II subunit A (POLR2A), regulatory factor X1 (RFX1), specificity protein 1 transcription factor (SP1), TATA-box-binding protein (TBP), upstream transcription factor 1 (USF1), and upstream transcription factor 2 (USF2) across 41 DIPG patients. Pairwise correlation coefficients were determined for all transcript combinations and visualized on a heatmap which was color-coded from positive correlations (red = +1) to negative correlations (blue = −1). The clustering algorithm identified the co-regulated sets of genes using the statistical package ggcorrplot_0.1.3 implemented in R. T-test was used to test the null hypothesis that the Pearson correlation coefficient was equal to zero. Significant correlations were identified for p-values less than 0.05 and FDR less than 0.10.
Normalized archived transcriptome profiling datasets acquired from the Gene Expression Omnibus web portal (https://www.ncbi.nlm.nih.gov/geo/ accessed 23 November 2021), including raw CEL files obtained using the Human Genome U133 Plus 2.0 Array platform for DIPG (N = 29; GSE26576), normal control samples (N = 2; GSE26576), and pediatric GBM patients harboring H3K27M mutations (N = 5, GSE34824; N = 7, GSE49822) [39 ] were also utilized as an independent validation dataset to compare the mRNA expression levels of TGFB1, TGFB2, and TGFB3 in normal control samples vs. brain tumor specimens from 41 patients with pediatric DIPG or H3K27M-mutant pediatric GBM. The normalization procedure to determine log₂-transformed mRNA expression levels employed the method of Robust Multi-array Averaging (RMA), as previously described [39 ]. mRNA expression levels were calculated using Aroma Affymetrix statistical packages (aroma.affymetrix_3.2.0, aroma.core_3.2.2 and aroma.light_3.24.0) run in the RStudio environment (R version 4.1.2 (1 November 2021), RStudio 2021.09.0 Build 351). Statistical comparisons were performed using an ANOVA statistical model. FDR-adjusted p-values less than 0.05 were deemed significant. mRNA expression levels in DIPG/H3K27M-mutant GBM patients for TGFB1, TGFB2, and TGFB3 were visualized using heatmaps, as described [39 ]. Expression levels of TGFB2 mRNA (log₂ RMA) were correlated to mRNA a two-way ANOVA model: expression levels for 11 transcription factor genes known to augment TGFB2 expression; ATF1, ATF2, CREB1, EP300, FOXO3, POLR2A, RFX1, SP1, TBP, USF1, and USF2.

Uckun F.M., Qazi S, & Trieu V. (2023). High Intra-Tumor Transforming Growth Factor Beta 2 Level as a Predictor of Poor Treatment Outcomes in Pediatric Diffuse Intrinsic Pontine Glioma. Cancers, 15(6), 1676.

Full text: Click here

Publication 2023

Most recents protocols related to «Activating transcription factor 6, human»

Selenoprotein Regulation by TFAP2C and Sp1

Not available on PMC !

Example 3

Since the data establish a link between Se-induced GPX4 transcription and protection from neuronal ferroptosis, we examined the GPX4 promoter and upstream region to better understand how transcription of GPX4 (and other genes in the protective selenome) are induced by Se. Transcription assays using a transiently transfected reporter construct containing the 4 kb region upstream from the GPX4 translation start site showed significant induction of GPX4 promoter activity in neurons exposed to protective Se concentrations. Targeted deletions of this upstream region revealed that the −1189 bp and −1467 bp sub-region was critical for Se-induced promoter activity (FIGS. 4A, 40, and 41). Analysis of this critical sub-region identified five motifs with similarity to Transcription Factor AP-2 family binding sites (5′-GCCNNN(NN)GGC-3′; (SEQ ID NO:27), FIG. 4B) (where sequences 1 to 5 are SEQ ID NOS:22, 23, 24, 25 and 26, respectively) (See Williamson et al., Genomics 35(1):262-4 (1996)). Mutation of three of these motifs blocked Se-induced GPX4 promoter activity (FIG. 4C). The TFAP-2 family is highly conserved in mice and humans and is represented by five isoforms (TFAP2A-E) (See Eckert et al., Genome Biol 6(13): 246 (2005)). Since TFAP2C was the only isoform we detected in mouse primary neurons (FIG. 11 (panels A and B), FIG. 42, FIG. 67A, and FIG. 67B), we focused on this isoform. Chromatin immunoprecipitation (ChIP) assays showed that Se exposure significantly increased TFAP2C occupancy on the Se-responsive region of GPX4 (FIGS. 4D and 43). This binding to the upstream region was significantly stronger at 4 vs. 6 hours post-Se treatment (FIG. 4D). Since GPX4 expression levels are elevated longer than 4 hours following Se exposure, additional transcription factors likely sustain the activation of GPX4 transcription. TFAP2C binding overlaps with Sp1 in other gene promoters (See Orso et al., BMC Genomics 11: 355 (2010); Yang et al., J Biol Chem 270(15): 8514-20 (1995)), and our lab previously showed that Sp1 DNA binding is significantly induced by ferroptotic oxidative stress (See Ryu et al., J Neurosci 23(9): 3597-606 (2003)). Thus, we examined whether Se exposure induced Sp1 binding to the Se-responsive region of the GPX4 upstream region. ChIP studies showed that Se exposure induced the Sp1 occupancy of this region, but unlike TFAP2C, this occupancy was significantly greater at 6 vs 4 hours post Se treatment (FIGS. 4E and 4F, 11 (panels C and D), and 43). To confirm that the Se-responsive region of GPX4 could mediate TFAP2C and Sp1 dependent regulation, human TFAP2C or Sp1 were overexpressed with either a wild-type GPX4 reporter or a mutant reporter lacking the TFAP2C binding sites. These studies showed that TFAP2C and Sp1 activated only wild-type reporter, which suggests that TFAP2C and Sp1 act via the same DNA binding site(s) (FIGS. 4G and 4H). In addition, over-expression of TFAP2C or Sp1 drove mRNA expression for several ferroptotic-stress response genes and protected neurons from hemin or HCA-induced ferroptosis (FIGS. 4I, 4J, 44A, and 44B). By contrast, overexpression of a mutant Sp1 protein with a zinc-finger DNA binding domain deletion (FIG. 45) did not induce expression of ferroptotic-stress response genes or provide protection from HCA (FIGS. 41, 4J, 44A, 44B, and 46). Overexpression of the mutant Sp1 protein also reduced protection mediated by Se treatment in primary neurons or HT1080 fibrosarcoma cells (FIGS. 4J, 4K, and 47). Together, these findings indicate that Se provides protection from ferroptosis, at least in part, by activating gene expression regulated by TFAP2C and Sp1.

US11878997B2. Methods of treatment comprising Selenoprotein P fragment-containing compounds (2024-01-23). Burke Neurological Institute [US], University of Vermont and State Agricultural College [US]. Inventors: Rajiv R. Ratan [US], Ishraq Alim [US], Saravanan Karuppagounder [US], Robert Hondal [US].

Full text: Click here

Patent 2024

Quantitative Immunohistochemical Analysis of Cellular Pathways

For IHC staining, paraffin-embedded tissues were cut into 4-µm-thick sections and transferred onto microscope slides using a semi-automated rotary microtome (Leica Biosystems, Wetzlar, Germany). Each microscope slide was evaluated under the BX41 light microscope (Olympus Co., Tokyo, Japan) to confirm the presence of all tissue structures. Antibodies involved in the UPR, inflammatory signaling, programmed cell death, angiogenesis, or epithelial–mesenchymal transition were selected for IHC analysis, as shown in Table 1.Table 1

Antibodies used in this study for immunohistochemistry analysis.

Pathways/signaling	Number	Antibodies
Unfolded protein response	3	PERK, ATF6, XBP1
Inflammatory related	1	NFκB
Programmed cell death
Apoptosis	1	CHOP/GADD153
Necroptosis	1	MLKL
Autophagy	1	LC3
Angiogenesis	2	IL-8, HIF-1
Epithelial-mesenchymal transition	3	TGF-β, αSMA, E-cadherin
Total	12

All antibodies were obtained from Santa Cruz Biotechnology, USA.

*αSMA, α-smooth muscle actin; ATF6, activating transcription factor 6; CHOP/GADD153, CCAAT-enhancer-binding protein homologous protein/growth arrest-and DNA damage-inducible gene 153; E-cadherin, epithelial cadherin; HIF-1, hypoxia-inducible factor 1; IL-8, interleukin 8; LC3, microtubule-associated protein 1 light chain 3; MLKL. Mixed lineage kinase domain-like protein; NFκB, nuclear factor kappa-light-chain-enhance of activated B cells; PERK, protein kinase RNA activated (PKR-like ER kinase); TGFβ, transforming growth factor beta; XBP1, xbox-binding protein 1.

In the IHC protocol, paraffin-embedded tissue sections underwent paraffin removal, which involved drying in the oven for 1 h, sequential immersion in xylene and ethanol with concentration reduction from 100 to 50%, and a final distilled water rinse. Antigen retrieval was achieved through Proteinase K treatment and washing with phosphate-buffered saline. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 min to remove the blood cells and avoid false-positive results, followed by background blocking using 10% donkey serum. Primary antibodies were applied and incubated overnight at refrigerated temperatures. Slides were then left at room temperature for 2 h. Universal secondary antibodies were applied, followed by avidin–biotin complex reagent and diaminobenzidine (DAB) substrate application for 5 min, with monitoring for brown coloration. Hematoxylin staining was performed, followed by cover glass application.
For quantitative IHC analysis, we employed an open-source digital pathology software program (QuPath^®; Northern Ireland Molecular Pathology Lab., Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, UK) as a fundamental component of our materials and methods^{33 (link)}. QuPath^® has proven to be an indispensable tool for research, providing a comprehensive platform for the accurate and efficient assessment of IHC staining patterns and biomarker expression within tissue samples^{34 (link)–36 (link)}. Automated IHC measurements offer a solution to the challenges associated with subjective visual scoring by pathologists. Whole-slide imaging systems readily transform glass slides into high-quality digital images. These automated measurements demonstrate precision, especially in detecting subtle staining that may be difficult for the human eye to discern. Additionally, they generate continuous and reliable data. When integrated into the visual scoring process, computer-aided IHC analysis significantly enhances both intra- and inter-observer agreement among pathologists^{37 (link),38 (link)}.
The analysis process involved importing IHC images into QuPath^® and creating a structured project workspace. Within this workspace, we established representative brightfield H-DAB stain vectors encompassing the representative area consisting of representative hematoxylin and DAB stains and a background area set to auto. Subsequently, we defined regions of interest (ROIs) using QuPath^®’s “create thresholder” annotation tool, enabling precise localization of target tissue regions. For angiogenesis using interleukin-8 and HIF antibodies, the reticulum dermis with abundant blood vessels was selected as an ROI using the rectangle annotation. To quantify staining characteristics, we used QuPath^®’s positive cell-detection algorithms to automatically identify and classify cells within the ROIs, facilitating the calculation of quantitative data for each cell (Supplementary Table S2). The Histoscore (H-score) of the positive slide was generated and subjected to further statistical analysis (Supplementary Fig. S3). The H-score serves as a quantitative metric for the transformation of traditional IHC into a more objective range. It is determined considering both the intensity of staining and the proportion of stained cells within a given sample^{37 (link)}. In our study, H-scores were interpreted as < 10 points (negative), 10–100 points (low), 101–200 points (moderate), and > 200 points (intense), as described in previous literature^{39 (link)}.

Mustakim K.R., Eo M.Y., Seo M.H., Yang H.C., Kim M.K., Myoung H, & Kim S.M. (2024). Ultrastructural and immunohistochemical evaluation of hyperplastic soft tissues surrounding dental implants in fibular jaws. Scientific Reports, 14, 10717.

Full text: Click here

Publication 2024

Autoantigen Profiling in Systemic Autoimmune Diseases

Not available on PMC !

Example 8

In a second screening with 6088 antigens, the antigens which differentiate between healthy controls and donors with rheumatoid arthritis were tested on patients with early rheumatoid arthritis, SSc and SPA., This is of importance in particular since patients with collagenoses and mixed collagenoses have an overlapping autoantibody profile and therefore are difficult to diagnose, particularly in the early phase

FIG. 3 shows a volcano plot of the antigen reactivities of SLE patients against a combined group of patients with various autoimmune diseases, such as SSc, SPA, early rheumatoid arthritis, and SPA.

Following univariate statistical evaluation, a threshold value of p<0.05 and a 1.5 times modified reactivity compared with the control group were applied. A final list of antigen reactivities over both screens was established (Table 2).

In order to analyse the frequency of the newly identified antigens in comparison with, known antigens, a threshold value of 3 standard deviations (SD) above the mean value of the healthy samples was defined.

Astonishingly, at least 4 additional antigens were identified of which the frequency in SLE patients lies above 15%. These include TMPO (19%) (SEQ ID No. 13), HNRNPA1 (26%) (SEQ ID No. 5), XRCC5 (15%) (SEQ ID No. 22) and MVP (15%) (SEQ ID No. 7).

FIG. 4 shows the frequency of 23 antigens in comparison to the healthy controls.

Table 2 summarises the identified antigen reactivities and different group comparisons.

TABLE 2

List of all antigen reactivities

Statistical Test

SEQGeneGeneGenePanelSLEENA-4 negL. Nephr.SLE

ID No.IDSymbolNameGroupSLEL. Nephr.Clustervs HVvs SLEvs control

11629DBTdihydrolipoamide1xxSLE

branched chainvs

transacylase E2AID

21737DLATdihydrolipoamide S-1xSLE

acetyltransferasevs

AID

37430EZRezrin1xxxSLE

AID

43017HIST1H2BDhistone cluster 1,1xxSLE

H2bdvs

AID

53178HNRNPA1heterogeneous1xxSLE

nuclearvs

ribonucleoprotein A1AID

63181HNRNPA2B1heterogeneous1xxSLE

nuclearvs

ribonucleoproteinAID

A2/B1

79961MVPmajor vault protein1xxxxSLE

AID

86175RPLP0ribosomal protein,1xxxSLE

large, P0vs

AID

96176RPLP1ribosomal protein,1xxxxSLE

large, P1vs

AID

106181RPLP2ribosomal protein,1xxxSLE

large, P2vs

AID

1130011SH3KBP1SH3-domain kinase1xxSLE

binding protein 1vs

AID

126625SNRNP70small nuclear1xSLE

ribonucleoproteinvs

70 kDa (U1)AID

136628SNRPBsmall nuclear1xxxSLE

ribonucleoproteinvs

polypeptides B andAID

146638SNRPNsmall nuclear1xSLE

ribonucleoproteinvs

polypeptide NAID

156672SP100SP100 nuclear1xxSLE

antigenvs

AID

166710SPTBspectrin, beta,1xxSLE

erythrocyticvs

AID

176741SSBSjogren syndrome1xxSLE

antigen Bvs

(autoantigen La)AID

187112TMPOthymopoietin1xxxSLE

AID

196737TRIM21tripartite motif-1xxxSLE

containing 21vs

AID

206738TROVE2TROVE domain family,1xxSLE

member 2vs

217431VIMvimentin1xxSLE

AID

227520XRCC5X-ray repair1xxSLE

complementingvs

defective repair inAID

Chinese hamster

cells 5 (double-

strand-break

re joining)

237764ZNF217zinc finger protein1xxSLE

217vs

AID

2464763ZNF574zinc finger protein1xxSLE

574vs

AID

25148741ANKRD35ankyrin repeat2xxSLE

domain 35vs

2684779ARD1BARD1 homolog B2xxSLE

(S. cerevisiae)vs

AID

27672BRCA1breast cancer 1,2xxSLE

early onsetvs

28134359C5orf37chromosome 5 open2xxxSLE

reading frame 37vs

299478CABP1calcium binding2xxSLE

protein 1vs

3090557CCDC74Acoiled-coil domain2xxSLE

containing 74Avs

319973CCScopper chaperone for2xxxxSLE

superoxide dismutasevs

AID

321410CRYABcrystallin, alpha B2xxSLE

3355802DCP1ADCP1 decapping2xxSLE

enzyme homolog Avs

(S. cerevisiae)HV

3479147FKRPfukutin related2xSLE

proteinvs

3526128KIAA1279KIAA12792xxSLE

3657608KIAA1462KIAA14622xxSLE

371939LGTNligatin2xxSLE

3884298LLPHLLP homolog, long-2xxSLE

term synapticvs

facilitationHV

(Aplysia)

3911253MAN1B1mannosidase, alpha,2xxSLE

class 1B, member 1vs

4084930MASTLmicrotubule2xxSLE

associatedvs

serine/threonineHV

kinase-like

4154531MIER2mesorm induction2xxxxSLE

early response 1,vs

family member 2RA

424594MUTmethylmalonyl2xxSLE

Coenzyme A mutasevs

43399687myO18Amyosin XVIIIA2xxSLE

448883NAE1NEDD8 activating2xxSLE

enzyme E1 andvs

subunit 1HV

4510458BAIAP2BAI1-associated2xxSLE

protein 2vs

464869NPM1nucleophosim2xxSLE

(nucleolarvs

phosphoprotein B23,HV

numatrin)

475223PGAM1phosphoglycerate2xxSLE

mutase 1 (brain)vs

4811040PIM2pim-2 oncogene2xxSLE

4954517PUS7pseudouridylate2xxSLE

synthase 7 homologvs

(S. cerevisiae)HV

506605SMARCE1SWI/snf related,2xxSLE

matrix associated,vs

actin dependentAID

regulator of

chromatin,

subfamily e, member

5123635SSBP2single-stranded DNA2xxxSLE

binding protein 2vs

5283660TLN2talin 22xxSLE

5351673TPPP3tubulin2xxSLE

polymerization-vs

promoting proteinHV

family member 3

547265TTC1tetratricopeptide2xxSLE

repeat domain 1vs

55124930ANKRD13Bankyrin repeat3xSLE

domain 13Bvs

56160AP2A1adaptor-related3xSLE

protein complex 2,vs

alpha 1 subunitHV

5753335BCL11AB-cell CLL/lymphoma3xx

11A (zinc finger

protein)

5879959CEP76centrosomal protein3x

76 kDA

591153CIRBPcold inducible RNA3xSLE

binding proteinvs

6051084CRYL1crystallin, lambda 13x

6155827DCAF6DDB1 and CUL43xxxSLE

associated factor 6vs

AID

626993DYNLT1dynein, light chain,3xSLE

Tctex-type 1vs

63283991FAM100Bfamily with sequence3xSLE

similarity 100,vs

member BHV

649815GIT2G protein-coupled3xSLE

receptor kinasevs

interacting ArfGAP 2HV

6584706GPT2glutamic pyruvate3x

transaminase

(alanine

aminotransferase) 2

663059HCLS1hematopoieti cell-3xxSLE

specific Lynvs

substrate 1AID

673329HSPD1heat shock 60 kDa3x

protein 1

(chaperonin)

683490IGFBP7insulin-like growth3xSLE

factor bindingvs

protein 7HV

6923392KIAA0368KIAA03683x

7084695LOXL3lysyl oxidase-like3x

714133MAP2microtubule-3xSLE

associated proteinvs

2HV

726837MED22mediator complex3x

subunit 22

7329079MED4mediator complex3xx

subunit 4

7410933MORF4L1mortality factor 43x

like 1

7564963MRPS11mitochondrial3xxSLE

ribosomal proteinvs

7681565NDEL1nudE nuclear3x

distribution gene E

homolog

(A. nidulans)-like 1

7757447NDRG2NDRG family member3xSLE

2vs

784744NEFHneurofilament, heavy3x

polypeptide

79153478PLEKHG4Bpleckstrin homology3x

domain containing,

family G (with

RhoGef domain)

member 4B [homo

sapiena (human)]

8011054OGFRopioid growth factor3xxSLE

receptorvs

AID

8156122PCDHB14protocadherin beta3xSLE

14vs

822923PDIA3protein disulfide3xSLE

isomerase family A,vs

member 3HV

8323646PLD3phospholipase D3xSLE

family, member 3vs

8423759PPIL2peptdylprolyl3xx

isomerase

(cyclophilin)-like

855557PRIM1primase, DNA,3xSLE

polypeptide 1vs

(49 kDa)HV

865682PSMA1proteasome (prosome,3xSLE

macropain) subunit,vs

alpha type, 1HV

875802PTPRSprotein tyrosine3xSLE

phosphatase,vs

receptor type, SHV

8881890QTRT1queuine tRNA-3xSLE

ribosyltransferase 1vs

89116362RBP7retinol binding3xSLE

protein 7, cellularvs

9010287RGS19regulator of G-3xx

protein signaling 19

9183642RP3-402G11.5selenoprotein O3xSLE

926389SDHAsuccinate3xxSLE

dehydrogenasevs

complex, subunit A,AID

flavoprotein (Fp)

9354437SEMA5Bsema domain, seven3x

thrombospondin

repeats (type 1 and

type 1-like),

transmembrane

domain (TM) and

short cytoplasmic

domain,

(semaphorin) 5B

9459343SENP2SUMO1/sentrin/SMT33xSLE

specific peptidasevs

2HV

956629SNRPB2small nuclear3xSLE

ribonucleoproteinvs

polypeptide B″AID

9627131SNX5sorting nexin 53xSLE

979021SOCS3suppressor of3xxSLE

cytokine signalingvs

3HV

983925STMN1stathmin 13xSLE

9981551STMN4stathmin-like 43xSLE

10027097TAFSLTAF5-like RNA3xSLE

polymerase II,vs

p300/CBP-associatedHV

factor (PCAF)-

associated factor,

65 kDa

10179521TCEAL4transcription3xSLE

elongation factor Avs

(SII)-like 4HV

10210040TOM1L1target of mybl3xSLE

(chicken)-like 1vs

10322974TPX2TPX2, microtubule-3xSLE

associated, homologvs

(Xenopus laevis)HV

10451567TTRAPTRAF and TNF3x

receptor associated

protein

1058615USO1US01 homolog,3xx

vescicle docking

protein (yeast)

10610869USP19ubiquitin specific3xSLE

peptidase 19vs

10729761USP25ubiquitin specific3x

peptidase 25

108375690WASH5PWAS protein family3xxSLE

homolog 5vs

pseudogeneHV

10910413YAP1Yes-associated3x

protein 1, 65 kDa

110653121ZBTB8Azinc finger and BTB3xxSLE

domain containingvs

BAHV

11155311ZNF444zinc finger protein3x

444

11229ABRactive BCR-related4xSLE

genevs

AID

113118ADD1adducin 1 (alpha)4xSLE

AID

11455256ADI1acireductone4xSLE

dioxygenase 1vs

1159255AIMP1aminoacyl tRNA4x

synthetase complex-

interacting

multifunctional

protein 1

11654522ANKRD16ankyrin repeat4xSLE

domain 16vs

117348APOEapolipoprotein E4xSLE

11864333ARHGAP9Rho GTPase4xSLE

activating proteinvs

9HV

11922994AZI15-azacytidine4SLE

induced 1vs

12055971BAIAP2L1BAI1-associated4x

protein 2-like 1

1217919BAT1HLA-B associated4xSLE

transcript 1vs

1226046BRD2bromodomain4x

containing 2

12356912C11orf60chromosome 11 open4x

reading frame 60

12479415C17orf62chromosome 17 open4x

reading frame 62

12551300C3orf1chromosome 3 open4xSLE

reading frame 1vs

126128866CHMP4Bchromatin modifying4xSLE

protein 4Bvs

AID

12723122CICcapicua homolog4xSLE

(Drosophila)vs

AID

12810970CKAP4cytoskeleton-4xSLE

associated proteinvs

4HV

12923122CLASP2cytoplasmic linker4x

associated protein

1301311COMPcartilage4x

oligomeric matrix

protein

1317812CSDE1cold shock domain4xSLE

containing E1, RNA-vs

bindingHV

1328642DCHS1dachsous 14xSLE

(Drosophila)vs

AID

1339909DENND4BDENN/MADD domain4xx

containing 4B

1341743DLSTdihydrolipoamide S-4x

succinyltransferase

(E2 component of 2-

oxo-glutarate

complex)

13584444DOT1LDOT1-like, histone4x

H3 methyltransferase

(S. cerevisiae)

13651143DYNC1LI1dynein, cytoplasmic4xSLE

1, lightvs

intermediate chain 1HV

13751011FAHD2Afumarylacetoacetate4x

hydrolase domain

containing 2A

13892689FAM114A1family with sequence4x

similarity 114,

member A1

13954463FAM134Bfamily with sequence4x

similarity 134,

member B

140100129583FAM47Efamily with sequence4xSLE

similarity 47,vs

member EHV

14193611FBXO44F-box protein 444x

14260681FKBP10FK506 binding4xSLE

protein 10, 65 kDavs

AID

14323360FNBP4formin binding4x

protein 4

1442300FOXL1forkhead box L14xSLE

14564689GORASP1golgi reassembly4xSLE

stacking protein 1,vs

65 kDaAID

1462934GSNgelsolin4xSLE

(amyloidosis,vs

Finnish type)HV

1473039HBA1hemoglobin, alpha4x

1483040HBA2hemoglobin, alpha 24x

14938858HES5hairy and enhancer4x

of split 5

(Drosophilia)

15010525HYOU1hypoxia up-regulated4x

1513608ILF2interleukin enhancer4xSLE

binding factor 2,vs

45 kDaRA

15223135KDM6Blysine (K)-specific4xSLE

demethylasae 6Bvs

AID

15356243KIAA1217KIAA12174xSLE

15457662KIAA1543KIAA15434x

15557498KIDINS220kinase D-interacting4x

substrate, 220 kDA

1563855KRT7keratin 74xSLE

157729970LOC729970similar to4x

hCG2028352

1589935MAFBv-maf4x

musculoaponeurotic

fibrosarcoma

oncogene homolog B

(avian)

15923764MAFFv-maf4xSLE

musculoaponeuroticvs

fibrosarcomaHV

oncogene homolog F

(avian)

16022924MAPRE3microtubule-4x

associated protein,

RP/EB family, member

1618079MLF2myeloid leukemia4x

factor 2

1624676NAP1L4nucleosome assembly4x

protein 1-like 4

1634688NCF2neutrophil cytosolic4xSLE

factor 2vs

1644780NFE2L2nuclear factor4x

(erythroid-derived

2)-like 2

16579840NHEJ1nonhomologous end-4xx

joining factor 1

16622861NLRP1NLR family, pyrin4xSLE

domain containing 1vs

16765009NDRG4NDRG family member 44xSLE

1684841NONOnon-POU domain4xSLE

containing, octamer-vs

bindingAID

16929982NRBF2nuclear receptor4xSLE

binding factor 2vs

AID

1708439NSMAFneutral4xSLE

sphingomyelinase (N-vs

SMase)activationHV

associated factor

1714926NUMA1nuclear mitotic4xSLE

apparatus protein 1vs

17284759PCGF1polycomb group ring4x

finger 1

17384306PDCD2Lprogrammed cell4xSLE

death 2-likevs

1745195PEX14peroxisomal4xSLE

biogenesis factor 14vs

1759091PIGQphosphatidylinositol4xSLE

glycan anchorvs

biosynthesis, classRA

176100137049PLA2G4Bphospholipase A2,4xSLE

group IVBvs

(cytosolic)RA

17710226PLIN3perilipin 34x

1785373PMM2phosphomannomutase 24x

17910450PPIEpeptidylprolyl4x

isomerase E

(cyclophilin E)

1805694PSMB6proteasome (prosome,4x

macropain) subunit,

beta type, 6

18122913RALYRNA binding protein,4xSLE

autoantigenicvs

(hnRNP-associatedHV

with lethal yellow

homolog (mouse))

1828241RBM10RNA binding motif4x

protein 10

1839904RBM19RNA binding motif4xSLE

protein 19vs

1849743RICSRho GTPase-4x

activating protein

1858780RIOK3RIO kinase 3 (yeast)4x

1868578SCARF1scavenger receptor4xSLE

class 4, member 1vs

AID

18723513SCRIBscribbled homolog4xSLE

(Drosophila)vs

188644096SDHAF1succinate4xSLE

dehydrogenasevs

complex assemblyRA

factor 1

18957794SF4splicing factor 44xSLE

1909814SFI1Sfi1 homolog,4x

spindle assembly

associated (yeast)

1916421SFPQsplicing factor4xSLE

proline/glutamine-vs

rich (polypyrimidineAID

tract binding

protein associated)

19283442SH3BGRL3SH3 domain binding4x

glutamic acid-rich

protein like 3

1936461SHBSrc homology 24xSLE

domain containingvs

adaptor protein BAID

19423381SMG5Smg-5 homolog,4xSLE

nonsense mediatedvs

mRNA decay factorHV

(C. elegans)

195112574SNX18sorting nexin 184xSLE

19684501SPIRE2spire homolog 24xSLE

(Drosophila)vs

19754961SSH3slingshot homolog 34xSLE

(Drosophila)vs

AID

1989263STK17Aserine/threonine4x

kinase 17a

19951111SUV420H1suppressor of4x

variegation 4-20

homolog 1

(Drosophila)

2006902TBCAtubulin folding4x

cofactor A

2017024TFCP2transcription factor4xSLE

CP2vs

2027030TFE3transcription factor4xSLE

binding to IGHMvs

enhancer 3HV

20390326THAP3THAP domain4xSLE

containing,vs

apoptosis associatedAID

protein 3

20410043TOM1target of myb14x

(chicken)

2057168TPM1tropomyosin 14xSLE

(alpha)vs

20654952TRNAU1APtRNA selenocysteine4x

1 associated protein

20726140TTLL3tubulin tyrosine4x

ligase-like family,

member 3

2087371UCK2uridine-cytidine4xSLE

kinase 2vs

2099277WDR46WD repeat domain 464xSLE

21055100WDR70WD repeat domain 704xSLE

AID

21123038WDTC1WD and4xSLE

tetratricopeptidevs

repeats 1HV

2129831ZNF623zinc finger protein4x

623

21379364ZXDCZXD family zinc4xxSLE

finger Cvs

AID

2147791ZYXzyxin4xSLE

AID

21555964SEPT3septin 35X

2165413SEPT5septin 55x

21726574AATFapoptosis5x

antagonizing

transcription factor

21891703ACY3aspartoacylase5x

(aminocyclase) 3

2199509ADAMTS2ADAM6SLE

metallopeptidasevs

with thrombospondinRA

type 1 motif, 2

22010939AFG3L2AFG3 ATPase family6SLE

gene 3-like 2vs

(yeast)HV

2211646AKR1C2aldo-keto reductase6SLE

family 1, member C2vs

(dihydrodiolRA

dehydrogenase 2;

bile acid binding

protein; 3-alpha

hydroxysteroid

dehydrogenase, type

III)

222267AMFRautocrine motility6SLE

factor receptorvs

22310777ARPP-21cyclic AMP-regulated6SLE

phosphoprotein, 21vs

kDRA

224421ARVCFarmadillo repeat6SLE

gene deletes invs

velocardiofacialRA

syndrome

22580150ASRGL1asparaginase like 16SLE

226539ATP50ATP synthase, H+6SLE

transporting,vs

mitochondrial F1RA

complex, O subunit

22779870BAALCbrain and acute6SLE

leukemia,vs

cytoplasmicRA

2289531BAG3BCL2-associated5x

athanogene 3

2299275BCL7BB-cell CLL/lymphoma6SLE

7Bvs

23055108BSDC1BSD domain6SLE

containing 1vs

AID

23154934C12orf41chromosome 12 open6SLE

reading frame 41vs

23255049C19orf60chromosome 19 open6SLE

reading frame 60vs

233388799C20orf107chromosome 20 open5x

reading frame 107

234149840C20orf196chromosome 20 open6SLE

reading frame 196vs

23551507C20orf43chromosome 20 open6SLE

reading frame 43vs

23655684C9orf86chromosome 9 open6SLE

reading frame 86vs

23723523CABIN1calcineurin binding6SLE

protein 1vs

238157922CAMSAP1calmodulin regulated6SLE

spectrin-associatedvs

protein 1RA

23923624CBLCCas-Br-M (murine)6SLE

ecotropic retroviralvs

transformingHV

sequence c

240124808CCDC43coiled-coil domain6SLE

containing 43vs

241100133941CD24CD24 molecule5x

24211140CDC37cell division cycle6SLE

37 homologvs

(S. cerevisiae)RA

24310153CEBPZCCAAT/enhancer6SLE

binding proteinvs

(C/EBP), zetaRA

24451510CHMP5chromatin modifying6SLE

protein 5vs

24563922CHTF18CTF18, chromosome5x

transmission

fidelity factor 18

homolog

(S. cerevisiae)

24651727CMPK1cytidine6SLE

monophosphate (UMP-vs

CMP) kinase 1,AID

cytosolic

24764708COPS7BCOP9 constitutive5x

photomorphogenic

homolog subunit 7B

(Arabidopsis)

24851117COQ4coenzyme Q4 homolog6SLE

(S. cerevisiae)vs

24927254CSDC2cold shock domain5x

containing C2, RNA

binding

250162989DEDD2death effector6SLE

domain containing 2vs

2519704DHX34DEAH (Asp-Glu-Ala-6SLE

His) box polypeptidevs

34RA

25255837EAPPE2F-associated6SLE

phosphoproteinvs

2531915EEF1A1eukaryotic6SLE

translationvs

elongation factor 1RA

alpha 1

2541936EEF1Deukaryotic6SLE

translationvs

elongation factor 1RA

delta (guanine

nucleotide exchange

protein)

2558669EIF3Jeukaryotic6SLE

translationvs

initiation factor 3,RA

subunit J

25655740ENAHenabled homolog6SLE

(Drosophila)vs

2572023ENO1enolase 1, (alpha)6SLE

25811124FAF1Fas (TNFRSF6)5x

associated factor 1

25911170FAM107Afamily with sequence6SLE

similarity 107,vs

member AHV

26084908FAM136Afamily with sequence6SLE

similarity 136,vs

member ARA

26110144FAM13Afamily with sequence6SLE

similarity 13,vs

member ARA

26226017FAM32Afamily with sequence6SLE

similarity 32,vs

member AHV

26364762FAM59Afamily with sequence6SLE

similarity 59,vs

member ARA

264150946FAM59Bfamily with sequence6SLE

similarity 59,vs

member BHV

26583706FERMT3fermitin family6SLE

homolog 3vs

(Drosophila)RA

26623307FKBP15FK506 binding6SLE

protein 15, 133 kDavs

2672670GFAPglial fibrillary6SLE

acidic proteinvs

26851031GLOD4glyoxalase domain6SLE

containing 4vs

AID

26981488GRINL1Aglutamate receptor,6SLE

ionotropic, N-methylvs

D-aspartate-like 1ARA

2702922GRPgastrin-releasing6SLE

peptidevs

2712935GSPT1G1 to S phase6SLE

transition 1vs

27293323HAUS8HAUS augmin-like6SLE

complex, subunit 8vs

2733054HCFC1host cell factor C16SLE

(VP16-accessoryvs

protein)AID

2743069HDLBPhigh density6SLE

lipoprotein bindingvs

proteinRA

2753184HNRNPDheterogenous nuclear6SLE

ribonucleoprotein Dvs

(AU-rich element RNAHV

binding protein 1,

37 kDa)

2763320HSP90AA1heat shock protein6SLE

90 kDa alphavs

(cytosolic), class ARA

member 1

2777184HSP90B1heat shock protein6SLE

90 kDa beta (Grp94),vs

member 1RA

2783304HSPA1Bheat shock 70 kDa6SLE

protein 1Bvs

2793315HSPB1heat shock 27 kDa4xxSLE

protein 1vs

2805654HTRA1HtrA serine6SLE

peptidase 1vs

2813382ICA1islet cell6SLE

autoantigen 1, 69 kDavs

2823550IKIK cytokine, down-6SLE

regulator of HLA IIvs

28380895ILKAPintegrin-linked6SLE

kinase-associatedvs

serine/threonineRA

phosphatase 2C

28484162KIAA1109KIAA11096SLE

AID

2853856KRT8keratin 86SLE

28623367LARP1La ribonucleoprotein6SLE

domain family,vs

member 1AID

2874001LMNB1lamin B16SLE

28879888LPCAT1lysophosphatidylcholine5xSLE

acyltransferasevs

1HV

28910916MAGED2melanoma antigen5x

family D, 2

29055700MAP7D1MAP7 domain6SLE

containing 1vs

2915602MAPK10mitogen-activated6SLE

protein kinase 10vs

29222919MAPRE1microtubule-6SLE

associated protein,vs

RP/EB family, memberAID

2934137MAPTmicrotubule-6SLE

associated protein,vs

tauRA

29423139MAST2microtubule6SLE

associatedvs

serine/threonineRA

kinase 2

29553615MBD3methyl-CpG binding6SLE

domain protein 3vs

29656922MCCC1methylcrotonoyl-6SLE

Coenzyme Avs

carboxylase 1HV

(alpha)

2971953MEGF6multiple EGF-like-6SLE

domains 6vs

2984302MLLT6myeloid/lymphoid or6SLE

mixed-lineagevs

leukemia (trithoraxRA

homolog,

Drosophila);

translocated to, 6

29910200MPHOSPH6M-phase6SLE

phosphoprotein 6vs

30010240MRPS31mitochondrial6SLE

ribosomal proteinvs

S31HV

30184939MUM1melanoma associated5x

antigen (mutated) 1

3024599MX1myxovirus (influenza6SLE

virus) resistance 1,vs

interferon-inducibleRA

protein p78 (mouse)

3034716NDUFB10NADH dehydrogenase6SLE

(ubiquinone) 1 betavs

subcomplex, 10,RA

22 kDa

3044796NFKBIL2nuclear factor of6SLE

kappa lightvs

polypeptide geneHV

enhancer in B-cells

inhibitor-like 2

30511188NISCHnischarin6SLE

30610381TUBB3tubulin, beta 36SLE

class IIIvs

3078602NOP14NOP14 nucleolar6SLE

protein homologvs

(yeast)RA

3089722NOS1APnitric oxice6

synthase 1

(neuronal) adaptor

protein

30929959NRBP1nuclear receptor5x

binding protein 1

310142PARP1poly (ADP-ribose)6SLE

polymerase 1vs

3115091PCpyruvate6SLE

carboxylasevs

31223024PDZRN3PDZ domain6SLE

containing ringvs

finger 3RA

3138682PEA15phosphoprotein6SLE

enriched invs

astrocytes 15RA

3145187PER1period homolog 16SLE

(Drosophila)vs

31557649PHF12PHD finger protein5x

31626227PHGDHphosphoglycerate5x

dehydrogenase

3171263PLK3polo-like kinase 36SLE

(Drosophila)vs

31823654PLXNB2plexin B26SLE

31956902PNO1partner of NOB16SLE

homologvs

(S. cerevisiae)RA

3205479PPIBpeptidylprolyl6SLE

isomerase Bvs

(cyclophilin B)HV

32156978PRDM8PR domain6SLE

containing 8vs

32255119PRPF38BPRP38 pre-mRNA6SLE

processing factorvs

38 (yeast) domainRA

containing B

3235764PTNpleiotrophin6SLE

3245819PVRL2poliovirus5x

receptor-related 2

(herpesvirus entry

mediator B)

3255831PYCR1pyrroline-5-6SLE

carboxylatevs

reductase 1RA

32665997RASL11BRAS-like, family6SLE

11, member Bvs

32755658RNF126ring finger protein6SLE

126vs

AID

328115992RNF166ring finger protein6SLE

166vs

3299025RNF8ring finger protein6SLE

8vs

3306092ROBO2roundabout, axon5x

guidance receptor,

homolog 2

(Drosophila)

33164221ROBO3roundabout, axonx

guidance receptor,

homolog 3

(Drosophila)

3324736RPL10Aribosomal protein6SLE

L10avs

3336152RPL24robosomal protein6SLE

L24vs

334148418SAMD13sterile alpha motif6SLE

domain containingvs

13HV

33557147SCYL3SCY1-like 36SLE

(S. cerevisiae)vs

AID

3366382SDC1syndecan 16SLE

33791461SGK493protein kinase-like5x

protein SgK493

3386449SGTAsmall glutamine-6SLE

richvs

tetratricopeptideHV

repeat (TPR)-

containing, alpha

3399627SNCAIPsynuclein, alpha5x

interacting protein

3409552SPAG7sperm associated6SLE

antigen 7vs

34157522SRGAP1SLIT-ROBO Rho6SLE

GTPase activatingvs

protein 1RA

3426744SSFA2sperm specific6SLE

antigen 2vs

3436487ST3GAL3ST3 beta-6SLE

galactoside alpa-vs

2,3-RA

sialyltransferase 3

34423345SYNE1spectrin repeat6SLE

containing, nuclearvs

envelope 1AID

3456879TAF7TAF7 RNA polymerase6SLE

II, TATA boxvs

binding proteinHV

(TBF)-associated

factor, 55 kDa

3466895TARBP2TAR (HIV-1) RNA6SLE

binding protein 2vs

3476949TCOF1Treacher Collins-6SLE

Franceschettivs

syndrome 1RA

3487980TFPI2tissue factor5x

pathway inhibitor 2

34956674TMEM9BTMEM9 domain6SLE

family, member Bvs

35011189TNRC4trinucleotide5x

repeat containing 4

35110155TRIM28tripartite motif-6SLE

containing 28vs

3527204TRIOtriple functional6SLE

domain (PTPRFvs

interacting)RA

353203068TUBBtubulin, beta6SLE

3547280TUBB2Atubulin, beta 2ASLE

35527229TUBGCP4tubulin, gamma6SLE

complex associatedvs

protein 4RA

35610422UBAC1UBA domain6SLE

containing 1vs

3577316UBCubiquitin C6SLE

35855585UBE2Q1ubiquitin-6SLE

conjugating enzymevs

E2Q familiy member 1HV

35965109UPF3BUPF3 regulator of5x

nonsense

transcripts homolog

B (yeast)

3607378UPP1uridine6SLE

phosphorylase 1vs

AID

36164856VWA1von Willebrand6SLE

factor A domainvs

containing 1RA

36255884WSB2WD repeat and SOCS5x

box-containing 2

3639877ZC3H11Azinc finger CCCH-5x

type containing 11A

36455854ZC3H15zinc finger CCCH-6SLE

type containing 15vs

3657592ZNF41zinc finger protein6SLE

41vs

366170959ZNF431zinc finger protein6SLE

431vs

367146542ZNF688zinc finger protein6SLE

688vs

3684670HNRNPMheterogeneous7SLE

nuclearvs

ribonucleoprotein MHV

36910540DCTN2dynactin 2 (p50)7SLE

37010938EHD1EH-domain7SLE

containing 1vs

37138ACAT1Acetyl-Coenzyme A7SLE

acetyltransferase 1vs

(acetoacetylHV

Coenzyme A

thiolase)

372684BST2bone marrow stromal7SLE

cell antigen 2vs

3731058CENPAcentromere protein A7SLE

3741665DHX15DEAH (Asp-Glu-Ala-7SLE

His) box polypeptidevs

15HV

3753092HIP1Huntingtin7SLE

interacting proteinvs

1HV

3763336HSPE1heating shock 10 kDa7SLE

protein 1vs

(chaperonin 10)HV

3775455POU3F3POU class 3 homeobox7SLE

3vs

3785918RARRES1retinoic acid7SLE

receptor respondervs

(tazarotene induced)HV

3796136RPL12ribosomalprotein L127SLE

3806626SNRPAsmall nuclear7SLE

ribonucleoproteinvs

polypeptide AHV

3816631SNRPCsmall nuclear7SLE

ribonucleoproteinvs

polypeptide CHV

3826757SSX2synovial sarcoma, X7SLE

breakpoint 2vs

3839788MTSS1metastasis7SLE

suppressor 1vs

38410134BCAP31B-cell receptor-7SLE

associated proteinvs

31HV

38510522DEAFldeformed epidermal7SLE

autoregulatoryvs

factor 1HV

(Drosophila)

38610633RASLl0ARAS-like, family 10,7SLE

member Avs

38754795TRPM4transient receptor7SLE

potential cationvs

channel, subfamilyHV

M, member 4

38854913RPP25ribonuclease P/MRP7SLE

25 kDa subunitvs

38954994C20orf11chromosome 20 open7SLE

reading frame 11vs

39055727BTBD7BTB (POZ) domain7SLE

containing 7vs

39179140CCDC28Bcoiled-coil domain7SLE

containing 28Bvs

39279613TMCO7transmembrane and7SLE

coiled-coil domainsvs

7HV

3935504PPP1R2protein phosphatase7SLE

1, regulatoryvs

(inhibitor subunit 2HV

3948349HIST2H2BEhistone cluster 2,7SLE

H2bevs

39511168PSIPlPC4 and SFRS17SLE

interacting proteinvs

1HV

396149986LSM14BLSM14B, SCD6 homolog7SLE

B (S. cerevisiae)vs

397655BMP7Bone morphogenetic7SLE

protein 7vs

(osteogenic proteinHV

3981676DFFADNA fragmentation7SLE

factor, 45 kDa, alphavs

polypeptideHV

3993071NCKAPlLNCK-associated7SLE

protein 1-likevs

4003727JUNDjun D proto-oncogene7SLE

4013960LGALS4lectin, galactoside-7SLE

binding, soluble, 4vs

4024920ROR2Receptor tyrosine7SLE

kinase-like orphanvs

receptor 2HV

4037424VEGFCvascular endothelial7SLE

growth factor Cvs

4048906AP1G2adaptor-related7SLE

protein complex 1,vs

gamma 2 subunitHV

40510297APC2adenomatosis7SLE

polyposis coli 2vs

40610841FTCDFormiminotransferase7SLE

cyclodeaminasevs

40711066SNRNP35small nuclear7SLE

ribonucleoproteinvs

35 kDa (Ull/U12)HV

40811345GABARAPL2GABA(A)receptor-7SLE

associated protein-vs

like 2HV

40925854FAM149Afamily with sequence7SLE

similarity 149,vs

member AHV

41026065LSM14ALSM14A, SCD6 homolog7SLE

A (S. cerevisiae)vs

41128998MRPL13mitochondrial7SLE

ribosomal proteinvs

L13HV

41251520LARSleucyl-tRNA7SLE

systhetasevs

41355747FAM21Bfamily with sequence7SLE

similarity 21,vs

member BHV

41464841GNPNAT1glucosamine-7SLE

phosphate N-vs

acetyltransferase 1HV

41583483PLVAPPlasmalemma vesicle7SLE

associated proteinvs

41684968PNMA6Aparaneoplastic7SLE

antigen like 6Avs

417118430MUCLlMucin-like 17SLE

418122830NAT12N-acetyltransferase7SLE

12vs

419221092HNRNPUL2heterogeneous7SLE

nuclearvs

ribonucleoprotein U-HV

like 2

420388962BOLA3bolA homolog 37SLE

(E. coli)vs

421729230FLJ78302Similar to c-c7SLE

chemokine receptorvs

type 2 (C-C CKR-2)HV

(CC-CKR-2) (CCR-2)

(CCR2) (Monocyte

chemoattractant

protein 1 receptor)

(MCP-1-R) (CD192

antigen)

422729447GAGE2AG antigen 2A7SLE

4231152CKBNo Gene Name;7SLE

creatine kinase,vs

brainHV

424972CD74CD74 molecule, major7SLE

histocompatibilityvs

complex, class IIHV

invariant chain

4251397CRIP2cysteine-rich7SLE

protein 2vs

4262040STOMstomatin7SLE

4272316FLNAfilamin A, alpha7SLE

4284000LMNAlamin A/C7SLE

4294582MUClmucin 1, cell7SLE

surface associatedvs

4305230PGKlPhosphoglycerate7SLE

kinase 1vs

4315340PLGplasminogen7SLE

4326525SMTNsmoothelin7SLE

4338936WASFlWAS protein family,7SLE

member 1vs

43423647ARFIP2ADP-ribosylation7SLE

factor interactingvs

protein 2HV

4356712SPTBN2spectrin, beta, non-7SLE

erythrocytic 2vs

4366729SRP54signal recognition7SLE

particle 54 kDavs

4379987HNRPDLheterogeneous7SLE

nuclearvs

ribonucleoprotein D-HV

438337APOA4Apolipoprotein A-IV7SLE

439950SCARB2scavenger receptor

class B, member 2

4403183HNRNPCheterogeneous7SLE

nuclearvs

ribonucleoprotein CHV

(Cl/C2)

4413185HNRPFHeterogeneous7SLE

nuclearvs

ribonucleoprotein FHV

4423313HSPA9heat shock 70 kDa7SLE

protein 9 (mortalin)vs

4433467IFNWlInterferon, omega 17SLE

4443799KIF5Bkinesin family7SLE

member 5Bvs

4457918BAT4HLA-B associated7SLE

transcript 4vs

4468337HIST2H2AA3histone cluster 2,7SLE

H2aa3vs

44710195ALG3asparagine-linked7SLE

glycosylation 3,vs

alpha-1,3-HV

mannosyltransferase

homolog

(S. cerevisiae)

44823299BICD2bicaudal D homolog 27SLE

(Drosophila)vs

44980184CEP290centrosomal protein7SLE

290 kDavs

45090861HNlLhematological and7SLE

neurologicalvs

expressed 1-likeHV

451349136WDR86WD repeat domain 867SLE

452no Gene IDdsDNAdsDNA7SLE

45360ACTBactin, beta8SLE

454498ATP4A1ATP synthase, H+8SLE

transporting,vs

mitochondrial FlHV

complex, alpha

subunit 1, cardiac

muscle

455506ATP5BATP synthase, H+8SLE

transporting,vs

mitochondrial FlHV

complex, beta

polypeptide

456563AZGPlalpha-2-8SLE

glycoprotein 1,vs

zinc-bindingHV

457602BCL3B-cell CLL/lymphoma8SLE

3vs

4581729DIAPHldiaphanous-related8SLE

formin 1vs

4591937EEFlGeukaryotic8SLE

translationvs

elongation factor 1HV

gamma

4601973EIF4Aleukaryotic8SLE

translationvs

initiation factorHV

4A1

4612280FKBPlAFK506 binding8SLE

protein lA, 12 kDavs

4622495FTHlferritin, heavy8SLE

polypeptide 1vs

4632597GAPDHglyceraldehyde-3-8SLE

phosphatevs

dehydrogenaseHV

4642819GPDlglycerol-3-8SLE

phosphatevs

dehydrogenase 1HV

(soluble)

4653295HSD17B4hydroxysteroid 17-8SLE

beta) dehydrogenasevs

4HV

4663305HSPAlLheat shock 70 kDa8SLE

protein 1-likevs

4673312HSPA8heat shock 70 kDa8SLE

protein 8vs

4684174MCM5minichromosome8SLE

maintenance complexvs

component 5HV

4694215MAP3K3mitogen-activated8SLE

protein kinasevs

kinase kinase 3HV

4704591TRIM37tripartite motif8SLE

containing 37vs

4714691NCLnucleolin8SLE

4724898NRDlnardilysin (N-8SLE

arginine dibasicvs

convertase)HV

4734904YBXlY box binding8SLE

protein 1vs

4745037PEBPlphosphatidylethanol8SLE

amine bindingvs

protein 1HV

4755315PKM2pyruvate kinase,8SLE

musclevs

4765481PPIDpeptidylprolyl8SLE

isomerase Dvs

4775684PSMA3proteasome8SLE

(prosome,vs

macropain)subunit,HV

alpha type, 3

4786128RPL6ribosomal protein L68SLE

4796129RPL7ribosomal protein L78SLE

4806130RPL7Aribosomal protein8SLE

L7avs

4816132RPL8ribosomal protein LB8SLE

4826187RPS2ribosomal protein S28SLE

4836189RPS3Aribosomal protein8SLE

S3Avs

4846249CLIPlCAP-GLY domain8SLE

containing linkervs

protein 1HV

4856793STKl0serine/threonine8SLE

kinase 10vs

4866880TAF9TAF9 RNA polymerase8SLE

II, TATA box bindingvs

protein (TBP)-HV

associated factor,

32 kDa

4877001PRDX2peroxiredoxin 28SLE

4887552ZNF711zinc finger protein8SLE

711vs

4898260ARDlAN(alpha)-8SLE

acetyltransferasevs

10, NatA catalyticHV

subunit

4908317CDC7cell division cycle8SLE

7vs

4918667EIF3Heukaryotic8SLE

translationvs

initiation factorHV

subunit H

4929223MAGilmembrane associated8SLE

guanylate kinase, WWvs

and PDZ domainHV

containing 1

4939230RABllBRABllB, member RAS8SLE

oncogene familyvs

4949425CDYLchromodomain8SLE

protein, Y-likevs

4959694EMC2ER membrane protein8SLE

complex subunit 2vs

49610075HUWElHECT, UBA and WWE8SLE

domain containing 1,vs

E3 ubiquitin proteinHV

ligase

49710109ARPC2actin related8SLE

protein 2/3 complex,vs

subunit 2, 34 kDaHV

49810180RBM6RNA binding motif8SLE

protein 6vs

49910273STUBlSTIPl homology and8SLE

U-box containingvs

protein 1, E3HV

ubiquitin protein

ligase

50010432RBM14RNA binding motif8SLE

protein 14vs

50110539GLRX3glutaredoxin 38SLE

50210806SDCCAG8serologically8SLE

defined colon cancervs

antigen 8HV

50311108PRDM4PR domain containing8SLE

4vs

50423002DAAMldishevelled8SLE

associated activatorvs

of morphogenesis 1HV

50523351KHNYNKH and NYN domain8SLE

containingvs

50623589CARHSP1calcium regulated8SLE

heat stable proteinvs

1, 24 kDaHV

50726986PABPClpoly (A) binding8SLE

protein, cytoplasmicvs

1HV

50827072VPS41vacuolar protein8SLE

sorting 41 homologvs

(S. cerevisiae)HV

50930836DNTTIP2deoxynucleotidyltransferase,8SLE

terminal,vs

interacting proteinHV

51051028VPS36vacuolar protein8SLE

sorting 36 homologvs

(S. cerevisiae)HV

51151082POLRlDpolymerase (RNA) I8SLE

polypeptide D, 16 kDavs

51251138COPS4COP9 signalosome8SLE

subunit 4vs

51351466EVLEnah/Vasp-like8SLE

51454869EPS8LlEPS8-like 18SLE

51554903MKSlMeckel syndrome,8SLE

type 1vs

51657017COQ9coenzyme Q98SLE

51757026PDXPpyridoxal8SLE

(pyridoxine, vitaminvs

B6) phosphataseHV

51857221ARFGEF3ARFGEF family8SLE

member 3vs

51964753CCDC136coiled-coil domain8SLE

containing 136vs

52080208SPGllspastic paraplegia8SLE

11 (autosomalvs

recessive)HV

52183858ATAD3BATPase family, AAA8SLE

domain containing 3Bvs

52284893FBXO18F-box protein,8SLE

helicase, 18vs

523129563DIS3L2DIS3 like 3′-5′8SLE

exoribonuclease 2vs

524144097Cllorf84chromosome 11 open8SLE

reading frame 84vs

525256364EML3echinorm microtubule8SLE

associated proteinvs

like 3HV

526347733TUBB2Btubulin, beta 2B8SLE

class IIbvs

5273303HSPAlAheat shock 70 kDa8SLE

protein 1Avs

5285163PDKlpyruvate8SLE

dehydrogenasevs

kinase, isozyme 1HV

5291001CDH3cadherin 3, type 1,8SLE

P-cadherinvs

(placental)HV

US11860162B2. Marker sequences for diagnosing and stratifying SLE patients (2024-01-02). Oncimmune Germany GmbH [DE]. Inventors: Angelika Luking [DE], Peter Schulz-Knappe [DE], Carmen Theek [DE], Petra Budde [DE], Anna Telaar [DE].

Full text: Click here

Patent 2024

Identification and Characterization of Cancer Stem Cell Markers

Not available on PMC !

Example 8

1. Preparation of Lgr5-Negative Adherent Cells by Irinotecan Treatment

Using a stem cell medium, Lgr5-positive adherent cells were seeded at 3×10⁵cells/well in a 6-well plate (BD, Cat. No. 353046). On the following day, irinotecan (Hospira, 61703-349-09) was added to cells at a final concentration of 10 μg/ml. After three-day culture, irinotecan-resistant cells were detected. The cells were harvested using Accutase, and suspended in FACS buffer. Then, the cells were incubated at 4° C. for 30 minutes with 7-AAD Viability Dye as dead cell staining and each of the following antibodies as cancer stem cell markers:

FITC-labeled mouse mAb to human CD326 (EpCAM), PE-labeled mouse mAb to human CD133/1 (AC133), PE-labeled mouse mAb to human CD44, PE-labeled mouse mAb to human CD166, PE-labeled mouse mAb to human CD24, PE-labeled mouse mAb to human CD26, or PE-labeled mouse mAb to human CD29. To detect Lgr5, the cells were incubated with the mouse mAb to human Lgr5 at 4° C. for 30 minutes. After washing once with FACS buffer, the cells were incubated with the PE-labeled goat Ab to mouse IgG2a at 4° C. for 30 minutes. Then, after washing once with FACS buffer, the cells were subjected to flow cytometry analysis. The ALDH activity was detected using AldeFluor Kit according to the procedure recommended by the manufacturer. Flow cytometry analysis was performed using EPICS ALTRA. Cells negative for 7-AAD Viability Dye were analyzed for cancer stem cell markers. The irinotecan-resistant cells were demonstrated to change from positive to negative for Lgr5.

2. Identification of Molecules Specifically Expressed in Cancer Stem Cells

Primary cells from PLR59 and PLR123, high proliferative Lgr5-positive cancer stem cells prepared by adherent culture of primary cells, and low proliferative Lgr5-negative cancer stem cells prepared by irinotecan treatment of the cells as described above were homogenized mechanically with QIAshredder (Qiagen, Cat. No. 79654), and RNAs were extracted from them using RNeasy Mini Kit (Qiagen, Cat. No. 74104) and RNase-Free DNase Set (Qiagen, Cat. No. 79254) according to the procedure recommended by the manufacturer. The extracted RNAs were analyzed for the purity and quality using Agilent 2100 Bioanalyzer. Following cRNA synthesis, gene expression information was obtained using GeneChip (HG-U133 plus2) of Affymetrix. Data analysis was performed with Microsoft Excel and Statistics software R. The three types of cells (primary cells, Lgr5-positive cells, and Lgr5-negative cells) were compared to each other to make a list of genes whose expression levels are significantly increased in each cell type. Specifically, raw data from GeneChip were normalized and log 2 transformed by GCRMA to calculate differences in the expression level between distinct sample types (three types: primary cells and Lgr5-positive cells, Lgr5-positive cells and Lgr5-negative cells, and Lgr5-negative cells and primary cells). The criteria used for selecting differently expressed genes were:

- (1) genes showing a twofold or more change in Lgr5-positive cells as compared to primary cells and a twofold or more change in Lgr5-negative cells as compared to primary cells (expressed at high levels in both Lgr5-positive and -negative cancer stem cells) (Table 6-1 to 6-10) (partial amino acid sequences of proteins encoded by the genes are shown in SEQ ID NOs: 1 to 7);
- (2) genes showing a twofold or more change in Lgr5-positive cells as compared to primary cells and a less than twofold change in Lgr5-negative cells as compared to primary cells and (expressed at a high level in Lgr5-positive cancer stem cells alone) (Table 7-1 to 7-5);
- (3) genes showing a less than twofold change in Lgr5-positive cells as compared to primary cells and a twofold or more change in Lgr5-negative cells as compared to primary cells (expressed at a high level in Lgr5-negative cancer stem cells alone) (Table 8-1 and 8-2) (partial amino acid sequences of proteins encoded by the genes are shown in SEQ ID NOs: 8 and 9).

Furthermore, to identify genes encoding proteins that are presented specifically on cell membrane of cancer stem cells, genes of GO:0005886 [plasma membrane] were extracted from GeneOntology (GO). Then, the present inventors extracted genes with GO:0005576 [extracellular region], GO:0009986 [cell surface], and GO:0016020 [membrane], or genes which are predicted to have a transmembrane region by membrane protein prediction software TMHMM and to have a signal peptide by signal peptide prediction software SignalP, and which do not have GO:0031090 [organelle membrane]. Furthermore, with the aid of GeneChip data from normal colorectal tissues, the present inventors exclude genes whose expression levels are relatively high in normal tissues or primary cells as well as gene only showing a small fold-change in Lgr5-positive or Lgr5-negative cells.

TABLE 6-1

DB PLR59PLR123PLR59PLR123SEQ

accessionprimaryprimaryprimaryprimary-ID

NO.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO.

NP_036438.2FAIM2Fas apoptotic inhibitory molecule 28.47.05.44.1—

NP_116265.1JUBjub, ajuba homolog (Xenopus laevis)5.85.76.25.5—

NP_116281.2FRMD5FERM domain containing 54.06.72.54.8—

NP_071731.1EDARectodysplasin A receptor7.07.65.34.61

NP_001240622.1MCOLN3mucolipin 33.75.10.82.5—

NP_001007098.1NTRK2neurotrophic tyrosine kinase, receptor, type 23.65.21.02.6—

NP_001243.1CD70CD70 molecule4.04.94.14.72

NP_062818.1SLCO1B3solute carrier organic anion transporter family, member 1B34.94.43.03.9—

NP_003606.3SLC4A7solute carrier family 4, sodium bicarbonate2.94.01.63.2—

cotransporter, member 7

NP_005836.2ABCC4ATP-binding cassette, sub-family C (CFTR/MRP),4.04.01.82.4—

member 4

NP_003920.1RAB7L1RAB7, member RAS oncogene family-like 15.55.35.66.0—

NP_009162.1SLC6A14solute carrier family 6 (amino acid transporter), member1.93.74.76.3—

NP_872631.1EFNA4ephrin-A42.63.61.42.5—

NP_001423.1EREGepiregulin2.53.32.63.13

NP_001127839.1SLC6A6solute carrier family 6 (neurotransmitter transporter,3.33.30.81.6—

taurine), member 6

NP_003497.2FZD6frizzled homolog 6 (Drosophila)3.73.24.64.0—

NP_003264.2TM7SF2transmembrane 7 superfamily member 24.04.26.86.2—

NP_001172024.1AIF1Lallograft inflammatory factor 1-like3.52.92.32.0—

NP_060033.3IL17RDinterleukin 17 receptor D3.12.82.43.0—

NP_000013.2ADAadenosine deaminase2.12.72.82.9—

NP_004834.1IL27RAinterleukin 27 receptor, alpha3.62.62.21.8—

Table 6-2 is a continuation of Table 6-1.

TABLE 6-2

DB PLR59PLR123PLR59PLR123SEQ

accessionprimaryprimaryprimaryprimaryID

NO.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_001731.2CALB2calbindin 23.82.52.40.5—

NP_065209.2ANK1ankyrin 1, erythrocytic5.82.54.92.4—

NP_001157412.1PYGLphosphorylase, glycogen, liver1.22.51.72.1—

NP_001097.2ACVR2Bactivin A receptor, type IIB2.42.51.51.2—

NP_001129141.1XPR1xenotropic and polytropic retrovirus receptor 13.42.53.52.4—

NP_001523.2SLC29A2solute carrier family 29 (nucleoside transporters),1.62.40.51.7—

member 2

NP_563615.3DCBLD2discoidin, CUB and LCCL domain containing 22.95.35.24.9—

NP_689522.2PIK3AP1phosphoinositide-3-kinase adaptor protein 13.52.41.31.0—

NP_054772.1FLVCR1feline leukemia virus subgroup C cellular receptor 13.52.41.71.2—

NP_006849.1TMED1transmembrane emp24 protein transport domain2.62.32.41.9—

containing 1

NP_116254.4TNS4tensin 41.92.31.91.5—

NP_001193874.1CSPG5chondroitin sulfate proteoglycan 5 (neuroglycan C)3.92.21.50.5—

NP_000667.1ADORA2Badenosine A2b receptor1.52.10.71.5—

NP_064423.2ACCN2amiloride-sensitive cation channel 2, neuronal1.81.91.71.3—

NP_001018000.1KAZkazrin2.51.82.01.8—

NP_004773.1SNAP29synaptosomal-associated protein, 29 kDa0.31.70.81.9—

NP_066292.2KCNJ12potassium inwardly-rectifying channel, 3.43.54.03.4—

subfamily J, member 12

NP_938205.1FLRT3fibronectin leucine rich transmembrane protein 32.81.57.15.0—

NP_115899.1PARD6Gpar-6 partitioning defective 6 homolog gamma (C.1.81.02.71.9—

elegans)

NP_066924.1CLDN1claudin 11.40.73.43.5—

NP_066939.1ADCY1adenylate cyclase 1 (brain)2.92.22.11.4—

Table 6-3 is a continuation of Table 6-2.

TABLE 6-3

DB PLR59PLR123PLR59PLR123SEQ

accessionprimaryprimaryprimaryprimaryID

NO.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_001127807.1AIMP2aminoacyl tRNA synthetase complex-interacting1.00.61.20.7—

multifunctional protein 2

NP_619539.1AKAP7A kinase (PRKA) anchor protein 72.01.31.41.2—

NP_064715.1ANKMY2ankyrin repeat and MYND domain containing 22.72.53.23.4—

NP_112591.2APH1Banterior pharynx defective 1 homolog B (C. elegans)0.81.61.13.1—

NP_658985.2AP0A1BPapolipoprotein A-I binding protein1.81.32.21.6—

NP_940852.3APOOLapolipoprotein O-like1.61.62.32.3—

NP_647537.1ATRNattractin1.52.11.11.8—

NP_001193.2BMP4bone morphogenetic protein 42.94.21.92.84

NP_001720.1BTCbetacellulin1.42.51.73.2—

NP_001224.1CAV2caveolin 21.92.93.74.2—

NP_001788.2CDH11cadherin 11, type 2, OB-cadherin (osteoblast)6.22.43.80.6—

NP_857592.1CKLFchemokine-like factor0.91.6-0.81.1—

NP_612419.1CMTM7CKLF-like MARVEL transmembrane domain 2.84.22.33.2—

containing 7

NP_054860.1CNTNAP2contactin associated protein-like 24.64.33.83.7—

NP_004738.3DLG5discs, large homolog 5 (Drosophila)1.51.01.51.2—

NP_001926.2DPP4dipeptidyl-peptidase 40.11.32.03.8—

NP_690611.1FASFas (TNF receptor superfamily, member 6)1.40.13.42.1—

NP_001099043.1FBX045F-box protein 451.01.30.31.2—

NP_001138390.1FGFR2fibroblast growth factor receptor 23.91.73.01.0—

NP_001457.1FZD2frizzled homolog 2 (Drosophila)5.25.42.62.9—

NP_031379.2GNA12guanine nucleotide binding protein (G protein) alpha 121.20.51.10.3—

Table 6-4 is a continuation of Table 6-3.

TABLE 6-4

DB PLR59PLR123PLR59PLR123SEQ

accessionprimaryprimaryprimaryprimaryID

NO.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_001243343.1GNAI1guanine nucleotide binding protein (G protein), alpha4.04.67.68.2—

inhibiting activity polypeptide 1

NP_002766.1HTRA1HtrA serine peptidase 15.17.05.06.3—

NP_001543.2IGEBP4insulin-like growth factor binding protein 43.42.70.51.1—

NP_001002915.2IGFL2IGF-like family member 23.03.32.32.5—

NP_001034659.2KREMEN1kringle containing transmembrane protein 13.03.71.32.3—

NP_005597.3LGMNlegumain1.11.80.51.4—

NP_002303.2LIG4ligase IV, DNA, ATP-dependent3.73.75.65.4—

NP_006024.1LIPGlipase, endothelial2.12.60.11.1—

NP_001392.2LPAR1lysophosphatidic acid receptor 12.93.41.32.4—

NP_036284.1LPAR3lysophosphatidic acid receptor 36.74.93.72.9—

NP_002327.2LRP6low density lipoprotein receptor-related protein 62.81.81.50.6—

NP_055414.2MAGED2melanoma antigen family D, 21.11.82.72.9—

NP_005922.2MICBMHC class I polypeptide-reiated sequence B2.63.62.33.7—

NP_001182555.1MLLT10myeloid/lymphoid or mixed-lineage leukemia (trithorax2.52.01.01.3—

homolog, Drosophila); translocated to, 10

NP_002435,1MSNmoesin3.01.72.20.0—

NP_001018169.1NAE1NEDD8 activating enzyme El subunit 11.81.71.21.8—

NP_056146.1NCSTNnicastrin1.50.42.30.8—

NP_060562.3NETO2neuropilin (NRP) and tolloid (TLL)-like 27.36.18.06.9—

NP_009014.2NUDT6nudix (nucleoside diphosphate linked moiety X)-type3.21.94.22.9—

motif 6

NP_689501.1ORAI3ORAI calcium release-activated calcium modulator 31.51.14.32.2—

NP_002605.2PDZK1PDZ domain containing 18.18.34.93.6—

Table 6-5 is a continuation of Table 6-4.

TABLE 6-5

DBPLR59PLR123PLR59PLR123SEQ

accessionprimaryprimaryprimaryprimaryID

NO.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_055824.1PDZRN3PDZ domain containing ring finger 32.62.51.40.6—

phospholipase A2, group IVA (cytosolic, calcium-

NP_077734.1PLA2G4Adependent)7.78.25.05.9—

NP_001123508.1PLEKHB1pleckstrin homology domain containing, family B4.05.35.16.3—

(evectins) member 1

NP_079501.2PNPLA3patatin-like phospholipase domain containing 34.44.91.73.5—

NP_004641.1PNPLA4patatin-like phospholipase domain containing 42.31.92.82.2—

NP_006395.2PROCRprotein C receptor, endothelial1.31.54.23.65

NP_001159449.1PROM2prominin 25.44.610.510.46

NP_077748.3PSTPIP2proline-serine-threonine phosphatase interacting protein2.22.75.14.9—

NP_002834.3PTPRJprotein tyrosine phosphatase, receptor type, J2.93.01.52.2—

NP_002861.1RAB13RAB13, member RAS oncogene family1.30.92.01.4—

NP_066361.1RAP2ARAP2A, member of RAS oncogene family2.01.72.41.7—

NP_001094058.1RC3H2ring finger and CCCH-type zinc finger domains 21.20.32.00.0—

NP_002897.1RDXradixin4.75.34.54.6—

NP_006502.1RSC1A1regulatory solute carrier protein, family 1, member 11.10.01.20.4—

(DDI2)(DNA-damage inducible 1 homolog 2)

NP_004162.2SLC1A2solute carrier family 1 (glial high affinity7.47.32.11.9—

glutamate transporter), member 2

NP_006349.1SLC25A17solute carrier family 25 (mitochondrial2.41.92.41.5—

carrier; peroxisomal membrane protein, 34 kDa), member 17

NP_075053.2SLC30A5solute carrier family 30 (zinc transporter), member 51.30.91.20.8—

NP_001070253.1SLC7A6solute carrier family 7 (cationic amino acid transporter,1.00.21.50.8—

y+ system), member 6

NP_071420.1SMOC1SPARC related modular calcium binding 12.65.4-0.12.4—

NP_001159884.1SMOC2SPARC related modular calcium binding 27.28.4-0.92.2—

NP_054730.1SOCS5suppressor of cytokine signaling 52.31.72.51.9—

Table 6-6 is a continuation of Table 6-5.

TABLE 6-6

DBPLR59PLR123PLR59PLR123SEQ

accessionprimaryprimaryprimaryprimaryID

NO.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_001030127.1SORBS1sorbin and SH3 domain containing 16.44.31.1−0.2—

NP_003095.2SORDsorbitol dehydrogenase1.51.81.02.1—

NP_003705.1STC2stanniocalcin 23.63.11.22.3—

NP_005810.1STX6syntaxin 63.42.54.43.2—

NP_003229.1TGFB2transforming growth factor, beta 25.35.14.23.6—

NP_001124388.1TGFBR1transforming growth factor, beta receptor 11.91.81.51.0—

NP_057635.1TM7SF3transmembrane 7 superfamily member 31.91.62.72.2—

NP_653233.3TMEM182transmembrane protein 1823.33.44.64.6—

NP_003802.1TNFSF9tumor necrosis factor (ligand) superfamiiy, member 94.54.45.24.97

NP_005714.2TSPAN5tetraspanin 51.82.90.01.5—

NP_068835.1UTS2urotensin 22.41.72.52.3—

Table 6-7 is a continuation of Table 6-6.

TABLE 6-7

PLR59 PLR123 PLR59 PLR123 SEQ

DB accession primaryprimaryprimary primary ID

No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_056222.2ABHD14Aabhydrolase domain containing 14A1.30.92.71.9—

NP_004449.1ACSL4acyl-CoA synthetase long-chain family member 44.54.53.64.2—

NP_665812.1AlFM1apoptosis-inducing factor, mitochondrion-associated, 11.91.62.01.9—

aldo-keto reductase family 1, member C1 (dihydrodiol

NP_001344.2AKR1C1dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid9.47.88.12.7—

dehydrogenase)

NP_940683.1ANKRD46ankyrin repeat domain 462.61.83.52.2—

NP_077027.1APOOapolipoprotein O1.41.51.21.0—

NP_004766.2B4GALT6UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase,2.01.50.31.1—

polypeptide 6

NP_004326.1BST2bone marrow stromal cell antigen 2−3.94.4−3.95.9—

NP_001185983.1C16orf5chromosome 16 open reading frame 53.12.66.95.4—

NP_115700.1C1orf57chromosome 1 open reading frame 571.51.33.03.0—

NP_653181.1C1orf85chromosome 1 open reading frame 851.10.82.92.4—

NP_001074293.1C2orf89chromosome 2 open reading frame 891.91.70.81.1—

NP_775823.1C3orf58chromosome 3 open reading frame 582.00.31.6−0.4—

NP_439896.1C6orf192chromosome 6 open reading frame 1924.02.54.53.3—

NP_001135942.1C6orf203chromosome 6 open reading frame 2031.21.02.01.8—

NP_620140.1C6orf72chromosome 6 open reading frame 721.31.81.21.7—

NP_001243894.1CCDC51coiled-coil domain containing 511.10.93.63.1—

NP_001157882.1CDK5cyclin-dependent kinase 51.92.03.53.3—

NP_055061.1CELSR1cadherin, EGF LAG seven-pass G-type receptor 10.51.50.61.1—

(flamingo homolog, Drosophila)

NP_004077.1COCHcoagulation factor C homolog, cochlin (Limulus2.12.30.52.2—

polyphemus)

NP_001896.2CTPSCTP synthase2.01.01.40.7—

NP_055191.2CYFIP2cytoplasmic FMR1 interacting protein 22.63.95.05.3—

NP_004393.1DFFBDNA fragmentation factor, 40kDa, beta polypeptide2.31.81.71.5—

(caspase-activated DNase)

NP_001077058.1E2F5E2F transcription factor 5, p130-binding2.82.22.72.2—

Table 6-8 is a continuation of Table 6-7.

TABLE 6-8

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_056067.2EHBP1EH domain binding protein 12.31.91.10.4—

NP_060682.2ENAHenabled homolog (Drosophila)4.43.51.51.2—

NP_001180289.1EXD2exonuclease 3′-5′ domain containing 22.31.22.71.9—

NP_660323.3FAM119Afamily with sequence similarity 119, member A5.45.13.03.7—

NP_620775.2FAM175Afamily with sequence similarity 175, member A3.42.83.82.8—

NP_937995.1FAM189Bfamily with sequence similarity 189, member B1.71.71.31.1—

NP_055679.1FAM2OBfamily with sequence similarity 20, member B1.60.81.50.8—

NP_942600.1FIBPfibroblast growth factor (acidic) intracellular binding protein1.01.22.01.7—

NP_000139.1FUT1fucosyltransferase 1 (galactoside 2-alpha-L-7.05.63.14.0—

fucosyltransferase, H blood group)

NP_000135.2FXNfrataxin1.71.12.01.6—

NP_000393.4G6PDglucose-6-phosphate dehydrogenase0.81.02.01.4—

NP_000143.2GAAglucosidase, alpha; acid1.21.43.32.2—

NP_000160.1GLAgalactosidase, alpha2.01.11.50.9—

NP_002072.2GPC1glypican 11.01.22.60.5—

NP_001008398.2GPX8glutathione peroxidase 8 (putative)5.05.57.17.0—

NP_005329.3HIP1huntingtin interacting protein 12.62.72.62.5—

NP_254274.1IL33interleukin 33−0.51.23.63.4—

NP_002262.3IPO5importin 53.62.31.90.4—

NP_060573.2LRRC8Dleucine rich repeat containing 8 family, member D2.01.01.91.2—

NP_067679.6MFAP3Lmicrofibrillar-associated protein 3-like1.50.23.31.2—

NP_612440.1MFSD3major facilitator superfamily domain containing 30.91.32.02.4—

NP_066014.1MOV10Mov10, Moloney leukemia virus 10, homolog (mouse)0.51.41.32.0—

NP_036351.3MRASmuscle RAS oncogene homolog1.10.64.31.3—

NP_057034.2MRPL2mitochondrial ribosomal protein L21.30.80.80.0—

NP_065972.3NINninein (GSK3B interacting protein)2.03.12.73.0—

Table 6-9 is a continuation of Table 6-8.

TABLE 6-9

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_065181.1NIPAL3NIPA-like domain containing 31.71.33.02.3—

NP_002504.2NME3non-metastatic cells 3, protein expressed in1.12.11.51.9—

NP_057672.1NRN1neuritin 18.55.77.76.3—

NP_689643.2OR51 Elolfactory receptor, family 51, subfamily E, member 16.99.80.33.0—

NP_071748.2OSGEPL1O-sialoglycoprotein endopeptidase-like 14.14.73.14.4—

NP_079431.1PAAF1proteasomal ATPase-associated factor 11.92.11.51.9—

NP_000523.2PCCBpropionyl CoA carboxylase, beta polypeptide1.41.61.42.0—

NP_061757.1PCDHB14protocadherin beta 141.40.51.40.1—

NP_002622.2PGDphosphogluconate dehydrogenase1.21.41.21.2—

NP_003550.1PIP4K2Bphosphatidylinositol-5-phosphate 4-kinase, type II, beta1.81.31.81.1—

NP_056530.2PLA2G3phospholipase A2, group III2.64.30.13.2—

NP_005038.1PSMD5proteasome (prosome, macropain) 26S subunit, 1.50.71.70.7—

non-ATPase, 5

NP_006255.1PTPN13protein tyrosine phosphatase, non-receptor type 130.11.10.72.2—

(APO-1/CD95 (Fas)-associated phosphatase)

NP_057161.1PTRH2peptidyl-tRNA hydrolase 21.41.41.51.8—

NP_037390.2PYCARDPYD and CARD domain containing0.21.31.12.1—

NP_055113.2QPRTquinolinate phosphoribosyltransferase2.53.63.14.5—

NP_060233.3RNF43ring finger protein 432.72.71.21.5—

NP_060616.1RNMTL1RNA methyltransferase like 11.50.91.71.1—

NP_003698.1RUVBL1RuvB-like 1 (E. coli)1.92.21.32.2—

NP_002949.2RYKRYK receptor-like tyrosine kinase1.51.01.21.2—

NP_116250.3SERAC1serine active site containing 11.80.82.71.5—

NP_005016.1SERPINI1serpin peptidase inhibitor, clade I (neuroserpin), member 15.58.72.14.2—

NP_008927.1SLC19A2solute carrier family 19 (thiamine transporter), member 23.82.72.01.2—

NP_065075.1SLC39A10solute carrier family 39 (zinc transporter), member 102.93.12.62.7—

NP_060306.3SLC41A3solute carrier family 41, member 31.91.51.30.8—

Table 6-10 is a continuation of Table 6-9.

TABLE 6-10

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_005692.1SNUPNsnurportin 12.22.42.32.5—

NP_055563.1SNX17sorting nexin 170.81.11.01.0—

NP_003156.1STXBP1syntaxin binding protein 14.35.74.95.3—

NP_003192.1TFAMtranscription factor A, mitochondrial2.11.61.11.6—

NP_444283.2THEM4thioesterase superfamily member 41.6−0.52.80.4—

NP_612472.1TLCD1TLC domain containing 12.72.13.94.2—

NP_057548,1TMEM138transmembrane protein 1381.82.22.02.7—

NP_085054,2TMEM177transmembrane protein 1773.13.23.54.1—

NP_056236.2TMEM186transmembrane protein 1862.42.02.02.3—

NP_078863.2TMEM53transmembrane protein 531.21.31.21.0—

NP_001008495.2TMEM64transmembrane protein 646.75.78.38.0—

NP_669630.1TMEM68transmembrane protein 681.81.83.42.9—

NP_861448.2TMTC3transmembrane and tetratricopeptide repeat containing 32.51.83.82.3—

NP_775107.1TRIM59tripartite motif-containing 593.74.33.04.0—

NP_003293.2TRIP6thyroid hormone receptor interactor 62.71.35.13.3—

NP_009215.1TWF2twinfilin, actin-binding protein, homolog 2 (Drosophila)0.61.51.41.8—

NP_079094.1UBA5ubiquitin-like modifier activating enzyme 51.82.12.42.4—

NP_060769.4UBE2Wubiquitin-conjugating enzyme E2W (putative)1.31.72.42.4—

NP_001017980.1VMA21VMA21 vacuolar H+-ATPase homolog (S. cerevisiae)3.02.62.92.6—

NP_660295.2ZG16Bzymogen granule protein 16 homolog B (rat)1.12.01.41.9—

NP_115549.2ZNRF3zinc and ring finger 34.74.12.22.6—

(The values in Tables 6-1 to 6-10 shown above represent the expression difference (log 2 ratio).)

TABLE 7-1

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_003658.1LGR5leucine-rich repeat-containing G protein-coupled receptor 53.03.8−1.50.4—

NP_066301.1SNTB1syntrophin, beta 1 (dystrophin-associated protein A1, 3.13.10.00.4—

59kDa, basic component 1)

NP_001123575.1COL13A1collagen, type XIII, alpha 12.02.9−0.20.3—

NP_001167538.1FGER1fibroblast growth factor receptor 11.32.3−0.70.6—

NP_004432.1EPHB1EPH receptor B11.02.3−0.30.7—

NP_002002.3FGFR4fibroblast growth factor receptor 42.02.2−1.9-0.5—

NP_004622.2LRP8low density lipoprotein receptor-related protein 8,2.32.10.50.7—

apolipoprotein e receptor

NP_002832.3PTPRGprotein tyrosine phosphatase, receptor type, G1.82.1−5.0−5.4—

NP_004727.2XPR1xenotropic and polytropic retrovirus receptor 11.52.10.70.9—

NP_859052.3QSOX2quiescin Q6 sulfhydryl oxidase 21.82.00.70.8—

NP_003876.1CDK5R1cyclin-dependent kinase 5, regulatory subunit 1 (p35)3.13.30.32.0—

NP_002821.1PTPN4protein tyrosine phosphatase, non-receptor type 41.71.90.30.7—

(megakaryocyte)

NP_001457.1FZD2frizzled homolog 2 (Drosophila)5.25.42.62.9—

NP_000674.2ADRA2Cadrenergic, alpha-2C-, receptor1.81.60.1−0.1—

NP_000334.1SLC5A1solute carrier tam ly 5 (sodium/glucose cotransporter),2.21.60.20.6—

member1

NP_005901.2MAPTmicrotubule-associated protein tau1.91.50.40.6—

NP_598328.1SYN2synapsin II0.31.70.20.5—

NP_005496.4SCARB1scavenger receptor class B, member 11.11.2−0.3−0.3—

NP_004434,2EPHB3EPH receptor B30.71.0−0.5−0.4—

NP_000259.1NF2neurofibromin 2 (merlin)1.20.90.60.1—

NP_003477.4SLC7A5solute carrier family 7 (cationic amino acid transporter,2.00.8−0.4−0.7—

y+ system), member 5

NP_054740.3SSX2IPsynovial sarcoma, X breakpoint 2 interacting protein1.80.70.9−0.3—

NP_055736.2LPHN1latrophilin 11.40.60.90.3—

NP_004435.3EPHB4EPH receptor B41.30.50.7−0.1—

Table 7-2 is a continuation of Table 7-1.

TABLE 7-2

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_116197.4LINGO1leucine rich repeat and Ig domain containing 11.20.00.6−1.3—

NP_004987.2ABCC1ATP-binding cassette, sub-family C (CFTR/MRP), 1.10.90.0−0.2—

member 1

NP_003174.3ADAM17ADAM metallopeptidase domain 171.00.3−0.3−0.4—

NP_620686.1ADAMTS15ADAM metallopeptidase with thrombospondin 6.14.6−3.6−4.2—

type 1 motif, 15

NP_005091.2AKAP12A kinase (PRKA) anchor protein 122.43.7−1.20.3—

NP_001618.2ALCAMactivated leukocyte cell adhesion molecule1.30.90.30.6—

NP_001648.1AREGBamphiregulin B1.70.40.0−0.5—

NP_001164.2ARHGAP5Rho GTPase activating protein 51.20.40.80.3—

NP_542172.2B3GALT6UDP-Gal:betaGal beta 1,3-galactosyltransferase1.10.60.60.3—

polypeptide 6

NP_001711.2BMP8Bbone morphogenetic protein 8b0.91.30.40.3—

NP_006560.3CGREF1cell growth regulator with EF-hand domain 10.91.00.30.8—

NP_058647.1CKLFchemokine-like factor0.91.5−0.50.9—

NP_849199.2CMTM8CKLF-like MARVEL transmembrane domain 1.31.40.60.7—

containing 8

NP_001422.1EPB41L2erythrocyte membrane protein band 4.1-like 21.30.9−0.4−0.4—

NP_004433.2EPHB2EPH receptor 621.00.5−3.1−2.8—

NP_000496.2F12coagulation factor XII (Hageman factor)1.21.60.21.0—

NP_057133.2FAM158Afamily with sequence similarity 158, member A0.81.20.10.4—

NP_001990.2FBN2fibrillin 22.62.5−0.40.1—

NP 068656,21FGGfibrinogen gamma chain1.30.60.60.5—

(OSMR)(oncostatin M receptor)

NP_001439.2GPC4glypican 42.02.1−0.90.0—

NP_065857.1GPHNgephyrin3.22.9−3.4−2.6—

NP_057399.1GULP1GULP, engulfment adaptor PTB domain containing 11.42.00.40.9—

NP_036616.2HMMRhyaluronan-mediated motility receptor (RHAMM)5.04.5−1.7−1.5—

NP_0008661IGF1Rinsulin-like growth factor 1 receptor0.71.1−1.9−1.2—

Table 7-3 is a continuation of Table 7-2.

TABLE 7-3

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_000588.2IGFBP2insulin-like growth factor binding protein 2, 36kDa1.40.70.3−0.4—

NP_0611952IL17RBinterleukin 17 receptor B1.01.2−2.7−1.9—

NP_002326.2LRP5low density lipoprotein receptor-related protein 51.21.7−0.8−0.2—

NP_006491.21MCAMmelanoma cell adhesion molecule2.11.5−0.2−0.4—

NP_055456.2MDC1mediator of DNA-damage checkpoint 12.62.2−1.3−1.7—

NP_055606.1MELKmaternal embryonic leucine zipper kinase1.51.6−2.0−1.1—

NP_000236.2METmet proto-oncogene (hepatocyte growth factor receptor)1.21.20.60.4—

NP_065825.1MIB1mindbomb homolog 1 (Drosophila)1.31.30.70.5—

NP_005952.2MUC6mucin 6, oligomeric mucus/gel-forming1.13.0−0.11.0—

NP_777596.2PCSK9proprotein convertase subtilisin/kexin type 91.51.8−1.6−0.4—

NP_003619.2PKP4plakophilin 41.51.40.0−0.1—

NP_003042.3SLC16A1solute carrier family 16, member 1 1.70.60.30.3—

(monocarboxylic acid transporter 1)

NP_057438.3SLCO4A1solute carrier organic anion transporter family, 0.51.2−1.4−0.2—

member 4A1

NP_003093.2SOD3superoxide dismutase 3, extracellular2.21.70.6−1.2—

NP_006425.2SORBS1sorbin and SH3 domain containing 13.23.4−0.90.2—

NP_003095.2SORDsorbitol dehydrogenase1.10.70.90.8—

NP_000342.2STSsteroid sulfatase (microsomal), isozyme S1.82.2−0.5−0.7—

NP_003234.2TGFBR3transforming growth factor, beta receptor III1.41.8−0.2−0.2—

NP_055388.2TMEM97transmembrane protein 972.62.11.0−0.3—

CAA26435.1TRACT cell receptor alpha constant0.01.10.00.0—

Table 7-4 is a continuation of Table 7-3.

TABLE 7-4

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_076417.2AACSacetoacetyl-CoA synthetase1.11.60.20.6—

NP_653191.2ANKRD22ankyrin repeat domain 222.01.5−1.8−1.7—

NP_056020.2ATP11AATPase, class VI, type 11A1.31.00.70.6—

NP_001159.2BIRC5baculoviral IAP repeat-containing 52.72.4−4.8−4.4—

NP_061189.2CDCA7Lcell division cycle associated 7-like2.11.8−0.60.4—

NP_005183.2CDKN3cyclin-dependent kinase inhibitor 34.85.3−3.1−1.8—

NP_004077.1COCHcoagulation factor C homolog, cochlin (Limulus1.21.2−0.40.7—

polyphemus)

NP_0053023GRB10growth factor receptor-bound protein 101.10.4−1.2−1.5—

NP_6717043HS6ST2heparan sulfate 6-O-sulfotransferase 21.81.6−1.1−0.2—

NP_002262.3IP05importin 52.61.70.5−0.3—

NP_114428.1ITFG3integrin alpha FG-GAP repeat containing 31.00.90.0−1.1—

NP_002241.1KCNN4potassium intermediate/small conductance calcium-1.20.9−0.7−0.7—

activated channel, subfamily N, member 4

NP_061159.1KIAA1199KIAA11991.11.1−1.5−2.4—

NP_115940.2KISS1RKISS1 receptor1.11.8−3.00.2—

NP_057034.2MRPL2mitochondrial ribosomal protein L21.30.80.80.0—

NP_000242.1MSH2mutS homolog 2, colon cancer, nonpolyposis type 1 (E.2.52.8−0.8−0.4—

coli)

NP_055452.3MTFR1mitochondrial fission regulator 11.60.90.2−0.1—

NP_005947.3MTHFD1methylenetetrahydrofolate dehydrogenase (NADP+1.71.60.30.1—

dependent) 1, methenyltetrahydrofolate cyclohydrolase,

formyitetrahydrofolate synthetase

NP_005366.2MYBv-myb myeloblastosis viral oncogene homolog (avian)2.72.3−4.40.2—

NP_078938.2NAT10N-acetyltransferase 10 (GCN5-related)1.91.30.1−0.1—

NP_777549.1NDUFAF2NADH dehydrogenase (ubiguinone) 1 alpha1.01.4−0.90.1—

subcomplex, assembly factor 2

NP_004280.5NFE2L3nuclear factor (erythroid-derived 2)-like 31.21.2−0.9−0.1—

NP_006672.1NMUneuromedin U0.21.6−4.70.2—

NP_055950.1NUP205nucleoporin 205kDa1.91.7−0.3−0.2—

Table 7-5 is a continuation of Table 7-4.

TABLE 7-5

PLR59 PLR123 PLR59 PLR123 SEQ

DB accessionprimaryprimaryprimary primary ID

No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_942590.1NUP43nucleoporin 43kDa1.30.90.00.0—

NP 0554843NUP93nucleoporin 93kDa2.12.40.10.8—

NP_006614.2PHGDHphosphoglycerate dehydrogenase3.03.4−2.0−0.4—

NP_060904.2RNF130ring finger protein 1300.91.00.40.3—

NP_060259.4SEMA4Csema domain, immunoglobulin domain (Ig),1.92.30.20.5—

transmembrane domain (TM) and short

cytoplasmic domain, (semaphorin) 40

NP_005857.1SIGMAR1sigma non-opioid intracellular receptor 11.01.10.80.9—

NP_055413.1SOCS7suppressor of cytokine signaling 71.00.60.70.1—

NP_005554.1STMN1stathmin 11.92.1−3.1−1.4—

NP_054897,4STXBP6syntaxin binding protein 6 (amisyn)2.12.1−3.4−0.1—

NP_001070884.1TMEM231transmembrane protein 2310.51.5−2.2−0.8—

NP_002537.3TNFRSF11Btumor necrosis factor receptor superfamily, 1.76.3−2.9−0.9—

member 11b

NP_443195.1TOP1MTtopoisomerase (DNA) I, mitochondrial1.10.40.5−0.1—

NP_001058.2TOP2Atopoisomerase (DNA) II alpha 170kDa2.22.7−6.1−3.1—

NP_006364.2VAT1vesicle amine transport protein 1 homolog 1.01.50.51.0—

(T. californica)

NP_612471.1ZMYND19zinc finger, MYND-type containing 191.20.70.70.3—

(The values in Tables 7-1 to 7-5 shown above represent the expression difference (log 2 ratio).)

TABLE 8-1

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_076404.3GPR87G protein-coupled receptor 870.00.07.17.4—

NP_005109.2TNFSF15tumor necrosis factor (ligand) superfamily, member 15−2.4−2.33.53.3—

NP_001926.2DPP4dipeptidyl-peptidase 40.11.32.03.8—

NP_149017.2BBS4Bardet-Biedl syndrome 40.20.61.72.6—

NP_542386.1C9orf30chromosome 9 open reading frame 300.20.21.51.7—

NP_001447.2FLNAfilamin A, alpha−0.40.80.71.7—

NP_000425.1NEU1sialidase 1 (lysosomal sialidase)0.30.41.91.7—

NP_002125.3HMOX2hems oxygenase (decycling) 20.30.41.51.6—

NP_001078.2AAMPangio-associated, migratory cell protein0.10.31.21.6—

NP_061985.2ABCA7ATP-binding cassette, sub-family A (ABC1), member 7−1.30.42.11.6—

NP_000401.1HFEhemochromatosis2.22.01.22.8—

NP_001142.2SLC25A4solute carrier family 25 (mitochondrial carrier: adenine0.60.81.31.5—

nucleotide translocator), member 4

NP_006569.1GNB5guanine nucleotide binding protein (G protein), beta 50.20.80.91.4—

NP_005846.1RAMP1receptor (G protein-coupled) activity modifying protein 1−0.5−0.51.11.4—

NP_598378.3RHOVras homolog gene family, member V0.1−0.20.51.4—

NP_112178.2PVRL4poliovirus receptor-related 4−0.3−2.86.11.87

NP_003686.1LY6Dlymphocyte antigen 6 complex, locus D−3.8−1.8−0.52.7—

NP_775876.1KCNRGpotassium channel regulator1.00.22.61.9—

NP_005063.19LC12A4solute carrier family 12 (potassium/chloride−0.2−0.11.21.2—

transporters), member 4

NP_005292.2GPR35G protein-coupled receptor 35−0.8−1.41.1−0.8—

NP_001607.1ACVR2Aactivin A receptor, type IIA0.2−0.51.41.0—

NP_001687.1ATP6V1E1ATPase, H+ transporting, lysosomal 31kDa, V1 subunit E1−0.10.31.11.2—

NP_036474.1BAMBIBMP and activin membrane-bound inhibitor homolog−0.80.71.32.4—

(Xenopus laevis)

NP_005177.2CAPN1calpain 1, (mu/l) large subunit−1.3−1.31.3−0.2—

Table 8-2 is a continuation of Table 8-1.

TABLE 8-2

PLR59 PLR123 PLR59 PLR123 SEQ

primaryprimaryprimary primary ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−NO

NP_001798.2CELcarboxyl ester lipase (bile salt-stimulated lipase)0.00.07.45.4—

NP_056034.2EXOC7exocyst complex component 7−0.2−0.21.91.2—

NP_000034.1FASFas (TNF receptor superfamily, member 6)0.80.42.82.39

NP_005802.1GDF11growth differentiation factor 110.70.91.31.9—

NP_060456.3GPR172BG protein-coupled receptor 17280.00.05.64.3—

NP_665895.1KLK10kallikrein-related peptidase 10−0.90.41.21.6—

NP_055414.2MAGED2melanoma antigen family D, 20.00.32.13.0—

NP_000519.2MAN2B1mannosidase, alpha, class 2B, member 1−0.2−0.22.31.5—

NP_002434.1MSMBmicroseminoprotein, beta-0.00.05.64.7—

NP_009014.2NUDT6nudix (nucleoside diphosphate linked moiety X)-type 0.00.01.30.8—

motif 6

NP_036528.1PHLDA3pleckstrin homology-like domain, family A, member 3−0.3−0.22.91.0—

NP_002629.1PI3peptidase inhibitor 3, skin-derived−7.6−7.91.4−0.6—

NP_597998.1SAT2spermidine/spermine N1-acetyltransferase family 0.70.91.71.2—

member 2

NP_006207.1SERPINE2serpin peptidase inhibitor, clade E (nexin, −3.2− 2.51.30.7—

plasminogen activator inhibitor type 1), member 2

NP_036303.1TSPAN17tetraspanin 170.60.41.71.3—

(The values in Tables 8-1 and 8-2 shown above represent the expression difference (log 2 ratio).)

Furthermore, genes that meet a criterion described below and have GO:0005886 [plasma membrane] from GeneOntology (GO) (Tables 9 and 10) were extracted in order to obtain genes encoding proteins that are specifically presented on cell membrane of cancer stem cells.

Markers common for both proliferating and quiescent CSCs:

genes whose expression levels are in average greater than 64 in Lgr5-negative and Lgr5-positive cells; which show a greater than four-fold change in both Lgr5-negative and Lgr5-positive cells relative to primary cells; and which show a significant difference by t-test (Table 9).

TABLE 9

PLR59 PLR123 PLR59 PLR123 PLR59PLR123SEQ

primaryprimaryprimary primary Lgr5+Lgr5+ID

DB accession No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−Lgr5−Lgr5−NO

NP_001423.1EREGepiregulin2.23.42.53.10.3−0.33

NP_001986.2ACSL1acyl-CoA synthetase long-chain family 3.03.92.93.7−0.2−0.2—

member 1

NP_005922.2MICBMHC class I polypeptide-related sequence B2.33.82.23.7−0.1−0.1—

NP_000296.2PON2paraoxonase 22.34.62.94.60.70.0—

NP_003458.1CXCR4chemokine (C-X-C motif) receptor 410.29.58.57.8−1.7−1.7—

NP_062818.1SLCO1B3solute carrier organic anion transporter family, 4.45.13.04.1−1.4−1.0—

member 183

NP_000280.1PF KMphosphofructokinase, muscle3.32.33.32.10.0−0.2—

NP_057672.1NRN1neuritin 18.88.57.86.5−1.0−2.0—

NP_060369.3TESCtescalcin3.35.02.036−1.3−1.3—

NP_000139.1FUT1fucosyltransferase 1 (galactoside 2-alpha-L-6.95.93.14.3−3.9−1.6—

fucosyltransferase, H blood group)

NP_003802.1TNFSF9tumor necrosis factor (ligand) superfamily, 4.54.45.45.10.90.77

member 9

NP_059108.1FZD3frizzled homolog 3 (Drosophila)2.82.62.63.2−0.20.6—

NP_005810.1STX6syntaxin 62.82.74.23.51.40.9—

NP_057635.1TM7SF3transmembrane 7 superfamily member 33.02.53.93.00.90.5—

NP_000013.2ADAadenosine deaminase2.02.72.82.90.80.2—

NP_071731.1EDARectodysplasin A receptor6.26.85.03.7−1.2−3.11

NP_003264.2TM7SF2transmembrane 7 superfamily member 23.64.36.96.73.22.4—

NP_116265.1JUBjab, ajuba homolog (Xenopus laevis)3.74.34.73.81.0−0.5—

NP_689740.2SLC16A14solute carrier family 16, member 14 5.46.65.86.10.3−0.4—

(monocarboxylic acid transporter 14)

NP_065209.2ANK1ankyrin 1, erythrocytic6.63.05.32.7−1.3−0.3—

Quiescent CSC-specific markers: genes whose expression levels are in average greater than 64 in Lgr5-negative cells and are in average less than 64 in both primary cells and Lgr5-positive cells; which show a greater than 20 fold change in Lgr5-negative cells relative to Lgr5-positive cells; and which show a significant difference by t-test (Table 10).

TABLE 10

PLR59 PLR123 PLR59 PLR123 PLR59PLR123SEQ

DB accession primaryprimaryprimary primary Lgr5+Lgr5+ID

No.AbbreviationMolecule nameLgr5+Lgr5+Lgr5−Lgr5−Lgr5−Lgr5−NO

NP_006111.2HLA-DMAmajor histocompatibility complex, 0.00.28.08.78.08.5—

class II, DM alpha

NP_862830.1AMIGO2adhesion molecule with Ig-like domain 20.00.07.07.57.07.5—

NP_001159449.1PROM2prominin 23.32.910.110.56.97.66

NP_076404.3GPR87G protein-coupled receptor 870.00.07.07.47.07.4—

NP_722582.2GPR110G protein-coupled receptor 1100.00.06.07.06.07.0—

NP_112178.2PVRL4poliovirus receptor-related 4−0.6−2.75.81.76.44.48

NP_938205.1FLRT3fibronectin leucine rich transmembrane 1.30.26.85.45.55.2—

protein 3

3. Expression Analysis by Flow Cytometry Analysis
3.1. Flow Cytometry Analysis of NOG-Established Cancer Cell Lines

After suspending in FACS buffer, cells of NOG-established cancer lines collected from mice were incubated at 4° C. for 30 minutes with rat mAb to mouse MHC I (Abcam; ab15680) and mAb to human EREG (EP27; WO2008/047723). Then, following washing once with FACS buffer, the cells were incubated at 4° C. for 30 minutes with 7-AAD Viability Dye (Beckman Coulter; A07704) as dead cell staining and secondary antibodies: PE-labeled goat F(ab′)2 fragment to mouse IgG (H+L) (Beckman Coulter; IM0855) and APC-labeled goat Ab to rat IgG (BioLegend; 405406). After washing once with FACS buffer, the cells were subjected to flow cytometry analysis. Flow cytometry analysis was performed using EPICS ALTRA. Cells negative for 7-AAD Viability Dye and mouse MHC were analyzed for EREG expression.

3.2. Flow Cytometry Analysis of In Vitro Cultured Cancer Cell Lines

Lgr5-positive adherent cells and Lgr5-negative adherent cells resulting from induction by irinotecan treatment were harvested using Accutase. The cells were suspended in FACS buffer, and then incubated at 4° C. for 30 minutes with mouse mAb to human EREG. After the cells were washed once with FACS buffer, 7-AAD Viability Dye as dead cell staining and a PE-labeled goat F(ab′)2 fragment to mouse IgG (H+L) as a secondary antibody were added thereto. The cells were incubated at 4° C. for 30 minutes. Then, the cells were washed once with FACS buffer, and subjected to flow cytometry analysis. Flow cytometry analysis was performed using EPICS ALTRA. Cells negative for 7-AAD Viability Dye were analyzed for EREG expression. The result showed that the corresponding protein was expressed at a high level on cell membrane surface.

The result obtained by EREG flow cytometry analysis of primary cells from PLR59 and PLR123, and Lgr5⁺ and Lgr5⁻ cancer stem cells is shown in FIG. 37. The cells were stained using an antibody against EREG and analyzed by flow cytometry. It was demonstrated that primary cells were negative for EREG while Lgr5⁺ and Lgr5⁻ cancer stem cells were homogeneous EREG-positive cell populations. Gray indicates fluorescence intensity after cell staining with an indicated antibody; and white indicates fluorescence intensity after cell staining with a control isotype antibody.

4. In Vitro Assessment of Drug Efficacy by ADCC Activity Measurement

4.1. Preparation of Effector Cell Suspension

A mononuclear cell fraction collected from human peripheral blood was used as human effector cells. Fifty ml of peripheral blood was collected from a healthy volunteer (adult male) of the inventors' company using a syringe loaded in advance with 200 μl of 1000 units/ml heparin solution (Novo-Heparin 5,000 units/5 ml for Injection; Novo Nordisk). The peripheral blood was diluted twofold with PBS(−), and then introduced into a Leucosep lymphocyte separation tube (Greiner bio-one) in advance loaded with Ficoll-Paque PLUS and subjected to centrifugation. After centrifugation (2150 rpm, room temperature, 10 minutes), the monocyte fraction layer was collected from the tube. The cells were washed once with 10% FBS/D-MEM, and then suspended at a cell density of 5×10⁶/ml in 10% FBS/D-MEM. The suspension was used as an effector cell suspension.

4.2. Preparation of Target Cell Suspensions

Target cell suspensions were prepared at the time of use. One×10⁶cells of cancer lines were centrifuged (1200 rpm, room temperature, 5 minutes). The cell pellets were suspended in 200 μl of 0.2 mg/ml calcein-AM (Nacalai Tesque)/DMEM (10% FBS) medium. Cell suspensions in calcein-AM solution were incubated for two hours in a CO₂incubator set to 37° C. and to a CO₂concentration of 5%. After washing once with 10% FBS/D-MEM, the cells were adjusted to a cell density of 2×10⁵/ml with 10% FBS/D-MEM to prepare target cell suspensions.

4.3. ADCC Activity Measurement

Anti-EREG antibody was prepared at a concentration of 0.5 mg/ml, which was further diluted with 10% FBS/D-MEM to give antibody solutions. The final concentration was adjusted to 0.4, 4, and 40 μg/ml. The antibody solutions of respective concentrations or 10% FBS/D-MEM were each added at 50 μl/well to a 96-well round-bottomed plate. Then, the target cell suspensions were added at 50 μl/well to every well. The plate was incubated at room temperature for 15 minutes. Next, 100 μl of the effector cell suspension was added to each well containing target cell suspension, and antibody solution or 10% FBS/D-MEM. One-hundred μl of 10% FBS/D-MEM or 2% NP-40 solution (NP-40 substitute; Wako Pure Chemical Industries) was added to each of other wells containing 10% FBS/D-MEM and target cell suspension. The plate was centrifuged (1200 rpm, room temperature, 5 minutes) and incubated for 4 hours in a CO₂incubator set to 37° C. and to a CO₂concentration of 5%. The plate was centrifuged (1200 rpm, room temperature, 5 minutes), and a 100-μl aliquot of supernatant was collected from each well. The fluorescence intensity (λ_ex=490 nm, λ_em=515 nm) was determined using a spectrofluorometer. The specific calcein release rate (cytotoxicity (%)) was determined according to the following formula.
cytotoxicity(%)=(A−C)×100/(B−C) Formula 1:where A represents the fluorescence intensity in each well; B represents the mean value of fluorescence intensity in a well where 50 μl of target cell suspension and 100 μl of NP-40 solution were added to 50 μl of 10% FBS/D-MEM; and C represents the mean value of fluorescence intensity in a well where 50 μl of target cell suspension and 100 μl of 10% FBS/D-MEM were added to 50 μl of 10% FBS/D-MEM. This assay was carried out in triplicate, and the cytotoxicity (%) at each antibody concentration was determined using Microsoft Office Excel 2007.

The anti-EREG antibody-mediated ADCC activities against Lgr5-positive and -negative cells derived from PLR59 cells, and those against Lgr5-positive and -negative cells derived from PLR123 cells are shown in FIG. 38. The result showed that the anti-EREG antibody exerted cytotoxic activity against both Lgr5-positive and -negative cancer stem cells from PRL59 or PLR123 in a dose dependent manner whereas the control antibody had no cytotoxic activity.

To assess in vivo EREG expression, the Lgr5-positive cells were administered into the peritoneal cavities of NOG mice. In the early stage of tumor generation, EREG was expressed at a high level. In the late stage where the tumor formed specific ductal structures, EREG expression was somewhat localized to the budding clusters rather than ductal structures. EREG-positive cells were detected even after irinotecan administration to tumor-bearing mice (FIG. 54). The anti-EREG antibody was assessed for anti-tumor activity after irinotecan treatment. Effector cells are essential for the anti-EREG antibody to mediate ADCC activity. Thus, SCID mice were used as a model to assess the pharmacological efficacy of the anti-EREG antibody. Tumor growth was suppressed when the antibody was administered at the time points of days 4 and 11 after the final irinotecan administration (FIG. 57).

As a first step to assess the pharmacological efficacy based on the metastasis model, it was tested whether EREG is expressed in the metastasis model. When Lgr5-positive cells were intravenously injected into NOG mice, tumors were formed in multiple tissues including lung. Cells of the tumors formed in lungs are mostly positive for EREG (FIG. 58A). The pharmacological efficacy of the anti-EREG antibody was assessed using SCID-Beige mice where macrophages and mononuclear cells can serve as effector cells to mediate ADCC. The anti-EREG antibody was administered to mice once a week for a total of five times starting at three days after the injection of Lgr5-positive cells. The number of tumor cells in distal locations was demonstrated to be markedly reduced as compared to that in control mice (FIG. 58B). In addition, the size of each tumor was also shown to be remarkably reduced in mice administered with the antibody (FIGS. 58C and 58D).

US11858987B2. Cancer stem cell-specific molecule (2024-01-02). CHUGAI SEIYAKU KABUSHIKI KAISHA [JP]. Inventors: Tatsumi Yamazaki [JP], Hisafumi Okabe [JP], Shinta Kobayashi [SG], Takeshi Watanabe [JP], Koichi Matsubara [SG], Osamu Natori [SG], Atsuhiko Kato [JP], Masami Suzuki [JP].

Full text: Click here

Patent 2024

Targeted Integration of CAR and IL-15 in T-cells

Not available on PMC !

Example 2

Materials

X-vivo-15 was obtained for Lonza (cat #BE04-418Q), IL-2 from Miltenyi Biotech (cat #130-097-748), human serum AB from Seralab (cat #GEM-100-318), human T activator CD3/CD28 from Life Technology (cat #11132D), QBEND10-APC from R&D Systems (cat #FAB7227A), vioblue-labeled anti-CD3, PE-labeled anti-LNGFR, APC-labeled anti-CD25 and PE-labeled anti-PD1 from Miltenyi (cat #130-094-363, 130-112-790, 130-109-021 and 130-104-892 respectively) 48 wells treated plates (CytoOne, cat #CC7682-7548), human IL-15 Quantikine ELISA kit from R&D systems (cat #S1500), ONE-Glo from Promega (cat #E6110). AAV6 batches containing the different matrices were obtained from Virovek, PBMC cells were obtained from Allcells, (cat #PB004F) and Raji-Luciferase cells were obtained after Firefly Luciferase-encoding lentiviral particles transduction of Raji cells from ATCC (cat #CCL-86).

Methods

2.1—Transfection-Transduction

The double targeted integration at TRAC and PD1 or CD25 loci were performed as follows. PBMC cells were first thawed, washed, resuspended and cultivated in X-vivo-15 complete media (X-vivo-15, 5% AB serum, 20 ng/mL IL-2). One day later, cells were activated by Dynabeads human T activator CD3/CD28 (25 uL of beads/1E⁶CD3 positive cells) and cultivated at a density of 1E⁶cells/mL for 3 days in X-vivo complete media at 37° C. in the presence of 5% CO₂. Cells were then split in fresh complete media and transduced/transfected the next day according to the following procedure. On the day of transduction-transfection, cells were first de-beaded by magnetic separation (EasySep), washed twice in Cytoporation buffer T (BTX Harvard Apparatus, Holliston, Massachusetts) and resuspended at a final concentration of 28E⁶cells/mL in the same solution. Cellular suspension was mixed with 5 μg mRNA encoding TRAC TALEN® arms (SEQ ID NO:16 and 17) in the presence or in the absence of 15 μg of mRNA encoding arms of either CD25 or PD1 TALEN® (SEQ ID NO:18 and 19 and SEQ ID NO:20 and 21 respectively) in a final volume of 200 μl. TALEN® is a standard format of TALE-nucleases resulting from a fusion of TALE with Fok-1 Transfection was performed using Pulse Agile technology, by applying two 0.1 mS pulses at 3,000 V/cm followed by four 0.2 mS pulses at 325 V/cm in 0.4 cm gap cuvettes and in a final volume of 200 μl of Cytoporation buffer T (BTX Harvard Apparatus, Holliston, Massachusetts). Electroporated cells were then immediately transferred to a 12-well plate containing 1 mL of prewarm X-vivo-15 serum-free media and incubated for 37° C. for 15 min. Cells were then concentrated to 8E⁶cells/mL in 250 μL of the same media in the presence of AAV6 particles (MOI=3E⁵vg/cells) comprising the donor matrices in 48 wells regular treated plates. After 2 hours of culture at 30° C., 250 μL of Xvivo-15 media supplemented by 10% AB serum and 40 ng/ml IL-2 was added to the cell suspension and the mix was incubated 24 hours in the same culture conditions. One day later, cells were seeded at 1E⁶cells/mL in complete X-vivo-15 media and cultivated at 37° C. in the presence of 5% CO₂.

2.2—Activation-Dependent Expression of ΔLNGFR and Secretion of IL15

Engineered T-cells were recovered from the transfection-transduction process described earlier and seeded at 1E⁶cells/mL alone or in the presence of Raji cells (E:T=1:1) or Dynabeads (12.5 uL/1E⁶cells) in 100 μL final volume of complete X-vivo-15 media. Cells were cultivated for 48 hours before being recovered, labeled and analyzed by flow cytometry. Cells were labeled with two independent sets of antibodies. The first sets of antibodies, aiming at detecting the presence of ΔLNGFR, CAR and CD3 cells, consisted in QBEND10-APC (diluted 1/10), vioblue-labeled anti CD3 (diluted 1/25) and PE-labeled anti-ΔLNGFR (diluted 1/25). The second sets of antibodies, aiming at detecting expression of endogenous CD25 and PD1, consisted in APC-labeled anti-CD25 (diluted 1/25) and vioblue-labeled anti PD1 (diluted 1/25).

The same experimental set up was used to study IL-15 secretion in the media. Cells mixture were kept in co-culture for 2, 4, 7 and 10 days before collecting and analyzing supernatant using an IL-15 specific ELISA kit.

2.3—Serial Killing Assay

To assess the antitumor activity of engineered CAR T-cells, a serial killing assay was performed. The principle of this assay is to challenge CAR T-cell antitumor activity everyday by a daily addition of a constant amount of tumor cells. Tumor cell proliferation, control and relapse could be monitored via luminescence read out thanks to a Luciferase marker stably integrated in Tumor cell lines.

Typically, CAR T-cells are mixed to a suspension of 2.5×10⁵Raji-luc tumor cells at variable E:T ratio (E:T=5:1 or 1:1) in a total volume of 1 mL of Xvivo 5% AB, 20 ng/uL IL-2. The mixture is incubated 24 hours before determining the luminescence of 25 uL of cell suspension using ONE-Glo reagent. Cells mixture are then spun down, the old media is discarded and substituted with 1 mL of fresh complete X-vivo-15 media containing 2.5×10⁵Raji-Luc cells and the resulting cell mixture is incubated for 24 hours. This protocol is repeated 4 days.

Experiments and Results

This example describes methods to improve the therapeutic outcome of CAR T-cell therapies by integrating an IL-15/soluble IL-15 receptor alpha heterodimer (IL15/sIL15rα) expression cassette under the control of the endogenous T-cell promoters regulating PD1 and CD25 genes. Because both genes are known to be upregulated upon tumor engagement by CAR T-cells, they could be hijacked to re-express IL-IL15/sIL15rα only in vicinity of a tumor. This method aims to reduce the potential side effects of IL15/sIL15rα systemic secretion while maintaining its capacity to reduced activation induced T-cell death (AICD), promote T-cell survival, enhance T-cell antitumor activity and to reverse T-cell anergy.

The method developed to integrate IL15/sIL15rα at PD1 and CD25 loci consisted in generating a double-strand break at both loci using TALEN in the presence of a DNA repair matrix vectorized by AAV6. This matrix consists of two homology arms embedding IL15/sIL15rα coding regions separated by a 2A cis acting elements and regulatory elements (stop codon and polyA sequences). Depending on the locus targeted and its involvement in T-cell activity, the targeted endogenous gene could be inactivated or not via specific matrix design. When CD25 gene was considered as targeted locus, the insertion matrix was designed to knock-in (KI) IL15/sIL15rα without inactivating CD25 because the protein product of this gene is regarded as essential for T-cell function. By contrast, because PD1 is involved in T-cell inhibition/exhaustion of T-cells, the insertion matrix was designed to prevent its expression while enabling the expression and secretion of IL15/sIL15rα.

To illustrate this approach and demonstrate the feasibility of double targeted insertion in primary T-cells, three different matrices were designed (FIGS. 2A, 2B and 2C). The first one named CARm represented by SEQ ID NO:36 was designed to insert an anti-CD22 CAR cDNA at the TRAC locus in the presence of TRAC TALEN® (SEQ ID NO:16 and 17). The second one, IL-15_CD25m (SEQ ID NO:37) was designed to integrate IL15, sIL15rα and the surface marker named ΔLNGFR cDNAs separated by 2A cis-acting elements just before the stop codon of CD25 endogenous coding sequence using CD25 TALEN® (SEQ ID NO:18 and 19). The third one, IL-15_PD1m (SEQ ID NO:38), contained the same expression cassette and was designed to integrate in the middle of the PD1 open reading frame using PD1 TALEN® (SEQ ID NO:20 and 21). The three matrices contained an additional 2A cis-acting element located upstream expression cassettes to enable co-expression of IL15/sIL15rα and CAR with the endogenous gene targeted.

We first assessed the efficiency of double targeted insertion in T-cells by transducing them with one of the AAV6 encoding IL15/sIL15rα matrices (SEQ ID NO:41; pCLS30519) along with the one encoding the CAR and subsequently transfected the corresponding TALEN®. AAV6-assisted vectorization of matrices in the presence of mRNA encoding TRAC TALEN® (SEQ ID NO:22 and 23) and PD1 TALEN® (SEQ ID NO:24 and 25) or CD25 TALEN® (SEQ ID NO:26 and 27) enabled expression of the anti CD22 CAR in up to 46% of engineered T-cells (FIG. 3).

To determine the extent of IL15m integration at CD25 and PD1 locus, engineered T-cells were activated with either antiCD3/CD28 coated beads or with CD22 expressing Raji tumor cells. 2 days post activation, cells were recovered and analyzed by FACS using LNGFR expression as IL15/sIL15rα secretion surrogate (FIGS. 4 and 5). Our results showed that antiCD3/CD28 coated beads induced expression of ΔLNGFR by T-cells containing IL-15m_CD25 or IL-15m_PD1, independently of the presence of the anti CD22 CAR (FIG. 4A-B). Tumor cells however, only induced expression of ΔLNGFR by T-cell treated by both CARm and IL-15m. This indicated that expression of ΔLNGFR could be specifically induced through tumor cell engagement by the CAR (FIGS. 5 and 6).

As expected the endogenous CD25 gene was still expressed in activated treated T-cells (FIGS. 7 and 8) while PD1 expression was strongly impaired (FIG. 12).

To verify that expression of ΔLNGFR correlated with secretion of IL15 in the media, T-cells expressing the anti-CD22 CAR and ΔLNGFR were incubated in the presence of CD22 expressing Raji tumor cells (E:T ratio=1:1) for a total of 10 days. Supernatant were recovered at day 2, 4, 7 and 10 and the presence of IL15 was quantified by ELISA assay. Our results showed that IL15 was secreted in the media only by T-cells that were co-treated by both CARm and IL15m matrices along with their corresponding TALEN® (FIG. 13). T-cell treated with either one of these matrices were unable to secrete any significant level of IL15 with respect to resting T-cells. Interestingly, IL-15 secretion level was found transitory, with a maximum peak centered at day 4 (FIG. 14).

To assess whether the level of secreted IL-15 (SEQ ID NO:59) could impact CAR T-cell activity, CAR T-cell were co-cultured in the presence of tumor cells at E:T ratio of 5:1 for 4 days. Their antitumor activity was challenged everyday by pelleting and resuspended them in a culture media lacking IL-2 and containing fresh tumor cells. Antitumor activity of CAR T-cell was monitored everyday by measuring the luminescence of the remaining Raji tumor cells expressing luciferase. Our results showed that CAR T-cells co-expressing IL-15 had a higher antitumor activity than those lacking IL15 at all time points considered (FIG. 15).

Thus, together our results showed that we have developed a method allowing simultaneous targeted insertions of CAR and IL15 cDNA at TRAC and 0025 or PD1 loci. This double targeted insertion led to robust expression of an antiCD22 CAR and to the secretion of IL15 in the media. Levels of secreted IL15 were sufficient to enhance the activity of CAR T-cells.

TABLE 5

Sequences referred to in example 2.

SEQ

IDSequence

NO#NamePolypeptide sequenceRVD sequence

16TALENMGDPKKKRKVIDYPYDVPDYAIDIADLRTLGYSQQQQEKIKPKVRSTVANG-NN-NG-HD-

right TRACQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVHD-HD-NI-HD-NI-

GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEANN-NI-NG-NI-NG-

VHAWRNALTGAPLNLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGHD-NG#

LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGG

GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVL

CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIA

SHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQAL

LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ

VVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALE

TVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGL

TPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK

QALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLC

QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIAS

NGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGL

GDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMK

VMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGY

NLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGH

FKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFN

NGEINFAAD

17TALENMGDPKKKRKVIDKETAAAKFERQHMDSIDIADLRTLGYSQQQQEKIKPKHD-NG-HD-NI-NN-

Left TRACVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEAHD-NG-NN-NN-

THEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVNG-NI-HD-NI-HD-

TAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQRLLPVLNN-NG#

CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAI

ASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQA

LLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPE

QVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQA

LETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA

HGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASN

GGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLP

VLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVV

AIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETV

QRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLT

PQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRP

ALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNS

TQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVD

TKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTE

FKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLT

LEEVRRKFNNGEINFAAD

18TALENMGDPKKKRKVIDYPYDVPDYAIDIADLRTLGYSQQQQEKIKPKVRSTVANN-NG-NG-HD-

right CD25QHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVNG-NG-NG-NG-

GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEANN-NN-NG-NG-

VHAWRNALTGAPLNLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGNG-NG-HD-NG#

LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGG

GKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVL

CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAI

ASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQ

RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP

QQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQ

ALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQ

AHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIAS

NGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRL

LPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQ

VVAIASNGGGRPALESIVAQLSRPDPSGSGSGGDPISRSQLVKSELEEK

KSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHL

GGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEEN

QTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITN

CNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD

19TALEN leftMGDPKKKRKVIDYPYDVPDYAIDIADLRTLGYSQQQQEKIKPKVRSTVANI-HD-NI-NN-NN-

CD25QHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVNI-NN-NN-NI-NI-

GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEANN-NI-NN-NG-NI-

VHAWRNALTGAPLNLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLNG#

TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK

QALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLC

QAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS

NIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLL

PVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQV

VAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASNIGGKQALETV

QALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLT

PEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQ

ALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQ

AHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASN

GGGRPALESIVAQLSRPDPSGSGSGGDPISRSQLVKSELEEKKSELRH

KLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRK

PDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNK

HINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAV

LSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFAAD

20TALENMGDPKKKRKVIDYPYDVPDYAIDIADLRTLGYSQQQQEKIKPKVRSTVAKL-HD-HD-NG-HD-

right PD1QHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVNG-YK-NG-NN-

GVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEANN-NN-NN-HD-

VHAWRNALTGAPLNLTPEQVVAIASKLGGKQALETVQALLPVLCQAHGLHD-NI-NG#

TPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGG

KQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVL

CQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAI

ASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASYKGGKQALETVQ

RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP

QQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQ

ALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQA

HGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHD

GGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLP

VLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVA

IASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVK

KGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRIL

EMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYS

GGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFV

SGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRR

KFNNGEINFAAD

21TALENMGDPKKKRKVIDKETAAAKFERQHMDSIDIADLRTLGYSQQQQEKIKPKHD-NG-HD-NG-

Left PD1VRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEANG-NG-NN-NI-NG-

THEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVHD-NG-NN-N-NN-

TAVEAVHAWRNALTGAPLNLTPEQVVAIASHDGGKQALETVQRLLPVLHD-NG#

CQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAI

ASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQ

RLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP

QQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQ

ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQA

HGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASH

DGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLL

PVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQV

VAIASNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETV

QRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLT

PQQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRP

ALDAVKKGLGDPISRSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNS

TQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVD

TKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTE

FKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLT

LEEVRRKFNNGEINFAAD

SEQ

IDSequence

NO#NamePolynucleotide sequence

22TALEN TRACATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATTACCCATACGATGTTCCAGATTACGCTAT

pCLS11370CGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAA

GGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGC

ACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACA

TGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCC

GGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACA

GTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGT

GCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTGACCCCCCAGCAGGTGGTGG

CCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTG

CTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGG

CAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGA

CCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGT

CCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCA

TCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTG

TGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAA

GCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCC

CGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCA

GCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCG

CCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGC

CAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCA

GGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCG

GAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGC

GCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCA

GCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAG

GCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGC

GCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGC

AGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCT

GTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCA

ATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCC

CACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCT

GGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAG

GTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTT

GCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGGTGGTGGCCATCGCCAGCAATG

GCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTG

GCCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGGCGTCCTGCGCTGGA

TGCAGTGAAAAAGGGATTGGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGA

GGAGAAGAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGAT

CGAGATCGCCCGGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCAT

GAAGGTGTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCT

ACACCGTGGGCTCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGC

TACAACCTGCCCATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAG

GAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAA

GTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCA

CATCACCAACTGCAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGA

TCAAGGCCGGCACCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAAC

TTCGCGGCCGACTGATAA

23TALEN TRACATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATAAGGAGACCGCCGCTGCCAAGTTCGAG

pCLS11369AGACAGCACATGGACAGCATCGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAA

CAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGG

CCACGGGTTTACACACGCGCACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGT

CGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGG

CGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGT

TGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCG

TGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTG

ACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGG

TCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCC

ATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCT

GTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGC

AAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGAC

CCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGC

AGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATC

GCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTG

CCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAG

CAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCC

CCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAG

CGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGC

CAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCC

AGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAG

GCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCA

GCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGG

CTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAG

CAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGG

CCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGC

GCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGC

AGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTG

TTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCA

CGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCC

CACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCT

GGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGG

TGGTGGCCATCGCCAGCAATGGCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTA

TCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTC

GGCGGGCGTCCTGCGCTGGATGCAGTGAAAAAGGGATTGGGGGATCCTATCAGCCGTTCCCA

GCTGGTGAAGTCCGAGCTGGAGGAGAAGAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCC

CCACGAGTACATCGAGCTGATCGAGATCGCCCGGAACAGCACCCAGGACCGTATCCTGGAGAT

GAAGGTGATGGAGTTCTTCATGAAGGTGTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCA

GGAAGCCCGACGGCGCCATCTACACCGTGGGCTCCCCCATCGACTACGGCGTGATCGTGGAC

ACCAAGGCCTACTCCGGCGGCTACAACCTGCCCATCGGCCAGGCCGACGAAATGCAGAGGTA

CGTGGAGGAGAACCAGACCAGGAACAAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACC

CCTCCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGG

CCCAGCTGACCAGGCTGAACCACATCACCAACTGCAACGGCGCCGTGCTGTCCGTGGAGGAG

CTCCTGATCGGCGGCGAGATGATCAAGGCCGGCACCCTGACCCTGGAGGAGGTGAGGAGGAA

GTTCAACAACGGCGAGATCAACTTCGCGGCCGACTGATAA

24TALEN CD25ATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATTACCCATACGATGTTCCAGATTACGCTAT

pCLS30480CGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAA

GGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGC

ACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACA

TGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCC

GGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACA

GTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGT

GCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTGACCCCCCAGCAGGTGGTGG

CCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTG

CTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGG

CAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGA

CCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGT

CCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCA

TCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTG

TGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAA

GCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCC

CCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCA

GCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCG

CCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGC

CAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCA

GGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCC

AGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGG

CTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAG

CAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGG

CCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCG

CTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCA

GGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTG

TTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAA

TGGCGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCC

CACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCT

GGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAG

GTGGTGGCCATCGCCAGCCACGATGGCGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTT

GCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGGTGGTGGCCATCGCCAGCAATG

GCGGCGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGAGTGGC

AGCGGAAGTGGCGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGAGGAGAA

GAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGAT

CGCCCGGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGT

GTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCTACACCG

TGGGCTCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGCTACAACC

TGCCCATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAGGAACAAG

CACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAAGTTCCTG

TTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACC

AACTGCAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGATCAAGGC

CGGCACCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAACTTCGCGG

CCGACTGATAA

25TALEN CD25ATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATTACCCATACGATGTTCCAGATTACGCTAT

pCLS30479CGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAA

GGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGC

ACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACA

TGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCC

GGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACA

GTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGT

GCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTGACCCCGGAGCAGGTGGTGG

CCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTG

CTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCCACGATGGCGG

CAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGA

CCCCGGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTG

CAGGCGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCAT

CGCCAGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGT

GCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAG

CAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCC

GGAGCAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGG

CGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCC

AGCAATAATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCA

GGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGG

CGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAG

CAGGTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCT

GTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGGTGGCCATCGCCAGCA

ATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCGGTGCTGTGCCAGGCC

CACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATAATGGTGGCAAGCAGGCGCT

GGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAG

GTGGTGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTT

GCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATA

ATGGTGGCAAGCAGGCGCTGGAGACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCAC

GGCTTGACCCCCCAGCAGGTGGTGGCCATCGCCAGCAATGGCGGTGGCAAGCAGGCGCTGGA

GACGGTCCAGCGGCTGTTGCCGGTGCTGTGCCAGGCCCACGGCTTGACCCCGGAGCAGGTGG

TGGCCATCGCCAGCAATATTGGTGGCAAGCAGGCGCTGGAGACGGTGCAGGCGCTGTTGCCG

GTGCTGTGCCAGGCCCACGGCTTGACCCCTCAGCAGGTGGTGGCCATCGCCAGCAATGGCGG

CGGCAGGCCGGCGCTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGAGTGGCAGCG

GAAGTGGCGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGAGGAGAAGAAAT

CCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCC

GGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACG

GCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCTACACCGTGGGC

TCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGCTACAACCTGCCC

ATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAGGAACAAGCACAT

CAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAAGTTCCTGTTCGT

GTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTG

CAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGATCAAGGCCGGCA

CCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAACTTCGCGGCCGAC

TGATAA

26TALEN PD1ATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATTACCCATACGATGTTCCAGATTACGCTAT

pCLS28959CGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAACAGGAGAAGATCAAACCGAA

GGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGGCCACGGGTTTACACACGCGC

ACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGACA

TGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGGCGTCGGCAAACAGTGGTCC

GGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGTTGAGAGGTCCACCGTTACA

GTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGT

GCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTGACCCCCGAGCAAGTGGTGG

CTATCGCTTCCAAGCTGGGGGGAAAGCAGGCCCTGGAGACCGTCCAGGCCCTTCTCCCAGTG

CTTTGCCAGGCTCACGGACTGACCCCTGAACAGGTGGTGGCAATTGCCTCACACGACGGGGG

CAAGCAGGCACTGGAGACTGTCCAGCGGCTGCTGCCTGTCCTCTGCCAGGCCCACGGACTCA

CTCCTGAGCAGGTCGTGGCCATTGCCAGCCACGATGGGGGCAAACAGGCTCTGGAGACCGTG

CAGCGCCTCCTCCCAGTGCTGTGCCAGGCTCATGGGCTGACCCCACAGCAGGTCGTCGCCATT

GCCAGTAACGGCGGGGGGAAGCAGGCCCTCGAAACAGTGCAGAGGCTGCTGCCCGTCTTGTG

CCAAGCACACGGCCTGACACCCGAGCAGGTGGTGGCCATCGCCTCTCATGACGGCGGCAAGC

AGGCCCTTGAGACAGTGCAGAGACTGTTGCCCGTGTTGTGTCAGGCCCACGGGTTGACACCCC

AGCAGGTGGTCGCCATCGCCAGCAATGGCGGGGGAAAGCAGGCCCTTGAGACCGTGCAGCGG

TTGCTTCCAGTGTTGTGCCAGGCACACGGACTGACCCCTCAACAGGTGGTCGCAATCGCCAGC

TACAAGGGCGGAAAGCAGGCTCTGGAGACAGTGCAGCGCCTCCTGCCCGTGCTGTGTCAGGC

TCACGGACTGACACCACAGCAGGTGGTCGCCATCGCCAGTAACGGGGGCGGCAAGCAGGCTT

TGGAGACCGTCCAGAGACTCCTCCCCGTCCTTTGCCAGGCCCACGGGTTGACACCTCAGCAGG

TCGTCGCCATTGCCTCCAACAACGGGGGCAAGCAGGCCCTCGAAACTGTGCAGAGGCTGCTG

CCTGTGCTGTGCCAGGCTCATGGGCTGACACCCCAGCAGGTGGTGGCCATTGCCTCTAACAAC

GGCGGCAAACAGGCACTGGAGACCGTGCAAAGGCTGCTGCCCGTCCTCTGCCAAGCCCACGG

GCTCACTCCACAGCAGGTCGTGGCCATCGCCTCAAACAATGGCGGGAAGCAGGCCCTGGAGA

CTGTGCAAAGGCTGCTCCCTGTGCTCTGCCAGGCACACGGACTGACCCCTCAGCAGGTGGTG

GCAATCGCTTCCAACAACGGGGGAAAGCAGGCCCTCGAAACCGTGCAGCGCCTCCTCCCAGT

GCTGTGCCAGGCACATGGCCTCACACCCGAGCAAGTGGTGGCTATCGCCAGCCACGACGGAG

GGAAGCAGGCTCTGGAGACCGTGCAGAGGCTGCTGCCTGTCCTGTGCCAGGCCCACGGGCTT

ACTCCAGAGCAGGTCGTCGCCATCGCCAGTCATGATGGGGGGAAGCAGGCCCTTGAGACAGT

CCAGCGGCTGCTGCCAGTCCTTTGCCAGGCTCACGGCTTGACTCCCGAGCAGGTCGTGGCCAT

TGCCTCAAACATTGGGGGCAAACAGGCCCTGGAGACAGTGCAGGCCCTGCTGCCCGTGTTGTG

TCAGGCCCACGGCTTGACACCCCAGCAGGTGGTCGCCATTGCCTCTAATGGCGGCGGGAGAC

CCGCCTTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCGCGTTGACCA

ACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGGCGTCCTGCGCTGGATGCAGTGAAAAAG

GGATTGGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGAGGAGAAGAAATCC

GAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAGATCGCCCGG

AACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAGGTGTACGGC

TACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCTACACCGTGGGCTC

CCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGCTACAACCTGCCCAT

CGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAGGAACAAGCACATCA

ACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAAGTTCCTGTTCGTGT

CCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATCACCAACTGCA

ACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGATCAAGGCCGGCACC

CTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAACTTCGCGGCCGACTG

ATAA

27TALEN PD1ATGGGCGATCCTAAAAAGAAACGTAAGGTCATCGATAAGGAGACCGCCGCTGCCAAGTTCGAG

pCLS18792AGACAGCACATGGACAGCATCGATATCGCCGATCTACGCACGCTCGGCTACAGCCAGCAGCAA

CAGGAGAAGATCAAACCGAAGGTTCGTTCGACAGTGGCGCAGCACCACGAGGCACTGGTCGG

CCACGGGTTTACACACGCGCACATCGTTGCGTTAAGCCAACACCCGGCAGCGTTAGGGACCGT

CGCTGTCAAGTATCAGGACATGATCGCAGCGTTGCCAGAGGCGACACACGAAGCGATCGTTGG

CGTCGGCAAACAGTGGTCCGGCGCACGCGCTCTGGAGGCCTTGCTCACGGTGGCGGGAGAGT

TGAGAGGTCCACCGTTACAGTTGGACACAGGCCAACTTCTCAAGATTGCAAAACGTGGCGGCG

TGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCCCGCTCAACTTG

ACCCCCGAGCAAGTCGTCGCAATCGCCAGCCATGATGGAGGGAAGCAAGCCCTCGAAACCGT

GCAGCGGTTGCTTCCTGTGCTCTGCCAGGCCCACGGCCTTACCCCTCAGCAGGTGGTGGCCAT

CGCAAGTAACGGAGGAGGAAAGCAAGCCTTGGAGACAGTGCAGCGCCTGTTGCCCGTGCTGT

GCCAGGCACACGGCCTCACACCAGAGCAGGTCGTGGCCATTGCCTCCCATGACGGGGGGAAA

CAGGCTCTGGAGACCGTCCAGAGGCTGCTGCCCGTCCTCTGTCAAGCTCACGGCCTGACTCCC

CAACAAGTGGTCGCCATCGCCTCTAATGGCGGCGGGAAGCAGGCACTGGAAACAGTGCAGAG

ACTGCTCCCTGTGCTTTGCCAAGCTCATGGGTTGACCCCCCAACAGGTCGTCGCTATTGCCTCA

AACGGGGGGGGCAAGCAGGCCCTTGAGACTGTGCAGAGGCTGTTGCCAGTGCTGTGTCAGGC

TCACGGGCTCACTCCACAACAGGTGGTCGCAATTGCCAGCAACGGCGGCGGAAAGCAAGCTCT

TGAAACCGTGCAACGCCTCCTGCCCGTGCTCTGTCAGGCTCATGGCCTGACACCACAACAAGT

CGTGGCCATCGCCAGTAATAATGGCGGGAAACAGGCTCTTGAGACCGTCCAGAGGCTGCTCCC

AGTGCTCTGCCAGGCACACGGGCTGACCCCCGAGCAGGTGGTGGCTATCGCCAGCAATATTG

GGGGCAAGCAGGCCCTGGAAACAGTCCAGGCCCTGCTGCCAGTGCTTTGCCAGGCTCACGGG

CTCACTCCCCAGCAGGTCGTGGCAATCGCCTCCAACGGCGGAGGGAAGCAGGCTCTGGAGAC

CGTGCAGAGACTGCTGCCCGTCTTGTGCCAGGCCCACGGACTCACACCTGAACAGGTCGTCGC

CATTGCCTCTCACGATGGGGGCAAACAAGCCCTGGAGACAGTGCAGCGGCTGTTGCCTGTGTT

GTGCCAAGCCCACGGCTTGACTCCTCAACAAGTGGTCGCCATCGCCTCAAATGGCGGCGGAAA

ACAAGCTCTGGAGACAGTGCAGAGGTTGCTGCCCGTCCTCTGCCAAGCCCACGGCCTGACTCC

CCAACAGGTCGTCGCCATTGCCAGCAACAACGGAGGAAAGCAGGCTCTCGAAACTGTGCAGCG

GCTGCTTCCTGTGCTGTGTCAGGCTCATGGGCTGACCCCCGAGCAAGTGGTGGCTATTGCCTC

TAATGGAGGCAAGCAAGCCCTTGAGACAGTCCAGAGGCTGTTGCCAGTGCTGTGCCAGGCCCA

CGGGCTCACACCCCAGCAGGTGGTCGCCATCGCCAGTAACAACGGGGGCAAACAGGCATTGG

AAACCGTCCAGCGCCTGCTTCCAGTGCTCTGCCAGGCACACGGACTGACACCCGAACAGGTGG

TGGCCATTGCATCCCATGATGGGGGCAAGCAGGCCCTGGAGACCGTGCAGAGACTCCTGCCA

GTGTTGTGCCAAGCTCACGGCCTCACCCCTCAGCAAGTCGTGGCCATCGCCTCAAACGGGGG

GGGCCGGCCTGCACTGGAGAGCATTGTTGCCCAGTTATCTCGCCCTGATCCGGCGTTGGCCG

CGTTGACCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGGCGTCCTGCGCTGGATGCA

GTGAAAAAGGGATTGGGGGATCCTATCAGCCGTTCCCAGCTGGTGAAGTCCGAGCTGGAGGAG

AAGAAATCCGAGTTGAGGCACAAGCTGAAGTACGTGCCCCACGAGTACATCGAGCTGATCGAG

ATCGCCCGGAACAGCACCCAGGACCGTATCCTGGAGATGAAGGTGATGGAGTTCTTCATGAAG

GTGTACGGCTACAGGGGCAAGCACCTGGGCGGCTCCAGGAAGCCCGACGGCGCCATCTACAC

CGTGGGCTCCCCCATCGACTACGGCGTGATCGTGGACACCAAGGCCTACTCCGGCGGCTACA

ACCTGCCCATCGGCCAGGCCGACGAAATGCAGAGGTACGTGGAGGAGAACCAGACCAGGAAC

AAGCACATCAACCCCAACGAGTGGTGGAAGGTGTACCCCTCCAGCGTGACCGAGTTCAAGTTC

CTGTTCGTGTCCGGCCACTTCAAGGGCAACTACAAGGCCCAGCTGACCAGGCTGAACCACATC

ACCAACTGCAACGGCGCCGTGCTGTCCGTGGAGGAGCTCCTGATCGGCGGCGAGATGATCAA

GGCCGGCACCCTGACCCTGGAGGAGGTGAGGAGGAAGTTCAACAACGGCGAGATCAACTTCG

CGGCCGACTGATAA

28TALE NTTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGA

target TRAC

29TALE NTACAGGAGGAAGAGTAGAAGAACAATCTAGAAAACCAAAAGAACA

target CD25

30TALE NTACCTCTGTGGGGCCATCTCCCTGGCCCCCAAGGCGCAGATCAAAGAGA

target PD1

31Matrice TRACTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGA

locus_CubiCARAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGT

CD22GGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGC

pCLS30056TTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTA

TAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCT

GGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTG

TCCCACAGATATCCAGTACCCCTACGACGTGCCCGACTACGCCTCCGGTGAGGGCAGAGGAAG

TCTTCTAACATGCGGTGACGTGGAGGAGAATCCGGGCCCCGGATCCGCTCTGCCCGTCACCGC

TCTGCTGCTGCCACTGGCACTGCTGCTGCACGCTGCTAGGCCCGGAGGGGGAGGCAGCTGCC

CCTACAGCAACCCCAGCCTGTGCAGCGGAGGCGGCGGCAGCGGCGGAGGGGGTAGCCAGGT

GCAGCTGCAGCAGAGCGGCCCTGGCCTGGTGAAGCCAAGCCAGACACTGTCCCTGACCTGCG

CCATCAGCGGCGATTCCGTGAGCTCCAACTCCGCCGCCTGGAATTGGATCAGGCAGTCCCCTT

CTCGGGGCCTGGAGTGGCTGGGAAGGACATACTATCGGTCTAAGTGGTACAACGATTATGCCG

TGTCTGTGAAGAGCAGAATCACAATCAACCCTGACACCTCCAAGAATCAGTTCTCTCTGCAGCT

GAATAGCGTGACACCAGAGGACACCGCCGTGTACTATTGCGCCAGGGAGGTGACCGGCGACC

TGGAGGATGCCTTTGACATCTGGGGCCAGGGCACAATGGTGACCGTGAGCTCCGGAGGCGGC

GGATCTGGCGGAGGAGGAAGTGGGGGCGGCGGGAGTGATATCCAGATGACACAGTCCCCATC

CTCTCTGAGCGCCTCCGTGGGCGACAGAGTGACAATCACCTGTAGGGCCTCCCAGACCATCTG

GTCTTACCTGAACTGGTATCAGCAGAGGCCCGGCAAGGCCCCTAATCTGCTGATCTACGCAGC

AAGCTCCCTGCAGAGCGGAGTGCCATCCAGATTCTCTGGCAGGGGCTCCGGCACAGACTTCAC

CCTGACCATCTCTAGCCTGCAGGCCGAGGACTTCGCCACCTACTATTGCCAGCAGTCTTATAGC

ATCCCCCAGACATTTGGCCAGGGCACCAAGCTGGAGATCAAGTCGGATCCCGGAAGCGGAGG

GGGAGGCAGCTGCCCCTACAGCAACCCCAGCCTGTGCAGCGGAGGCGGCGGCAGCGAGCTG

CCCACCCAGGGCACCTTCTCCAACGTGTCCACCAACGTGAGCCCAGCCAAGCCCACCACCACC

GCCTGTCCTTATTCCAATCCTTCCCTGTGTGCTCCCACCACAACCCCCGCTCCAAGGCCCCCTA

CCCCCGCACCAACTATTGCCTCCCAGCCACTCTCACTGCGGCCTGAGGCCTGTCGGCCCGCTG

CTGGAGGCGCAGTGCATACAAGGGGCCTCGATTTCGCCTGCGATATTTACATCTGGGCACCCC

TCGCCGGCACCTGCGGGGTGCTTCTCCTCTCCCTGGTGATTACCCTGTATTGCAGACGGGGCC

GGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTCATGCGGCCAGTGCAGACAACCCAAGAGGA

GGATGGGTGTTCCTGCAGATTCCCTGAGGAAGAGGAAGGCGGGTGCGAGCTGAGAGTGAAGT

TCTCCAGGAGCGCAGATGCCCCCGCCTATCAACAGGGCCAGAACCAGCTCTACAACGAGCTTA

ACCTCGGGAGGCGCGAAGAATACGACGTGTTGGATAAGAGAAGGGGGCGGGACCCCGAGATG

GGAGGAAAGCCCCGGAGGAAGAACCCTCAGGAGGGCCTGTACAACGAGCTGCAGAAGGATAA

GATGGCCGAGGCCTACTCAGAGATCGGGATGAAGGGGGAGCGGCGCCGCGGGAAGGGGCAC

GATGGGCTCTACCAGGGGCTGAGCACAGCCACAAAGGACACATACGACGCCTTGCACATGCAG

GCCCTTCCACCCCGGGAATAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTG

CCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTG

CCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCAT

TCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAG

GCATGCTGGGGATGCGGTGGGCTCTATGACTAGTGGCGAATTCCCGTGTACCAGCTGAGAGAC

TCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA

AAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCA

AGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAA

CAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCA

GGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAA

CTCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAA

32Matrice CD25GTTTATTATTCCTGTTCCACAGCTATTGTCTGCCATATAAAAACTTAGGCCAGGCACAGTGGCTC

locus_IL15_ACACCTGTAATCCCAGCACTTTGGAAGGCCGAGGCAGGCAGATCACAAGGTCAGGAGTTCGAG

2A_sIL15RaACCAGCCTGGCCAACATAGCAAAACCCCATCTCTACTAAAAATACAAAAATTAGCCAGGCATGG

pCLS0519TGGCGTGTGCACTGGTTTAGAGTGAGGACCACATTTTTTTGGTGCCGTGTTACACATATGACCG

TGACTTTGTTACACCACTACAGGAGGAAGAGTAGAAGAACAATCGGTTCTGGCGTGAAACAGAC

TTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCGGTACCGG

GTCCGCCACCATGGACTGGACCTGGATTCTGTTCCTCGTGGCTGCTGCTACAAGAGTGCACAG

CGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGG

GTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACT

TTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGA

GTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCA

TCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAG

GAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATC

AACACTTCTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAG

AACCCTGGACCTGGGACCGGCTCTGCAACCATGGATTGGACGTGGATCCTGTTTCTCGTGGCA

GCTGCCACAAGAGTTCACAGTATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATC

TGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTA

AAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGG

ACAACCCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGCGCCACCC

TCCACAGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGAAAAGAG

CCCGCAGCTTCATCTCCCAGCTCAAACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGC

TCCCAGCTGATGCCTTCAAAATCACCTTCCACAGGAACCACAGAGATAAGCAGTCATGAGTCCT

CCCACGGCACCCCCTCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCACC

AGCCGCCAGGTGTGTATCCACAGGGCCACAGCGACACCACTGAGGGCAGAGGCAGCCTGCTG

ACCTGCGGCGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCA

TGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAG

GCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGA

GGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGA

CGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTC

CAGAGCATGTCGGCGCCGTGCGTGGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTA

CTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGC

CTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTAT

TCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCG

CCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGA

TTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAG

GCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGG

CAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCAT

CCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGAAAAACCAAAAGA

ACAAGAATTTCTTGGTAAGAAGCCGGGAACAGACAACAGAAGTCATGAAGCCCAAGTGAAATCA

AAGGTGCTAAATGGTCGCCCAGGAGACATCCGTTGTGCTTGCCTGCGTTTTGGAAGCTCTGAA

GTCACATCACAGGACACGGGGCAGTGGCAACCTTGTCTCTATGCCAGCTCAGTCCCATCAGAG

AGCGAGCGCTACCCACTTCTAAATAGCAATTTCGCCGTTGAAGAGGAAGGGCAAAACCACTAGA

ACTCTCCATCTTATTTTCATGTATATGTGTTCAT

33MatriCe PD1GACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAA

locus_IL15_GGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGG

2A_sIL15RaTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCA

pCLS30513GCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAG

CGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCGGTTCTGGCGTGA

AACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCG

GTACCGGGTCCGCCACCATGGACTGGACCTGGATTCTGTTCCTCGTGGCTGCTGCTACAAGAG

TGCACAGCGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGC

CAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGA

TGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTC

TCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAAT

CTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGA

ATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAAT

GTTCATCAACACTTCTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGT

GGAGGAGAACCCTGGACCTGGGACCGGCTCTGCAACCATGGATTGGACGTGGATCCTGTTTCT

CGTGGCAGCTGCCACAAGAGTTCACAGTATCACGTGCCCTCCCCCCATGTCCGTGGAACACGC

AGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTC

AAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGC

CCACTGGACAACCCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGC

GCCACCCTCCACAGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTG

GAAAAGAGCCCGCAGCTTCATCTCCCAGCTCAAACAACACAGCGGCCACAACAGCAGCTATTG

TCCCGGGCTCCCAGCTGATGCCTTCAAAATCACCTTCCACAGGAACCACAGAGATAAGCAGTCA

TGAGTCCTCCCACGGCACCCCCTCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGC

CTCCCACCAGCCGCCAGGTGTGTATCCACAGGGCCACAGCGACACCACTGAGGGCAGAGGCA

GCCTGCTGACCTGCGGCGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTGCCACCGG

CCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTG

CCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAAC

CTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGA

CAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCG

TGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGATGACGCCGTGTGCCGCTGCGC

CTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGG

GCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGAC

GGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGA

CACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCT

GGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCA

GGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCA

CAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCT

ATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGATC

TAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTT

GTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT

AAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGG

GCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCT

CTATGACTAGTGGCGAATTCGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACA

GGTGCGGCCTCGGAGGCCCCGGGGCAGGGGTGAGCTGAGCCGGTCCTGGGGTGGGTGTCCC

CTCCTGCACAGGATCAGGAGCTCCAGGGTCGTAGGGCAGGGACCCCCCAGCTCCAGTCCAGG

GCTCTGTCCTGCACCTGGGGAATGGTGACCGGCATCTCTGTCCTCTAGCTCTGGAAGCACCCC

AGCCCCTCTAGTCTGCCCTCACCCCTGACCCTGACCCTCCACCCTGACCCCGTCCTAACCCCT

GACCTTTG

34Matrice CD25GTTTATTATTCCTGTTCCACAGCTATTGTCTGCCATATAAAAACTTAGGCCAGGCACAGTGGCTC

locus_IL12a_ACACCTGTAATCCCAGCACTTTGGAAGGCCGAGGCAGGCAGATCACAAGGTCAGGAGTTCGAG

2A_IL12bACCAGCCTGGCCAACATAGCAAAACCCCATCTCTACTAAAAATACAAAAATTAGCCAGGCATGG

pCLS30520TGGCGTGTGCACTGGTTTAGAGTGAGGACCACATTTTTTTGGTGCCGTGTTACACATATGACCG

TGACTTTGTTACACCACTACAGGAGGAAGAGTAGAAGAACAATCGGTTCTGGCGTGAAACAGAC

TTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCATGTGGCC

CCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCATCCAGCGG

CTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCCTCCTTGTGG

CTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCCACTCCAGACC

CAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCA

GAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCA

CAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTG

CCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCT

TTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAA

GACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTG

GCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCT

CCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAA

TTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCGGAAGCGGAGCTACTAA

CTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGTGTCACCAGCA

GTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGA

AGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCT

CACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTT

AGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGT

CACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTT

GGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGC

CAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACATTCA

GTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCT

CTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGAC

AGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAG

CTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCA

AGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTG

ACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAG

CAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAA

AAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATGGGC

ATCTGTGCCCTGCAGTGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTCGAGGAGAACC

CCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTT

GCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACAC

ACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCC

AACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGAC

CGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTG

GAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCG

CTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGC

AGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGAC

CCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTG

GGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGG

GCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATA

GCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCG

AGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTCTT

GTGGCCTACATAGCCTTCAAGAGGTGAAAAACCAAAAGAACAAGAATTTCTTGGTAAGAAGCCG

GGAACAGACAACAGAAGTCATGAAGCCCAAGTGAAATCAAAGGTGCTAAATGGTCGCCCAGGA

GACATCCGTTGTGCTTGCCTGCGTTTTGGAAGCTCTGAAGTCACATCACAGGACACGGGGCAG

TGGCAACCTTGTCTCTATGCCAGCTCAGTCCCATCAGAGAGCGAGCGCTACCCACTTCTAAATA

GCAATTTCGCCGTTGAAGAGGAAGGGCAAAACCACTAGAACTCTCCATCTTATTTTCATGTATAT

GTGTTCAT

35Matrice PD1GACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAA

locus_IL12a_GGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGG

2A_IL12bTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCA

pCLS30511GCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAG

CGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCGGTTCTGGCGTGA

AACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCAT

GTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCATC

CAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCCTC

CTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCCACT

CCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAAC

ATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGA

AGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAAT

GAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAA

AGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTG

GAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAA

CATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAA

AAATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCT

TTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCGGAAGCGGAG

CTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGTGTC

ACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGG

GAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGG

TGGTCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTG

AGGTCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTA

CACCTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGAT

GGAATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATG

CGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTG

ACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCT

ACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAG

GAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTT

CACAAGCTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACC

CACCCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGT

ACCCTGACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGG

CAAGAGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTG

CCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGA

ATGGGCATCTGTGCCCTGCAGTGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTCGAGG

AGAACCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCT

GCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTA

CACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTG

GAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGC

GCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGT

GCGTGGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACT

GGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGG

ACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCAC

GTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCAC

ACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCC

CAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGAC

CTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGT

GACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGT

GGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGATCTAGAGGGCCCGTTTAAACCCGCTGATCA

GCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA

CCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCT

GAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGG

AAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGACTAGTGGCGAATTCGGCGCAG

ATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGGTGCGGCCTCGGAGGCCCCGGGGC

AGGGGTGAGCTGAGCCGGTCCTGGGGTGGGTGTCCCCTCCTGCACAGGATCAGGAGCTCCAG

GGTCGTAGGGCAGGGACCCCCCAGCTCCAGTCCAGGGCTCTGTCCTGCACCTGGGGAATGGT

GACCGGCATCTCTGTCCTCTAGCTCTGGAAGCACCCCAGCCCCTCTAGTCTGCCCTCACCCCT

GACCCTGACCCTCCACCCTGACCCCGTCCTAACCCCTGACCTTTG

36InsertedATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCCTGTTGCT

matrice TRACGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATC

locus_CubiCARCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCA

CD22 (60GGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGT

nucleotidesGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAA

upstream andGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGAC

downstream)TCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCA

CAGATATCCAGTACCCCTACGACGTGCCCGACTACGCCTCCGGTGAGGGCAGAGGAAGTCTTC

TAACATGCGGTGACGTGGAGGAGAATCCGGGCCCCGGATCCGCTCTGCCCGTCACCGCTCTG

CTGCTGCCACTGGCACTGCTGCTGCACGCTGCTAGGCCCGGAGGGGGAGGCAGCTGCCCCTA

CAGCAACCCCAGCCTGTGCAGCGGAGGCGGCGGCAGCGGCGGAGGGGGTAGCCAGGTGCAG

CTGCAGCAGAGCGGCCCTGGCCTGGTGAAGCCAAGCCAGACACTGTCCCTGACCTGCGCCAT

CAGCGGCGATTCCGTGAGCTCCAACTCCGCCGCCTGGAATTGGATCAGGCAGTCCCCTTCTCG

GGGCCTGGAGTGGCTGGGAAGGACATACTATCGGTCTAAGTGGTACAACGATTATGCCGTGTC

TGTGAAGAGCAGAATCACAATCAACCCTGACACCTCCAAGAATCAGTTCTCTCTGCAGCTGAAT

AGCGTGACACCAGAGGACACCGCCGTGTACTATTGCGCCAGGGAGGTGACCGGCGACCTGGA

GGATGCCTTTGACATCTGGGGCCAGGGCACAATGGTGACCGTGAGCTCCGGAGGCGGCGGAT

CTGGCGGAGGAGGAAGTGGGGGCGGCGGGAGTGATATCCAGATGACACAGTCCCCATCCTCT

CTGAGCGCCTCCGTGGGCGACAGAGTGACAATCACCTGTAGGGCCTCCCAGACCATCTGGTCT

TACCTGAACTGGTATCAGCAGAGGCCCGGCAAGGCCCCTAATCTGCTGATCTACGCAGCAAGC

TCCCTGCAGAGCGGAGTGCCATCCAGATTCTCTGGCAGGGGCTCCGGCACAGACTTCACCCTG

ACCATCTCTAGCCTGCAGGCCGAGGACTTCGCCACCTACTATTGCCAGCAGTCTTATAGCATCC

CCCAGACATTTGGCCAGGGCACCAAGCTGGAGATCAAGTCGGATCCCGGAAGCGGAGGGGGA

GGCAGCTGCCCCTACAGCAACCCCAGCCTGTGCAGCGGAGGCGGCGGCAGCGAGCTGCCCA

CCCAGGGCACCTTCTCCAACGTGTCCACCAACGTGAGCCCAGCCAAGCCCACCACCACCGCCT

GTCCTTATTCCAATCCTTCCCTGTGTGCTCCCACCACAACCCCCGCTCCAAGGCCCCCTACCCC

CGCACCAACTATTGCCTCCCAGCCACTCTCACTGCGGCCTGAGGCCTGTCGGCCCGCTGCTGG

AGGCGCAGTGCATACAAGGGGCCTCGATTTCGCCTGCGATATTTACATCTGGGCACCCCTCGC

CGGCACCTGCGGGGTGCTTCTCCTCTCCCTGGTGATTACCCTGTATTGCAGACGGGGCCGGAA

GAAGCTCCTCTACATTTTTAAGCAGCCTTTCATGCGGCCAGTGCAGACAACCCAAGAGGAGGAT

GGGTGTTCCTGCAGATTCCCTGAGGAAGAGGAAGGCGGGTGCGAGCTGAGAGTGAAGTTCTC

CAGGAGCGCAGATGCCCCCGCCTATCAACAGGGCCAGAACCAGCTCTACAACGAGCTTAACCT

CGGGAGGCGCGAAGAATACGACGTGTTGGATAAGAGAAGGGGGCGGGACCCCGAGATGGGA

GGAAAGCCCCGGAGGAAGAACCCTCAGGAGGGCCTGTACAACGAGCTGCAGAAGGATAAGAT

GGCCGAGGCCTACTCAGAGATCGGGATGAAGGGGGAGCGGCGCCGCGGGAAGGGGCACGAT

GGGCTCTACCAGGGGCTGAGCACAGCCACAAAGGACACATACGACGCCTTGCACATGCAGGC

CCTTCCACCCCGGGAATAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCC

TTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC

ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTC

TATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGC

ATGCTGGGGATGCGGTGGGCTCTATGACTAGTGGCGAATTCCCGTGTACCAGCTGAGAGACTC

TAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAA

GTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAG

AGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACA

GCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGGTGCCTTCGCAG

GCTGTTTCCTTGCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAAC

TCCTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGT

GAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATGAAGA

37InsertedAGTGCTGGCTAGAAACCAAGTGCTTTACTGCATGCACATCATTTAGCACAGTTAGTTGCTGTTTA

matrice CD25TTATTCCTGTTCCACAGCTATTGTCTGCCATATAAAAACTTAGGCCAGGCACAGTGGCTCACACC

locus_IL15_TGTAATCCCAGCACTTTGGAAGGCCGAGGCAGGCAGATCACAAGGTCAGGAGTTCGAGACCAG

2A_sIL15RaCCTGGCCAACATAGCAAAACCCCATCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGCG

(60TGTGCACTGGTTTAGAGTGAGGACCACATTTTTTTGGTGCCGTGTTACACATATGACCGTGACTT

nucleotidesTGTTACACCACTACAGGAGGAAGAGTAGAAGAACAATCGGTTCTGGCGTGAAACAGACTTTGAA

upstream andTTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCGGTACCGGGTCCGC

downstream)CACCATGGACTGGACCTGGATTCTGTTCCTCGTGGCTGCTGCTACAAGAGTGCACAGCGGCAT

TCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAAT

GTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATA

CGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACA

AGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAG

CAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACT

GGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACAC

TTCTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCC

TGGACCTGGGACCGGCTCTGCAACCATGGATTGGACGTGGATCCTGTTTCTCGTGGCAGCTGC

CACAAGAGTTCACAGTATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGT

CAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCC

GGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAAC

CCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGCGCCACCCTCCAC

AGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGAAAAGAGCCCG

CAGCTTCATCTCCCAGCTCAAACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCC

AGCTGATGCCTTCAAAATCACCTTCCACAGGAACCACAGAGATAAGCAGTCATGAGTCCTCCCA

CGGCACCCCCTCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCACCAGCC

GCCAGGTGTGTATCCACAGGGCCACAGCGACACCACTGAGGGCAGAGGCAGCCTGCTGACCT

GCGGCGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGA

CGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCAT

GCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGT

GTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTT

CTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGA

GCATGTCGGCGCCGTGCGTGGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTAC

CAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCG

TGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCG

ACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAG

CTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTAC

ACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCAC

CTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCT

CCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGG

CTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGAAAAACCAAAAGAACAAG

AATTTCTTGGTAAGAAGCCGGGAACAGACAACAGAAGTCATGAAGCCCAAGTGAAATCAAAGGT

GCTAAATGGTCGCCCAGGAGACATCCGTTGTGCTTGCCTGCGTTTTGGAAGCTCTGAAGTCACA

TCACAGGACACGGGGCAGTGGCAACCTTGTCTCTATGCCAGCTCAGTCCCATCAGAGAGCGAG

CGCTACCCACTTCTAAATAGCAATTTCGCCGTTGAAGAGGAAGGGCAAAACCACTAGAACTCTC

CATCTTATTTTCATGTATATGTGTTCATTAAAGCATGAATGGTATGGAACTCTCTCCACCCTATAT

GTAGTATAAAGAAAAGTAGGTT

38InsertedGGTGGCCGGGGAGGCTTTGTGGGGCCACCCAGCCCCTTCCTCACCTCTCTCCATCTCTCAGAC

matrice PD1TCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGG

locus_IL15_GGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTAC

2A_sIL15RaCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCC

(60CGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGT

nucleotidesGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCGGTTCTGGCGTGAAAC

upstream andAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCGGTA

downstream)CCGGGTCCGCCACCATGGACTGGACCTGGATTCTGTTCCTCGTGGCTGCTGCTACAAGAGTGC

ACAGCGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAA

CTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGC

TACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCT

TGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTG

ATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATG

TGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTT

CATCAACACTTCTGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGA

GGAGAACCCTGGACCTGGGACCGGCTCTGCAACCATGGATTGGACGTGGATCCTGTTTCTCGT

GGCAGCTGCCACAAGAGTTCACAGTATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGA

CATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAG

CGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCA

CTGGACAACCCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGCGCC

ACCCTCCACAGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGAAA

AGAGCCCGCAGCTTCATCTCCCAGCTCAAACAACACAGCGGCCACAACAGCAGCTATTGTCCC

GGGCTCCCAGCTGATGCCTTCAAAATCACCTTCCACAGGAACCACAGAGATAAGCAGTCATGAG

TCCTCCCACGGCACCCCCTCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGCCTCC

CACCAGCCGCCAGGTGTGTATCCACAGGGCCACAGCGACACCACTGAGGGCAGAGGCAGCCT

GCTGACCTGCGGCGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGC

GCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAA

GGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGG

GCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGC

GTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGG

GCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACG

GCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTC

GGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCA

CGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACC

GAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGCC

GTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAG

CCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTG

ATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGC

TCCATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGATCTAGAG

GGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTG

CCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAAT

GAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG

GACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTAT

GACTAGTGGCGAATTCGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGGT

GCGGCCTCGGAGGCCCCGGGGCAGGGGTGAGCTGAGCCGGTCCTGGGGTGGGTGTCCCCTC

CTGCACAGGATCAGGAGCTCCAGGGTCGTAGGGCAGGGACCCCCCAGCTCCAGTCCAGGGCT

CTGTCCTGCACCTGGGGAATGGTGACCGGCATCTCTGTCCTCTAGCTCTGGAAGCACCCCAGC

CCCTCTAGTCTGCCCTCACCCCTGACCCTGACCCTCCACCCTGACCCCGTCCTAACCCCTGAC

CTTTGTGCCCTTCCAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCCAGG

39InsertedAGTGCTGGCTAGAAACCAAGTGCTTTACTGCATGCACATCATTTAGCACAGTTAGTTGCTGTTTA

matrice CD25TTATTCCTGTTCCACAGCTATTGTCTGCCATATAAAAACTTAGGCCAGGCACAGTGGCTCACACC

locus_IL12a_TGTAATCCCAGCACTTTGGAAGGCCGAGGCAGGCAGATCACAAGGTCAGGAGTTCGAGACCAG

2A_IL12b (60CCTGGCCAACATAGCAAAACCCCATCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGCG

nucleotidesTGTGCACTGGTTTAGAGTGAGGACCACATTTTTTTGGTGCCGTGTTACACATATGACCGTGACTT

upstream andTGTTACACCACTACAGGAGGAAGAGTAGAAGAACAATCGGTTCTGGCGTGAAACAGACTTTGAA

downstream)TTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCATGTGGCCCCCTGG

GTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCATCCAGCGGCTCGCC

CTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCCTCCTTGTGGCTACCC

TGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCCACTCCAGACCCAGGAA

TGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGG

CCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAAA

GATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCTAA

ATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATG

ATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCA

TGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTT

ATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCCCTTG

AAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGG

CAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCGGAAGCGGAGCTACTAACTTCAG

CCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGTGTCACCAGCAGTTGGT

CATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAACTGAAGAAA

GATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCT

GTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCT

CTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAA

AGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCC

ACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGGCCAAGAA

TTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACATTCAGTGTCA

AAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACACTCTCTGCAG

AGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCC

TGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAG

TATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTT

GCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTG

GAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAGA

GAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCAAAAATGCCA

GCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATGGGCATCTGTGC

CCTGCAGTGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTCGAGGAGAACCCCGGGCC

CATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTC

TGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGT

GAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGAC

CGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGT

GCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGA

TGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGG

CGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACC

GTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCT

GCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGAC

GCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGA

CAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCA

CGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACC

ACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCT

ACATAGCCTTCAAGAGGTGAAAAACCAAAAGAACAAGAATTTCTTGGTAAGAAGCCGGGAACAG

ACAACAGAAGTCATGAAGCCCAAGTGAAATCAAAGGTGCTAAATGGTCGCCCAGGAGACATCC

GTTGTGCTTGCCTGCGTTTTGGAAGCTCTGAAGTCACATCACAGGACACGGGGCAGTGGCAAC

CTTGTCTCTATGCCAGCTCAGTCCCATCAGAGAGCGAGCGCTACCCACTTCTAAATAGCAATTT

CGCCGTTGAAGAGGAAGGGCAAAACCACTAGAACTCTCCATCTTATTTTCATGTATATGTGTTCA

TGAATGGTATGGAACTCTCTCCACCCTATATGTAGTATAAAGAAAAGTAGGTT

40InsertedGGTGGCCGGGGAGGCTTTGTGGGGCCACCCAGCCCCTTCCTCACCTCTCTCCATCTCTCAGAC

matrice PD1TCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGG

locus_IL12a_GGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGGTAC

2A_IL12b (60CGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACCGCAGCCAGCC

nucleotidesCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCGTGACTTCCACATGAGCGT

upstream andGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCTGTGGGGCCGGTTCTGGCGTGAAAC

downstream)AGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCATGT

GGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCATCCA

GCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCCTCCTT

GTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCCACTCCA

GACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATG

CTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGA

TATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAG

AGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGA

CCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAG

TTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACAT

GCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAA

TCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTC

AGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCGGAAGCGGAGCTA

CTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGTGTCACC

AGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCCTCGTGGCCATATGGGAA

CTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATCCGGATGCCCCTGGAGAAATGGTGG

TCCTCACCTGTGACACCCCTGAAGAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGG

TCTTAGGCTCTGGCAAAACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACAC

CTGTCACAAAGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGA

ATTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA

GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATTTGACA

TTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGCGGAGCTGCTACA

CTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTACTCAGTGGAGTGCCAGGAG

GACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCCATTGAGGTCATGGTGGATGCCGTTCAC

AAGCTCAAGTATGAAAACTACACCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCAC

CCAAGAACTTGCAGCTGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACC

CTGACACCTGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAA

GAGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCG

CAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGCGAATG

GGCATCTGTGCCCTGCAGTGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTCGAGGAGA

ACCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCT

GTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACAC

ACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAG

CCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCG

ACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGT

GGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGGGC

GCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAG

CAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGA

CCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCT

GGGCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAG

GGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCAT

AGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCC

GAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTCT

TGTGGCCTACATAGCCTTCAAGAGGTGATCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTC

GACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG

GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTA

GGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAC

AATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGACTAGTGGCGAATTCGGCGCAGATCAA

AGAGAGCCTGCGGGCAGAGCTCAGGGTGACAGGTGCGGCCTCGGAGGCCCCGGGGCAGGGG

TGAGCTGAGCCGGTCCTGGGGTGGGTGTCCCCTCCTGCACAGGATCAGGAGCTCCAGGGTCG

TAGGGCAGGGACCCCCCAGCTCCAGTCCAGGGCTCTGTCCTGCACCTGGGGAATGGTGACCG

GCATCTCTGTCCTCTAGCTCTGGAAGCACCCCAGCCCCTCTAGTCTGCCCTCACCCCTGACCCT

GACCCTCCACCCTGACCCCGTCCTAACCCCTGACCTTTGTGCCCTTCCAGAGAGAAGGGCAGA

AGTGCCCACAGCCCACCCCAGCCCCTCACCCAGGCC

41upstreamATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACC

TRAC locusCTG

polynucleotide

sequence

42downstreamGAAACAGTGAGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGATG

TRAC locusAAGA

polynucleotide

sequence

43upstreamAGTGCTGGCTAGAAACCAAGTGCTTTACTGCATGCACATCATTTAGCACAGTTAGTT

CD25 locusGCT

polynucleotide

sequence

44downstreamGAATGGTATGGAACTCTCTCCACCCTATATGTAGTATAAAGAAAAGTAGGTT

CD25 locus

polynucleotide

sequence

45upstream PD1GGTGGCCGGGGAGGCTTTGTGGGGCCACCCAGCCCCTTCCTCACCTCTCTCCATCT

locusCTCA

polynucleotide

sequence

46downstreamTGCCCTTCCAGAGAGAAGGGCAGAAGTGCCCACAGCCCACCCCAGCCCCTCACCC

PD1 locusAGGCC

polynucleotide

sequence

47IL-12aATGTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAG

polynucleotideGTCTGCATCCAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCA

GCGCGCAGCCTCCTCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGTTTGGC

CAGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACT

CCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGGCCAGACAAACTCTA

GAATTTTACCCTTGCACTTCTGAAGAGATTGATCATGAAGATATCACAAAAGATAAAA

CCAGCACAGTGGAGGCCTGTTTACCATTGGAATTAACCAAGAATGAGAGTTGCCTAA

ATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCCTCCAGAAAGACCT

CTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGT

GGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCT

AGATCAAAACATGCTGGCAGTTATTGATGAGCTGATGCAGGCCCTGAATTTCAACAG

TGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGATTTTTATAAAACTAAAATC

AAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGA

TGAGCTATCTGAATGCTTCC

48IL12bATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTCCCC

polynucleotideTCGTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGTATC

CGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGATGGT

ATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCTGAC

CATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAGGCG

AGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGTCCA

CTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGAGG

CCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGATT

TGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGTGC

GGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAGTA

CTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGCCC

ATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCAGC

TTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGCCA

TTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGTAC

TCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAGAG

AGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCGCA

AAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGAGC

GAATGGGCATCTGTGCCCTGCAGT

49IL15GGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCC

polynucleotideAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGC

ATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGC

AATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGT

ATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATG

GGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTA

AAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT

50sIL15raATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTA

polynucleotideCAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGG

CACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGA

CAACCCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTCACCAAAGGCCAGCG

CCACCCTCCACAGTAACGACGGCAGGGGTGACCCCACAGCCAGAGAGCCTCTCCC

CTTCTGGAAAAGAGCCCGCAGCTTCATCTCCCAGCTCAAACAACACAGCGGCCACA

ACAGCAGCTATTGTCCCGGGCTCCCAGCTGATGCCTTCAAAATCACCTTCCACAGGA

ACCACAGAGATAAGCAGTCATGAGTCCTCCCACGGCACCCCCTCTCAGACAACAGC

CAAGAACTGGGAACTCACAGCATCCGCCTCCCACCAGCCGCCAGGTGTGTATCCAC

AGGGCCACAGCGACACCACT

51solubleATGCTGACACTGCAGACTTGGCTGGTGCAGGCACTGTTTATTTTTCTGACTACTGAA

GP130TCAACTGGCGAACTGCTGGACCCTTGTGGCTACATCAGCCCTGAGTCCCCAGTGGT

polynucleotideGCAGCTGCACAGCAACTTCACCGCCGTGTGCGTGCTGAAGGAGAAGTGTATGGACT

ACTTTCACGTGAACGCCAATTATATCGTGTGGAAAACCAACCACTTCACAATCCCCAA

GGAGCAGTACACCATCATCAATAGGACAGCCAGCTCCGTGACCTTTACAGACATCG

CCTCCCTGAACATCCAGCTGACCTGCAATATCCTGACATTCGGCCAGCTGGAGCAG

AACGTGTATGGCATCACCATCATCTCTGGCCTGCCCCCTGAGAAGCCTAAGAACCTG

AGCTGCATCGTGAATGAGGGCAAGAAGATGCGGTGTGAGTGGGACGGCGGCAGAG

AGACACACCTGGAGACAAACTTCACCCTGAAGTCCGAGTGGGCCACACACAAGTTT

GCCGACTGCAAGGCCAAGCGCGATACCCCAACATCCTGTACCGTGGATTACTCTAC

AGTGTATTTTGTGAACATCGAAGTGTGGGTGGAGGCCGAGAATGCCCTGGGCAAGG

TGACCTCCGACCACATCAACTTCGATCCCGTGTACAAGGTGAAGCCTAACCCACCCC

ACAATCTGAGCGTGATCAATTCCGAGGAGCTGTCTAGCATCCTGAAGCTGACCTGGA

CAAACCCATCTATCAAGAGCGTGATCATCCTGAAGTACAATATCCAGTATCGGACCA

AGGACGCCTCCACATGGAGCCAGATCCCTCCAGAGGATACCGCCAGCACAAGATCC

TCTTTCACCGTGCAGGACCTGAAGCCCTTCACAGAGTACGTGTTTCGGATCAGATGT

ATGAAGGAGGACGGCAAGGGCTACTGGAGCGATTGGTCCGAGGAGGCCAGCGGCA

TCACCTATGAGGACAGGCCTTCTAAGGCCCCCAGCTTCTGGTACAAGATCGATCCAT

CCCACACCCAGGGCTATCGCACAGTGCAGCTGGTGTGGAAAACCCTGCCCCCTTTC

GAGGCCAACGGCAAGATCCTGGACTACGAGGTGACCCTGACACGGTGGAAGTCCC

ACCTGCAGAACTATACCGTGAATGCCACCAAGCTGACAGTGAACCTGACAAATGATC

GGTACCTGGCCACCCTGACAGTGAGAAACCTGGTGGGCAAGTCTGACGCCGCCGT

GCTGACCATCCCTGCCTGCGATTTCCAGGCCACACACCCAGTGATGGACCTGAAGG

CCTTTCCCAAGGATAATATGCTGTGGGTGGAGTGGACCACACCTAGAGAGTCCGTG

AAGAAGTACATCCTGGAGTGGTGCGTGCTGTCTGACAAGGCCCCATGTATCACCGA

CTGGCAGCAGGAGGATGGCACCGTGCACAGGACATATCTGCGCGGCAACCTGGCC

GAGTCTAAGTGTTACCTGATCACCGTGACACCCGTGTATGCAGACGGACCAGGCTC

TCCTGAGAGCATCAAGGCCTACCTGAAGCAGGCACCACCAAGCAAGGGACCAACCG

TGCGGACAAAGAAGGTCGGCAAGAATGAGGCCGTGCTGGAGTGGGACCAGCTGCC

TGTGGATGTGCAGAACGGCTTCATCAGGAATTACACCATCTTTTATCGCACAATCATC

GGCAACGAGACAGCCGTGAATGTGGACAGCTCCCACACCGAGTATACACTGTCTAG

CCTGACCTCCGATACACTGTACATGGTGAGGATGGCCGCCTATACAGACGAGGGCG

GCAAGGATGGCCCCGAGTTT

52IgE signalGGTACCGGGTCCGCCACCATGGACTGGACCTGGATTCTGTTCCTCGTGGCTGCTGC

sequenceTACAAGAGTGCACAGC

53F2AGGTTCTGGCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTG

GAGTCCAACCCAGGGCCC

54P2AGGAAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGA

ACCCTGGACCT

55T2AGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTCGAGGAGAACCCCGGGCCC

56LNGFRATGGGGGCAGGTGCCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTG

CTGCTTCTGGGGGTGTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGT

ACACACACAGCGGTGAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCA

GCCTTGTGGAGCCAACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCT

CCGACGTGGTGAGCGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGC

TCCAGAGCATGTCGGCGCCGTGCGTGGAGGCCGATGACGCCGTGTGCCGCTGCGC

CTACGGCTACTACCAGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGC

GAGGCGGGCTCGGGCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCG

AGGAGTGCCCCGACGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCT

GCCCTGCACCGTGTGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGG

GCCGACGCCGAGTGCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCC

CAGAGGGCTCGGACAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGA

ACAAGACCTCATAGCCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCT

CCCAGCCCGTGGTGACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCC

ATCCTGGCTGCTGTGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGA

57IL-12aMWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNL

polypeptidePVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEA

CLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAK

LLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRA

VTIDRVMSYLNAS

58IL12bMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGIT

polypeptideWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKD

QKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAE

RVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPP

KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSA

TVICRKNASISVRAQDRYYSSSWSEWASVPCS

59IL15GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKC

polypeptideFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV

HIVQMFINTS

60sIL15raITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPS

polypeptideLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGS

QLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT

61soluble gp130MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHV

NANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGL

PPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSC

TVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLT

WTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKE

DGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGK

ILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQA

THPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTY

LRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWD

QLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGG

KDGPEF

62soluble gp130MLTLQTWLVQALFIFLTTESTGELLDPCGYISPESPVVQLHSNFTAVCVLKEKCMDYFHV

fused to a FcNANYIVWKTNHFTIPKEQYTIINRTASSVTFTDIASLNIQLTCNILTFGQLEQNVYGITIISGL

PPEKPKNLSCIVNEGKKMRCEWDGGRETHLETNFTLKSEWATHKFADCKAKRDTPTSC

TVDYSTVYFVNIEVWVEAENALGKVTSDHINFDPVYKVKPNPPHNLSVINSEELSSILKLT

WTNPSIKSVIILKYNIQYRTKDASTWSQIPPEDTASTRSSFTVQDLKPFTEYVFRIRCMKE

DGKGYWSDWSEEASGITYEDRPSKAPSFWYKIDPSHTQGYRTVQLVWKTLPPFEANGK

ILDYEVTLTRWKSHLQNYTVNATKLTVNLTNDRYLATLTVRNLVGKSDAAVLTIPACDFQA

THPVMDLKAFPKDNMLWVEWTTPRESVKKYILEWCVLSDKAPCITDWQQEDGTVHRTY

LRGNLAESKCYLITVTPVYADGPGSPESIKAYLKQAPPSKGPTVRTKKVGKNEAVLEWD

QLPVDVQNGFIRNYTIFYRTIIGNETAVNVDSSHTEYTLSSLTSDTLYMVRMAAYTDEGG

KDGPEFRSCDKTHTCPPCPAPEAEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED

PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP

ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP

63Matrice TRACGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACA

locus_CubiCARTTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAA

CD22AAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGC

pCLS30056ATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAA

full sequenceGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGAT

CCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTG

CTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCG

CATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTT

ACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAAC

ACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTT

TTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGA

ATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA

TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG

GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGTTCTCGCGGTAT

CATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGA

CGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC

TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA

TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT

GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA

GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA

AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT

TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTG

TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT

CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG

GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGG

GGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT

ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG

TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG

GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTC

GATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG

GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGGTCTTTCCTGCGT

TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC

GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAGAGCG

CCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC

ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGT

TAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGT

GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACG

CCAAGCGCGTCAATTAACCCTCACTAAAGGGAACAAAAGCTGTTAATTAATTGCTGG

GCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGA

AGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTT

CCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTG

GCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGG

TTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAG

AGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGA

GGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGTACCC

CTACGACGTGCCCGACTACGCCTCCGGTGAGGGCAGAGGAAGTCTTCTAACATGCG

GTGACGTGGAGGAGAATCCGGGCCCCGGATCCGCTCTGCCCGTCACCGCTCTGCT

GCTGCCACTGGCACTGCTGCTGCACGCTGCTAGGCCCGGAGGGGGAGGCAGCTGC

CCCTACAGCAACCCCAGCCTGTGCAGCGGAGGCGGCGGCAGCGGCGGAGGGGGT

AGCCAGGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTGAAGCCAAGCCAGACAC

TGTCCCTGACCTGCGCCATCAGCGGCGATTCCGTGAGCTCCAACTCCGCCGCCTGG

AATTGGATCAGGCAGTCCCCTTCTCGGGGCCTGGAGTGGCTGGGAAGGACATACTA

TCGGTCTAAGTGGTACAACGATTATGCCGTGTCTGTGAAGAGCAGAATCACAATCAA

CCCTGACACCTCCAAGAATCAGTTCTCTCTGCAGCTGAATAGCGTGACACCAGAGGA

CACCGCCGTGTACTATTGCGCCAGGGAGGTGACCGGCGACCTGGAGGATGCCTTT

GACATCTGGGGCCAGGGCACAATGGTGACCGTGAGCTCCGGAGGCGGCGGATCTG

GCGGAGGAGGAAGTGGGGGCGGCGGGAGTGATATCCAGATGACACAGTCCCCATC

CTCTCTGAGCGCCTCCGTGGGCGACAGAGTGACAATCACCTGTAGGGCCTCCCAGA

CCATCTGGTCTTACCTGAACTGGTATCAGCAGAGGCCCGGCAAGGCCCCTAATCTG

CTGATCTACGCAGCAAGCTCCCTGCAGAGCGGAGTGCCATCCAGATTCTCTGGCAG

GGGCTCCGGCACAGACTTCACCCTGACCATCTCTAGCCTGCAGGCCGAGGACTTCG

CCACCTACTATTGCCAGCAGTCTTATAGCATCCCCCAGACATTTGGCCAGGGCACCA

AGCTGGAGATCAAGTCGGATCCCGGAAGCGGAGGGGGAGGCAGCTGCCCCTACAG

CAACCCCAGCCTGTGCAGCGGAGGCGGCGGCAGCGAGCTGCCCACCCAGGGCAC

CTTCTCCAACGTGTCCACCAACGTGAGCCCAGCCAAGCCCACCACCACCGCCTGTC

CTTATTCCAATCCTTCCCTGTGTGCTCCCACCACAACCCCCGCTCCAAGGCCCCCTA

CCCCCGCACCAACTATTGCCTCCCAGCCACTCTCACTGCGGCCTGAGGCCTGTCGG

CCCGCTGCTGGAGGCGCAGTGCATACAAGGGGCCTCGATTTCGCCTGCGATATTTA

CATCTGGGCACCCCTCGCCGGCACCTGCGGGGTGCTTCTCCTCTCCCTGGTGATTA

CCCTGTATTGCAGACGGGGCCGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTCA

TGCGGCCAGTGCAGACAACCCAAGAGGAGGATGGGTGTTCCTGCAGATTCCCTGAG

GAAGAGGAAGGCGGGTGCGAGCTGAGAGTGAAGTTCTCCAGGAGCGCAGATGCCC

CCGCCTATCAACAGGGCCAGAACCAGCTCTACAACGAGCTTAACCTCGGGAGGCGC

GAAGAATACGACGTGTTGGATAAGAGAAGGGGGCGGGACCCCGAGATGGGAGGAA

AGCCCCGGAGGAAGAACCCTCAGGAGGGCCTGTACAACGAGCTGCAGAAGGATAA

GATGGCCGAGGCCTACTCAGAGATCGGGATGAAGGGGGAGCGGCGCCGCGGGAA

GGGGCACGATGGGCTCTACCAGGGGCTGAGCACAGCCACAAAGGACACATACGAC

GCCTTGCACATGCAGGCCCTTCCACCCCGGGAATAGTCTAGAGGGCCCGTTTAAAC

CCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC

CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAA

TGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGT

GGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAT

GCGGTGGGCTCTATGACTAGTGGCGAATTCCCGTGTACCAGCTGAGAGACTCTAAA

TCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCAC

AAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTAT

GGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTG

CAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTA

AGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCCAGGTTC

TGCCCAGAGCTCTGGTCAATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTT

ATCCATTGCCACCAAAACCCTCTTTTTACTAAGCGATCGCTCCGGTGCCCGTCAGTG

GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAAT

TGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGT

ACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTC

GCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCG

AGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCA

CGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTC

CGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCC

CTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCT

CAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGAC

CGGCGCCTACCTGAGATCACCGGCGCCACCATGGCTTCTTACCCTGGACACCAGCA

TGCTTCTGCCTTTGACCAGGCTGCCAGATCCAGGGGCCACTCCAACAGGAGAACTG

CCCTAAGACCCAGAAGACAGCAGGAAGCCACTGAGGTGAGGCCTGAGCAGAAGAT

GCCAACCCTGCTGAGGGTGTACATTGATGGACCTCATGGCATGGGCAAGACCACCA

CCACTCAACTGCTGGTGGCACTGGGCTCCAGGGATGACATTGTGTATGTGCCTGAG

CCAATGACCTACTGGAGAGTGCTAGGAGCCTCTGAGACCATTGCCAACATCTACACC

ACCCAGCACAGGCTGGACCAGGGAGAAATCTCTGCTGGAGATGCTGCTGTGGTGAT

GACCTCTGCCCAGATCACAATGGGAATGCCCTATGCTGTGACTGATGCTGTTCTGGC

TCCTCACATTGGAGGAGAGGCTGGCTCTTCTCATGCCCCTCCACCTGCCCTGACCC

TGATCTTTGACAGACACCCCATTGCAGCCCTGCTGTGCTACCCAGCAGCAAGGTAC

CTCATGGGCTCCATGACCCCACAGGCTGTGCTGGCTTTTGTGGCCCTGATCCCTCC

AACCCTCCCTGGCACCAACATTGTTCTGGGAGCACTGCCTGAAGACAGACACATTGA

CAGGCTGGCAAAGAGGCAGAGACCTGGAGAGAGACTGGACCTGGCCATGCTGGCT

GCAATCAGAAGGGTGTATGGACTGCTGGCAAACACTGTGAGATACCTCCAGTGTGG

AGGCTCTTGGAGAGAGGACTGGGGACAGCTCTCTGGAACAGCAGTGCCCCCTCAA

GGAGCTGAGCCCCAGTCCAATGCTGGTCCAAGACCCCACATTGGGGACACCCTGTT

CACCCTGTTCAGAGCCCCTGAGCTGCTGGCTCCCAATGGAGACCTGTACAATGTGT

TTGCCTGGGCTCTGGATGTTCTAGCCAAGAGGCTGAGGTCCATGCATGTGTTCATCC

TGGACTATGACCAGTCCCCTGCTGGATGCAGAGATGCTCTGCTGCAACTAACCTCTG

GCATGGTGCAGACCCATGTGACCACCCCTGGCAGCATCCCCACCATCTGTGACCTA

GCCAGAACCTTTGCCAGGGAGATGGGAGAGGCCAACTAAGGCGCGCCACTCGAGC

GCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGA

ATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAAC

CATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGG

TTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTA

TGGAAGGCGCGCCCAATTCGCCCTATAGTGAGTCGTATTACGTCGCGCTCACTGGC

CGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCT

TGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAAA

CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGAGCGCCCTGTAGCGG

CGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCC

AGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCC

GGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT

TTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTGGCCTGTAGTGGG

CCATAGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATA

GTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGA

TTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAA

AAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTAG

64Matrice CD25GTTTATTATTCCTGTTCCACAGCTATTGTCTGCCATATAAAAACTTAGGCCAGGCACA

locus_IL15_GTGGCTCACACCTGTAATCCCAGCACTTTGGAAGGCCGAGGCAGGCAGATCACAAG

2A_sIL15RaGTCAGGAGTTCGAGACCAGCCTGGCCAACATAGCAAAACCCCATCTCTACTAAAAAT

pCLS30519ACAAAAATTAGCCAGGCATGGTGGCGTGTGCACTGGTTTAGAGTGAGGACCACATTT

full sequenceTTTTGGTGCCGTGTTACACATATGACCGTGACTTTGTTACACCACTACAGGAGGAAG

AGTAGAAGAACAATCGGTTCTGGCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGT

TGGCGGGAGACGTGGAGTCCAACCCAGGGCCCGGTACCGGGTCCGCCACCATGGA

CTGGACCTGGATTCTGTTCCTCGTGGCTGCTGCTACAAGAGTGCACAGCGGCATTC

ATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGG

TGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGAT

GCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAG

TGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATG

ATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGT

AACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATT

TTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTGGAAGCGGAGCT

ACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGG

GACCGGCTCTGCAACCATGGATTGGACGTGGATCCTGTTTCTCGTGGCAGCTGCCA

CAAGAGTTCACAGTATCACGTGCCCTCCCCCCATGTCCGTGGAACACGCAGACATC

TGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTC

AAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGA

ATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGACCCTGCCCTGGTTC

ACCAAAGGCCAGCGCCACCCTCCACAGTAACGACGGCAGGGGTGACCCCACAGCC

AGAGAGCCTCTCCCCTTCTGGAAAAGAGCCCGCAGCTTCATCTCCCAGCTCAAACAA

CACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTGATGCCTTCAAAAT

CACCTTCCACAGGAACCACAGAGATAAGCAGTCATGAGTCCTCCCACGGCACCCCC

TCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCACCAGCCGCC

AGGTGTGTATCCACAGGGCCACAGCGACACCACTGAGGGCAGAGGCAGCCTGCTG

ACCTGCGGCGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTGCCACCGGC

CGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTG

GAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTG

CAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACC

GTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCG

AGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTG

CGTGGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAG

ACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTG

TTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGT

ATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGA

CACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGA

GATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACA

GCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCA

CGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCG

AGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGT

GGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGAAAAACCAAAAGAACAAGAATTTC

TTGGTAAGAAGCCGGGAACAGACAACAGAAGTCATGAAGCCCAAGTGAAATCAAAG

GTGCTAAATGGTCGCCCAGGAGACATCCGTTGTGCTTGCCTGCGTTTTGGAAGCTCT

GAAGTCACATCACAGGACACGGGGCAGTGGCAACCTTGTCTCTATGCCAGCTCAGT

CCCATCAGAGAGCGAGCGCTACCCACTTCTAAATAGCAATTTCGCCGTTGAAGAGGA

AGGGCAAAACCACTAGAACTCTCCATCTTATTTTCATGTATATGTGTTCATGCGATCG

CTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTG

GGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACT

GGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCG

TATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGA

ACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTAC

CTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGT

GCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGG

CCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTT

GCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTA

CAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGCGCCACCATGGCTTCT

TACCCTGGACACCAGCATGCTTCTGCCTTTGACCAGGCTGCCAGATCCAGGGGCCA

CTCCAACAGGAGAACTGCCCTAAGACCCAGAAGACAGCAGGAAGCCACTGAGGTGA

GGCCTGAGCAGAAGATGCCAACCCTGCTGAGGGTGTACATTGATGGACCTCATGGC

ATGGGCAAGACCACCACCACTCAACTGCTGGTGGCACTGGGCTCCAGGGATGACAT

TGTGTATGTGCCTGAGCCAATGACCTACTGGAGAGTGCTAGGAGCCTCTGAGACCA

TTGCCAACATCTACACCACCCAGCACAGGCTGGACCAGGGAGAAATCTCTGCTGGA

GATGCTGCTGTGGTGATGACCTCTGCCCAGATCACAATGGGAATGCCCTATGCTGT

GACTGATGCTGTTCTGGCTCCTCACATTGGAGGAGAGGCTGGCTCTTCTCATGCCC

CTCCACCTGCCCTGACCCTGATCTTTGACAGACACCCCATTGCAGCCCTGCTGTGCT

ACCCAGCAGCAAGGTACCTCATGGGCTCCATGACCCCACAGGCTGTGCTGGCTTTT

GTGGCCCTGATCCCTCCAACCCTCCCTGGCACCAACATTGTTCTGGGAGCACTGCC

TGAAGACAGACACATTGACAGGCTGGCAAAGAGGCAGAGACCTGGAGAGAGACTG

GACCTGGCCATGCTGGCTGCAATCAGAAGGGTGTATGGACTGCTGGCAAACACTGT

GAGATACCTCCAGTGTGGAGGCTCTTGGAGAGAGGACTGGGGACAGCTCTCTGGAA

CAGCAGTGCCCCCTCAAGGAGCTGAGCCCCAGTCCAATGCTGGTCCAAGACCCCAC

ATTGGGGACACCCTGTTCACCCTGTTCAGAGCCCCTGAGCTGCTGGCTCCCAATGG

AGACCTGTACAATGTGTTTGCCTGGGCTCTGGATGTTCTAGCCAAGAGGCTGAGGT

CCATGCATGTGTTCATCCTGGACTATGACCAGTCCCCTGCTGGATGCAGAGATGCTC

TGCTGCAACTAACCTCTGGCATGGTGCAGACCCATGTGACCACCCCTGGCAGCATC

CCCACCATCTGTGACCTAGCCAGAACCTTTGCCAGGGAGATGGGAGAGGCCAACTA

AGGCGCGCCACTCGAGCGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTT

GGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATG

CTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGC

ATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAA

ACCTCTACAAATGTGGTATGGAAGGCGCGCCCAATTCGCCCTATAGTGAGTCGTATT

ACGTCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGT

TACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGA

AGAGGCCCGCACCGAAACGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATG

GGAGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCG

TGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCT

TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAG

GGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATG

GTTGGCCTGTAGTGGGCCATAGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGA

GTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATC

TCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAA

TGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTT

AGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATA

CATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATT

GAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC

GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGC

TGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTA

AGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGT

TCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTC

GCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGC

ATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTG

ATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACC

GCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAG

CTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGC

AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACA

ATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCC

TTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGTTCTCGC

GGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC

ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGG

TGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAG

ATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT

CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTA

GAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC

AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAA

CTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCT

AGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCT

CGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTAC

CGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG

GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA

CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGAC

AGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG

GGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC

GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAAC

GCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGGTCTTTCCT

GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC

GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAG

AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGC

TGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGT

GAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTAT

GTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGA

TTACGCCAAGCGCGTCAATTAACCCTCACTAAAGGGAACAAAAGCTGTTAATTAA

65Matrice PD1GACTCCCCAGACAGGCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGT

locus_IL15_GACCGAAGGGGACAACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCT

2A_sIL15RaTCGTGCTAAACTGGTACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCC

pCLS30513TTCCCCGAGGACCGCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAAC

full sequenceTGCCCAACGGGCGTGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAG

CGGCACCTACCTCTGTGGGGCCGGTTCTGGCGTGAAACAGACTTTGAATTTTGACCT

TCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCAGGGCCCGGTACCGGGTCCGCC

ACCATGGACTGGACCTGGATTCTGTTCCTCGTGGCTGCTGCTACAAGAGTGCACAG

CGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGC

CAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATG

CATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAG

CAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAG

TATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAAT

GGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATT

AAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTGGAA

GCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCT

GGACCTGGGACCGGCTCTGCAACCATGGATTGGACGTGGATCCTGTTTCTCGTGGC

AGCTGCCACAAGAGTTCACAGTATCACGTGCCCTCCCCCCATGTCCGTGGAACACG

CAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACT

CTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAG

GCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGCATTAGAGACCCTGC

CCTGGTTCACCAAAGGCCAGCGCCACCCTCCACAGTAACGACGGCAGGGGTGACC

CCACAGCCAGAGAGCCTCTCCCCTTCTGGAAAAGAGCCCGCAGCTTCATCTCCCAG

CTCAAACAACACAGCGGCCACAACAGCAGCTATTGTCCCGGGCTCCCAGCTGATGC

CTTCAAAATCACCTTCCACAGGAACCACAGAGATAAGCAGTCATGAGTCCTCCCACG

GCACCCCCTCTCAGACAACAGCCAAGAACTGGGAACTCACAGCATCCGCCTCCCAC

CAGCCGCCAGGTGTGTATCCACAGGGCCACAGCGACACCACTGAGGGCAGAGGCA

GCCTGCTGACCTGCGGCGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTG

CCACCGGCCGCGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGT

GTCCCTTGGAGGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGT

GAGTGCTGCAAAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCA

ACCAGACCGTGTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAG

CGCGACCGAGCCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCG

GCGCCGTGCGTGGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTACC

AGGATGAGACGACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGG

GCCTCGTGTTCTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGA

CGGCACGTATTCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTG

TGCGAGGACACCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGT

GCGAGGAGATCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGA

CAGCACAGCCCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAG

CCAGCACGGTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGT

GACCCGAGGCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTG

TGGTTGTGGGTCTTGTGGCCTACATAGCCTTCAAGAGGTGATCTAGAGGGCCCGTTT

AAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCC

CCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT

AAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTG

GGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGG

GGATGCGGTGGGCTCTATGACTAGTGGCGAATTCGGCGCAGATCAAAGAGAGCCTG

CGGGCAGAGCTCAGGGTGACAGGTGCGGCCTCGGAGGCCCCGGGGCAGGGGTGA

GCTGAGCCGGTCCTGGGGTGGGTGTCCCCTCCTGCACAGGATCAGGAGCTCCAGG

GTCGTAGGGCAGGGACCCCCCAGCTCCAGTCCAGGGCTCTGTCCTGCACCTGGGG

AATGGTGACCGGCATCTCTGTCCTCTAGCTCTGGAAGCACCCCAGCCCCTCTAGTCT

GCCCTCACCCCTGACCCTGACCCTCCACCCTGACCCCGTCCTAACCCCTGACCTTT

GGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCG

AGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGG

GGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGG

GAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTG

CCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGC

CGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCG

CCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCG

AGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTC

CACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTG

CGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGCGCCACCA

TGGCTTCTTACCCTGGACACCAGCATGCTTCTGCCTTTGACCAGGCTGCCAGATCCA

GGGGCCACTCCAACAGGAGAACTGCCCTAAGACCCAGAAGACAGCAGGAAGCCAC

TGAGGTGAGGCCTGAGCAGAAGATGCCAACCCTGCTGAGGGTGTACATTGATGGAC

CTCATGGCATGGGCAAGACCACCACCACTCAACTGCTGGTGGCACTGGGCTCCAGG

GATGACATTGTGTATGTGCCTGAGCCAATGACCTACTGGAGAGTGCTAGGAGCCTCT

GAGACCATTGCCAACATCTACACCACCCAGCACAGGCTGGACCAGGGAGAAATCTC

TGCTGGAGATGCTGCTGTGGTGATGACCTCTGCCCAGATCACAATGGGAATGCCCT

ATGCTGTGACTGATGCTGTTCTGGCTCCTCACATTGGAGGAGAGGCTGGCTCTTCTC

ATGCCCCTCCACCTGCCCTGACCCTGATCTTTGACAGACACCCCATTGCAGCCCTG

CTGTGCTACCCAGCAGCAAGGTACCTCATGGGCTCCATGACCCCACAGGCTGTGCT

GGCTTTTGTGGCCCTGATCCCTCCAACCCTCCCTGGCACCAACATTGTTCTGGGAG

CACTGCCTGAAGACAGACACATTGACAGGCTGGCAAAGAGGCAGAGACCTGGAGAG

AGACTGGACCTGGCCATGCTGGCTGCAATCAGAAGGGTGTATGGACTGCTGGCAAA

CACTGTGAGATACCTCCAGTGTGGAGGCTCTTGGAGAGAGGACTGGGGACAGCTCT

CTGGAACAGCAGTGCCCCCTCAAGGAGCTGAGCCCCAGTCCAATGCTGGTCCAAGA

CCCCACATTGGGGACACCCTGTTCACCCTGTTCAGAGCCCCTGAGCTGCTGGCTCC

CAATGGAGACCTGTACAATGTGTTTGCCTGGGCTCTGGATGTTCTAGCCAAGAGGCT

GAGGTCCATGCATGTGTTCATCCTGGACTATGACCAGTCCCCTGCTGGATGCAGAG

ATGCTCTGCTGCAACTAACCTCTGGCATGGTGCAGACCCATGTGACCACCCCTGGC

AGCATCCCCACCATCTGTGACCTAGCCAGAACCTTTGCCAGGGAGATGGGAGAGGC

CAACTAAGGCGCGCCACTCGAGCGCTAGCTGGCCAGACATGATAAGATACATTGAT

GAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTT

GTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAAC

AATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCA

AGTAAAACCTCTACAAATGTGGTATGGAAGGCGCGCCCAATTCGCCCTATAGTGAGT

CGTATTACGTCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCT

GGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAAT

AGCGAAGAGGCCCGCACCGAAACGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC

GAATGGGAGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG

CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCC

CTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCC

CTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGG

GTGATGGTTGGCCTGTAGTGGGCCATAGCCCTGATAGACGGTTTTTCGCCCTTTGAC

GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAAC

CCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGT

TAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTT

ACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTT

CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAA

TAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCT

TTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA

AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACA

GCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT

TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAAC

TCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG

AAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCA

TGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAG

CTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAA

CCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC

AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG

GCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCT

CGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGT

TCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGT

TATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTG

AGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATAT

ACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTT

TTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA

CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGC

TGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAG

CTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACT

GTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCT

ACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCG

TGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGG

CTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAA

CTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAA

GGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGA

GCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTG

ACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACG

CCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGG

TCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC

TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAA

GCGGAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAA

TGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAA

TTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGC

TCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGA

CCATGATTACGCCAAGCGCGTCAATTAACCCTCACTAAAGGGAACAAAAGCTGTTAA

TTAA

66Matrice CD25GTTTATTATTCCTGTTCCACAGCTATTGTCTGCCATATAAAAACTTAGGCCAGGCACA

locus_IL12a_GTGGCTCACACCTGTAATCCCAGCACTTTGGAAGGCCGAGGCAGGCAGATCACAAG

2A_IL12bGTCAGGAGTTCGAGACCAGCCTGGCCAACATAGCAAAACCCCATCTCTACTAAAAAT

pCLS30520ACAAAAATTAGCCAGGCATGGTGGCGTGTGCACTGGTTTAGAGTGAGGACCACATTT

full sequenceTTTTGGTGCCGTGTTACACATATGACCGTGACTTTGTTACACCACTACAGGAGGAAG

AGTAGAAGAACAATCGGTTCTGGCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGT

TGGCGGGAGACGTGGAGTCCAACCCAGGGCCCATGTGGCCCCCTGGGTCAGCCTC

CCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCATCCAGCGGCTCGCCCT

GTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCCTCCTTGTGGC

TACCCTGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCCACTC

CAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTC

AGCAACATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAA

GAGATTGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTA

CCATTGGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAA

CTAATGGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTA

GTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAA

AGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTA

TTGATGAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCT

CCCTTGAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGC

TTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCGG

AAGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACC

CTGGACCTATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGG

CATCTCCCCTCGTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGG

ATTGGTATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAA

GAAGATGGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAA

AACCCTGACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAA

AGGAGGCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAA

TTTGGTCCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAG

ATGCGAGGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAG

TACTGATTTGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGT

GACGTGCGGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAG

TATGAGTACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGA

GTCTGCCCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACA

CCAGCAGCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGC

TGAAGCCATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACC

TGGAGTACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAG

AGCAAGAGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCAT

CTGCCGCAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCAT

CTTGGAGCGAATGGGCATCTGTGCCCTGCAGTGAGGGCAGAGGCAGCCTGCTGAC

CTGCGGCGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTGCCACCGGCCG

CGCCATGGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGA

GGTGCCAAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCA

AAGCCTGCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGT

GTGTGAGCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAG

CCGTGCAAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCG

TGGAGGCCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGAC

GACTGGGCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTT

CTCCTGCCAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTAT

TCCGACGAGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACA

CCGAGCGCCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGA

TCCCTGGCCGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGC

CCCCAGCACCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACG

GTGGCAGGTGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAG

GCACCACCGACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGG

GTCTTGTGGCCTACATAGCCTTCAAGAGGTGAAAAACCAAAAGAACAAGAATTTCTT

GGTAAGAAGCCGGGAACAGACAACAGAAGTCATGAAGCCCAAGTGAAATCAAAGGT

GCTAAATGGTCGCCCAGGAGACATCCGTTGTGCTTGCCTGCGTTTTGGAAGCTCTG

AAGTCACATCACAGGACACGGGGCAGTGGCAACCTTGTCTCTATGCCAGCTCAGTC

CCATCAGAGAGCGAGCGCTACCCACTTCTAAATAGCAATTTCGCCGTTGAAGAGGAA

GGGCAAAACCACTAGAACTCTCCATCTTATTTTCATGTATATGTGTTCATGCGATCGC

TCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGG

GGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTG

GGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGT

ATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAA

CACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACC

TGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTG

CCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGC

CTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTG

CCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTAC

AGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGCGCCACCATGGCTTCTT

ACCCTGGACACCAGCATGCTTCTGCCTTTGACCAGGCTGCCAGATCCAGGGGCCAC

TCCAACAGGAGAACTGCCCTAAGACCCAGAAGACAGCAGGAAGCCACTGAGGTGAG

GCCTGAGCAGAAGATGCCAACCCTGCTGAGGGTGTACATTGATGGACCTCATGGCA

TGGGCAAGACCACCACCACTCAACTGCTGGTGGCACTGGGCTCCAGGGATGACATT

GTGTATGTGCCTGAGCCAATGACCTACTGGAGAGTGCTAGGAGCCTCTGAGACCAT

TGCCAACATCTACACCACCCAGCACAGGCTGGACCAGGGAGAAATCTCTGCTGGAG

ATGCTGCTGTGGTGATGACCTCTGCCCAGATCACAATGGGAATGCCCTATGCTGTGA

CTGATGCTGTTCTGGCTCCTCACATTGGAGGAGAGGCTGGCTCTTCTCATGCCCCTC

CACCTGCCCTGACCCTGATCTTTGACAGACACCCCATTGCAGCCCTGCTGTGCTACC

CAGCAGCAAGGTACCTCATGGGCTCCATGACCCCACAGGCTGTGCTGGCTTTTGTG

GCCCTGATCCCTCCAACCCTCCCTGGCACCAACATTGTTCTGGGAGCACTGCCTGA

AGACAGACACATTGACAGGCTGGCAAAGAGGCAGAGACCTGGAGAGAGACTGGAC

CTGGCCATGCTGGCTGCAATCAGAAGGGTGTATGGACTGCTGGCAAACACTGTGAG

ATACCTCCAGTGTGGAGGCTCTTGGAGAGAGGACTGGGGACAGCTCTCTGGAACAG

CAGTGCCCCCTCAAGGAGCTGAGCCCCAGTCCAATGCTGGTCCAAGACCCCACATT

GGGGACACCCTGTTCACCCTGTTCAGAGCCCCTGAGCTGCTGGCTCCCAATGGAGA

CCTGTACAATGTGTTTGCCTGGGCTCTGGATGTTCTAGCCAAGAGGCTGAGGTCCAT

GCATGTGTTCATCCTGGACTATGACCAGTCCCCTGCTGGATGCAGAGATGCTCTGCT

GCAACTAACCTCTGGCATGGTGCAGACCCATGTGACCACCCCTGGCAGCATCCCCA

CCATCTGTGACCTAGCCAGAACCTTTGCCAGGGAGATGGGAGAGGCCAACTAAGGC

GCGCCACTCGAGCGCTAGCTGGCCAGACATGATAAGATACATTGATGAGTTTGGAC

AAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTAT

TGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTC

ATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACC

TCTACAAATGTGGTATGGAAGGCGCGCCCAATTCGCCCTATAGTGAGTCGTATTACG

TCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTAC

CCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGA

GGCCCGCACCGAAACGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGA

GCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGA

CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTC

TCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGG

TTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGT

TGGCCTGTAGTGGGCCATAGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGT

CCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTC

GGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT

GAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTA

GGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATAC

ATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGA

AAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGG

CATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA

AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGA

TCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCT

GCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCC

GCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCT

TACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAA

CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTT

TTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGA

ATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACA

ACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA

TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG

GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGTTCTCGCGGTAT

CATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGA

CGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCC

TCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGA

TTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCAT

GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAA

GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACA

AAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTT

TTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTG

TAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCT

CTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGG

GTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGG

GGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCT

ACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGG

TATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGG

GAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTC

GATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG

GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGGTCTTTCCTGCGT

TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC

GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAGAGCG

CCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC

ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGT

TAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGT

GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACG

CCAAGCGCGTCAATTAACCCTCACTAAAGGGAACAAAAGCTGTTAATTAA

67Matrice PD1TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG

locus_IL12a_GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGT

2A_IL12bCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGAT

pCLS30511TGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAA

full sequenceAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGA

TCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAA

GGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACG

GCCAGTGAATTCGAGCTCGGTACCTCGCGAATGCATCTAGATGACTCCCCAGACAG

GCCCTGGAACCCCCCCACCTTCTCCCCAGCCCTGCTCGTGGTGACCGAAGGGGAC

AACGCCACCTTCACCTGCAGCTTCTCCAACACATCGGAGAGCTTCGTGCTAAACTGG

TACCGCATGAGCCCCAGCAACCAGACGGACAAGCTGGCCGCCTTCCCCGAGGACC

GCAGCCAGCCCGGCCAGGACTGCCGCTTCCGTGTCACACAACTGCCCAACGGGCG

TGACTTCCACATGAGCGTGGTCAGGGCCCGGCGCAATGACAGCGGCACCTACCTCT

GTGGGGCCGGTTCTGGCGTGAAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCG

GGAGACGTGGAGTCCAACCCAGGGCCCATGTGGCCCCCTGGGTCAGCCTCCCAGC

CACCGCCCTCACCTGCCGCGGCCACAGGTCTGCATCCAGCGGCTCGCCCTGTGTC

CCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCGCAGCCTCCTCCTTGTGGCTACCC

TGGTCCTCCTGGACCACCTCAGTTTGGCCAGAAACCTCCCCGTGGCCACTCCAGAC

CCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAA

CATGCTCCAGAAGGCCAGACAAACTCTAGAATTTTACCCTTGCACTTCTGAAGAGAT

TGATCATGAAGATATCACAAAAGATAAAACCAGCACAGTGGAGGCCTGTTTACCATT

GGAATTAACCAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAAT

GGGAGTTGCCTGGCCTCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGT

ATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTT

CTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGAT

GAGCTGATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCCCTT

GAAGAACCGGATTTTTATAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCA

GAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCGGAAGCG

GAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGA

CCTATGTGTCACCAGCAGTTGGTCATCTCTTGGTTTTCCCTGGTTTTTCTGGCATCTC

CCCTCGTGGCCATATGGGAACTGAAGAAAGATGTTTATGTCGTAGAATTGGATTGGT

ATCCGGATGCCCCTGGAGAAATGGTGGTCCTCACCTGTGACACCCCTGAAGAAGAT

GGTATCACCTGGACCTTGGACCAGAGCAGTGAGGTCTTAGGCTCTGGCAAAACCCT

GACCATCCAAGTCAAAGAGTTTGGAGATGCTGGCCAGTACACCTGTCACAAAGGAG

GCGAGGTTCTAAGCCATTCGCTCCTGCTGCTTCACAAAAAGGAAGATGGAATTTGGT

CCACTGATATTTTAAAGGACCAGAAAGAACCCAAAAATAAGACCTTTCTAAGATGCGA

GGCCAAGAATTATTCTGGACGTTTCACCTGCTGGTGGCTGACGACAATCAGTACTGA

TTTGACATTCAGTGTCAAAAGCAGCAGAGGCTCTTCTGACCCCCAAGGGGTGACGT

GCGGAGCTGCTACACTCTCTGCAGAGAGAGTCAGAGGGGACAACAAGGAGTATGAG

TACTCAGTGGAGTGCCAGGAGGACAGTGCCTGCCCAGCTGCTGAGGAGAGTCTGC

CCATTGAGGTCATGGTGGATGCCGTTCACAAGCTCAAGTATGAAAACTACACCAGCA

GCTTCTTCATCAGGGACATCATCAAACCTGACCCACCCAAGAACTTGCAGCTGAAGC

CATTAAAGAATTCTCGGCAGGTGGAGGTCAGCTGGGAGTACCCTGACACCTGGAGT

ACTCCACATTCCTACTTCTCCCTGACATTCTGCGTTCAGGTCCAGGGCAAGAGCAAG

AGAGAAAAGAAAGATAGAGTCTTCACGGACAAGACCTCAGCCACGGTCATCTGCCG

CAAAAATGCCAGCATTAGCGTGCGGGCCCAGGACCGCTACTATAGCTCATCTTGGA

GCGAATGGGCATCTGTGCCCTGCAGTGAGGGCAGAGGCAGCCTGCTGACCTGCGG

CGACGTCGAGGAGAACCCCGGGCCCATGGGGGCAGGTGCCACCGGCCGCGCCAT

GGACGGGCCGCGCCTGCTGCTGTTGCTGCTTCTGGGGGTGTCCCTTGGAGGTGCC

AAGGAGGCATGCCCCACAGGCCTGTACACACACAGCGGTGAGTGCTGCAAAGCCT

GCAACCTGGGCGAGGGTGTGGCCCAGCCTTGTGGAGCCAACCAGACCGTGTGTGA

GCCCTGCCTGGACAGCGTGACGTTCTCCGACGTGGTGAGCGCGACCGAGCCGTGC

AAGCCGTGCACCGAGTGCGTGGGGCTCCAGAGCATGTCGGCGCCGTGCGTGGAGG

CCGATGACGCCGTGTGCCGCTGCGCCTACGGCTACTACCAGGATGAGACGACTGG

GCGCTGCGAGGCGTGCCGCGTGTGCGAGGCGGGCTCGGGCCTCGTGTTCTCCTGC

CAGGACAAGCAGAACACCGTGTGCGAGGAGTGCCCCGACGGCACGTATTCCGACG

AGGCCAACCACGTGGACCCGTGCCTGCCCTGCACCGTGTGCGAGGACACCGAGCG

CCAGCTCCGCGAGTGCACACGCTGGGCCGACGCCGAGTGCGAGGAGATCCCTGGC

CGTTGGATTACACGGTCCACACCCCCAGAGGGCTCGGACAGCACAGCCCCCAGCA

CCCAGGAGCCTGAGGCACCTCCAGAACAAGACCTCATAGCCAGCACGGTGGCAGG

TGTGGTGACCACAGTGATGGGCAGCTCCCAGCCCGTGGTGACCCGAGGCACCACC

GACAACCTCATCCCTGTCTATTGCTCCATCCTGGCTGCTGTGGTTGTGGGTCTTGTG

GCCTACATAGCCTTCAAGAGGTGATCTAGAGGGCCCGTTTAAACCCGCTGATCAGC

CTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTC

CTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGC

ATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACA

GCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTC

TATGACTAGTGGCGAATTCGGCGCAGATCAAAGAGAGCCTGCGGGCAGAGCTCAGG

GTGACAGGTGCGGCCTCGGAGGCCCCGGGGCAGGGGTGAGCTGAGCCGGTCCTG

GGGTGGGTGTCCCCTCCTGCACAGGATCAGGAGCTCCAGGGTCGTAGGGCAGGGA

CCCCCCAGCTCCAGTCCAGGGCTCTGTCCTGCACCTGGGGAATGGTGACCGGCAT

CTCTGTCCTCTAGCTCTGGAAGCACCCCAGCCCCTCTAGTCTGCCCTCACCCCTGA

CCCTGACCCTCCACCCTGACCCCGTCCTAACCCCTGACCTTTGATCGGATCCCGGG

CCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTT

TCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCAT

AAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCG

CTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCG

GCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCT

CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA

AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGA

GCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTT

CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT

GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTC

GTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCT

TCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTA

GGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT

GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC

CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT

ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGT

ATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCC

GGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACG

CGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT

CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC

TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGA

GTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGAT

CTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA

CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTC

ACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAA

GTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTA

GAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCA

TCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGAT

CAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTC

CTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAG

CACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGA

GTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC

GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCAT

TGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG

TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC

GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGC

GACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC

AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAAT

AGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTAT

TATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTC

TABLE 6

Preferred human endogenous gene loci responsive to T-cell activation

inductionRatio12T.8Eff.Sp.OT1.T.8Eff.Sp.OT1.T.8Eff.Sp.OT1.

symboldescriptionhrT.8Nve.Sp.OT1 12 hr.LisOva48 hr.LisOvad6.LisOva

Il3interleukin 2116.412.8208.918.413.6

Il2interleukin 397.016.01554.417.718.1

CcI4isopentenyl-diphosphate delta isomerase 22.116.835.617.619.7

Il21granzyme C9.217.4160.520.424.9

Gp49achemokine (C-C motif) receptor 85.918.5108.431.520.9

Cxcl10interleukin 258.421.11229.632.717.9

Nr4a3interleukin 1 receptor, type I2.621.254.635.521.7

Lilrb4tumor necrosis factor (ligand) superfamily,4.121.888.829.320.0

member 4

Cd200neuronal calcium sensor 14.524.1109.646.323.2

Cdkn1aCDK5 and Abl enzyme substrate 13.126.280.949.132.8

Gzmctransmembrane and tetratricopeptide repeat2.026.853.926.229.4

containing 2

Nr4a2LON peptidase N-terminal domain and ring3.228.490.450.428.3

finger 1

Cishglycoprotein 49 A15.031.6472.430.6212.5

Nr4a1polo-like kinase 23.631.7114.339.032.5

Tnflipase, endothelial2.132.466.735.933.3

Ccr8cyclin-dependent kinase inhibitor 1A (P21)9.734.6335.454.471.0

Lad1grainyhead-like 1 (Drosophila)2.135.173.452.044.1

Slamf1cellular retinoic acid binding protein II5.335.4187.243.336.3

Crabp2adenylate kinase 42.235.980.458.539.8

Furinmicrotubule-associated protein 1B2.136.277.736.438.4

Gadd45gacyl-CoA synthetase long-chain family2.037.276.045.241.3

member 6

Bcl2l1zinc finger E-box binding homeobox 22.138.680.744.9455.4

Ncs1CD200 antigen9.841.2404.370.436.8

Ciartcarboxypeptidase D3.141.6127.771.471.6

Ahrthioredoxin reductase 33.643.4157.861.728.8

Spry1myosin IE2.343.6100.261.377.0

Tnfsf4RNA binding protein with multiple splicing 22.143.691.549.836.5

Myo10mitogen-activated protein kinase kinase 3,2.944.8127.966.443.1

opposite strand

Dusp5PERP, TP53 apoptosis effector2.844.9127.278.472.4

Mycmyosin X4.145.5184.981.657.5

Psrc1immediate early response 32.745.6121.663.966.2

St6galnac4folliculin interacting protein 22.647.5124.287.496.6

Nfkbidleukocyte immunoglobulin-like receptor,9.948.9483.364.5179.1

subfamily B, member 4

Bst2circadian associated repressor of4.550.6225.5100.333.8

transcription

Txnrd3RAR-related orphan receptor gamma2.151.7106.747.552.8

Plk2proline/serine-rich coiled-coil 13.952.9205.992.379.6

Gfi1cysteine rich protein 22.454.2127.790.3182.9

Pim1cAMP responsive element modulator2.055.7112.654.457.3

Pvt1chemokine (C-C motif) ligand 420.255.81125.8103.189.0

Nfkbibnuclear receptor subfamily 4, group A,7.858.5457.678.772.0

member 2

Gnl2transglutaminase 2, C polypeptide2.358.7132.169.864.7

Cd69synapse defective 1, Rho GTPase, homolog 2

(C, elegans)2.162.5132.7111.331.0

Dgat2sprouty homolog 1 (Drosophila)4.263.8268.576.861.4

Atf3activating transcription factor 33.265.8210.388.375.8

Tnfrsf21pogo transposable element with KRAB

domain2.968.6196.991.1293.2

Lonrfltumor necrosis factor receptor superfamily,3.270.6224.5126.572.9

member 21

Cables1cytokine inducible SH2-containing protein7.574.3558.782.5133.9

Cpdlymphotoxin A2.674.6197.293.458.6

QtrtdlFBJ osteosarcoma oncogene3.074.9224.189.061.1

Polr3dsignaling lymphocytic activation molecule5.475.6412.0108.4190.4

family member 1

Kcnq5syndecan 32.476.0180.077.285.3

Fosmitochondrial ribosomal protein L472.177.2161.7152.072.3

Slc19a2ladinin5.577.3423.2152.570.4

Hif1aE2F transcription factor 52.577.7198.092.065.2

Il15raISG15 ubiquitin-like modifier2.877.9221.088.945.1

Nfkb1aryl-hydrocarbon receptor4.278.7333.2145.791.4

PhIda3diacylglycerol O-acyltransferase 23.281.0259.2150.084.4

MtrrFBJ osteosarcoma oncogene B2.081.3163.7139.398.5

Pogkpleckstrin homology-like domain, family A,2.984.8244.5126.983.8

member 3

Map2k3ospotassium voltage-gated channel, subfamily3.086.3261.0118.163.4

Q, member 5

Egr2tumor necrosis factor receptor superfamily,2.588.6219.0106.151.0

member 10b

Isg15Mir17 host gene 1 (non-protein coding)2.190.4190.1120.051.2

Perpglucose-fructose oxidoreductase domain

containing 12.292.9208.5168.7237.4

Ipo4plexin Al2.194.8200.7118.090.3

Mphosph10heat shock factor 22.496.8233.2191.0104.8

Plk3carbohydrate sulfotransferase 112.496.8235.1180.8385.7

Ifitm3growth arrest and DNA-damage-inducible 45

gamma4.8104.6504.8109.395.0

Polr1bsolute carrier family 5 (sodium-dependent2.1107.0227.3192.875.8

vitamin transporter), member 6

Usp18interferon induced transmembrane protein 32.8109.2302.643.9106.4

Top1mtDENN/MADD domain containing 5A2.6109.5279.9102.0517.4

Dkc1plasminogen activator, urokinase receptor2.1112.4234.855.757.3

Polr1csolute carrier family 19 (thiamine3.0115.4343.1221.7138.4

transporter), member 2

Cdk6ubiquitin domain containing 22.2117.4255.7198.9122.2

Ier3nuclear receptor subfamily 4, group A,

member 311.8118.01394.1114.269.6

Ltazinc finger protein 522.5118.8295.6160.9167.4

PtprsSH3 domain containing ring finger 12.4119.3280.9116.5156.5

Fnip2dihydrouridine synthase 22.1122.7260.3237.7202.8

Asna1cyclin-dependent kinase 5, regulatory subunit

1 (p35)2.1122.7259.3168.4124.0

Mybbp1aprocessing of precursor 7, ribonuclease P

family, (S, cerevisiae)2.1125.9264.9235.7150.6

Il1r1growth factor independent 13.5126.8437.7212.0156.6

Dennd5ainterleukin 15 receptor, alpha chain2.9130.9380.1144.3167.8

E2f5BCL2-like 14.7133.7627.4257.4231.2

Rcl1protein tyrosine phosphatase, receptor type,2.6136.6358.8157.5125.0

FosI2plasmacytoma variant translocation 13.4136.7465.5179.8140.7

Atad3afos-like antigen 22.5137.0347.5107.2177.8

BaxBCL2-associated X protein2.5138.0347.3260.1150.2

Phf6solute carrier family 4, sodium bicarbonate

cotransporter, member 72.3140.3328.2258.7397.5

Zfp52tumor necrosis factor receptor superfamily,

member 42.2141.7311.1161.7111.6

Crtamchemokine (C—X—C motif) ligand 1012.7141.71798.3242.159.4

Nop14polo-like kinase 32.8144.8406.3200.1119.9

RelCD3E antigen, epsilon polypeptide associated2.2158.7350.2260.9111.4

protein

Gramd1btumor necrosis factor (ligand) superfamily,2.1162.4342.1242.1169.7

member 11

Ifi27l2apolymerase (RNA) III (DNA directed)3.0166.3503.7296.1121.6

polypeptide D

Tnfrsf10bearly growth response 22.8173.5494.0136.368.2

Rpl7l1DnaJ (Hsp40) homolog, subfamily C, member2.1173.6369.4346.2254.3

Eif1aDNA topoisomerase 1, mitochondrial2.7182.2498.2338.6114.4

Nfkb2tripartite motif-containing 30D2.3182.6423.465.890.6

Heatr1DnaJ (Hsp40) homolog, subfamily C, member

212.0190.1389.4285.5228.2

SAM domain, SH3 domain and nuclear

Utp20localization signals, 12.2191.5422.1222.8304.1

Chst11solute carrier family 5 (inositol transporters),2.1191.6400.2210.0123.4

member 3

Ddx21mitochondrial ribosomal protein L152.1191.6396.3329.8137.7

Hsf2dual specificity phosphatase 54.0203.5818.1307.5560.7

Bccipapoptosis enhancing nuclease2.3211.1478.5288.2137.9

Tagapets variant 62.3218.3508.1220.5297.3

Sdc3DIM1 dimethyladenosine transferase 1-like

(S, cerevisiae)2.2218.4486.0356.0129.7

SytI32'-5′ oligoadenylate synthetase-like 12.1229.0473.3130.7124.3

Gtpbp4UTP18, small subunit (SSU) processome2.1232.0494.3384.9189.5

component, homolog (yeast)

Crip2BRCA2 and CDKN1A interacting protein2.4234.6563.3437.5269.8

Sh3rf1synaptotagmin-like 32.4242.4572.9316.7700.7

Nsfl1c5-methyltetrahydrofolate-homocysteine2.9245.7706.5334.6150.6

methyltransferase reductase

Gtf2f1URB2 ribosome biogenesis 2 homolog2.0245.7500.2489.8184.6

(S, cerevisiae)

Slc4a7ubiquitin-conjugating enzyme E2C binding2.1251.2530.5288.285.2

protein

Etv6lysine (K)-specific demethylase 2B2.2251.8547.1332.7262.1

Trim30dqueuine tRNA-ribosyltransferase domain3.0260.3788.7358.075.5

containing 1

Ddx27ubiquitin specific peptidase 312.0265.2533.2277.1176.2

Pwp2eukaryotic translation initiation factor 2-2.0267.7540.5260.8244.8

alpha kinase 2

Chchd2ATPase family, AAA domain containing 3A2.5268.8679.7523.1147.1

Myo1eadhesion molecule, interacts with CXADR

antigen 12.3269.5610.9272.9182.8

Eif5bSUMO/sentrin specific peptidase 32.0272.5548.7544.5298.4

Stat5aESF1, nucleolar pre-rRNA processing protein,2.2276.3610.4482.2266.5

homolog (S, cerevisiae)

Cops6deoxynucleotidyltransferase, terminal,2.1282.9600.4359.9326.1

interacting protein 2

D19Bwg1357eTGFB-induced factor homeobox 12.1300.5618.9217.5210.6

Aatfeukaryotic translation initiation factor 1A2.5300.8738.7597.7262.8

Aeninterferon-stimulated protein2.1305.7651.2144.3138.4

Amica1pleiomorphic adenoma gene-like 22.1311.5651.9376.2405.9

Wdr43PWP2 periodic tryptophan protein homolog2.3321.8743.3586.5189.3

(yeast)

Cct4furin (paired basic amino acid cleaving5.2329.71728.3271.7421.5

enzyme)

Nifktumor necrosis factor6.6330.72188.4489.9213.3

Tgm2apoptosis antagonizing transcription factor2.3331.4754.8523.1221.5

Ero1linterferon, alpha-inducible protein 27 like 2A2.5334.0828.1296.0221.4

Gfod1ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-3.9338.41311.3636.0298.2

galactosyl-1,3)-N-acetylgalactosaminide

alpha-2,6-sialyltransferase 4

Ak4methyltransferase like 12.2339.4744.7662.894.5

Sdad1notchless homolog 1 (Drosophila)2.0339.4690.3610.3158.1

Dimt1mitochondrial ribosomal protein L32.1340.0725.5651.4359.8

Esf1UBX domain protein 2A2.1343.8732.9532.1428.5

Cd3eapguanine nucleotide binding protein-like 23.2347.61124.7647.4227.5

(nucleolar)

Samsn1programmed cell death 112.0353.9711.8435.9287.4

Tnfrsf4cyclin-dependent kinase 82.0364.0731.1702.5346.2

Mettl1eukaryotic translation initiation factor 5B2.3365.1838.2544.5355.5

Cd274RNA terminal phosphate cyclase-like 12.5373.3948.8746.4155.8

Ubtd2NSFL1 (p97) cofactor (p47)2.3374.1876.1725.9369.7

Icosnuclear factor of kappa light polypeptide

gene enhancer in B cells inhibitor, delta3.9378.51465.1389.9224.0

Kdm2bM-phase phosphoprotein 10 (U3 small

nucleolar ribonucleoprotein)2.8379.81069.3738.4290.8

Larp4GRAM domain containing 1B2.5382.7949.6363.4659.2

Eif3dERO1-like (S, cerevisiae)2.2387.7872.3773.0520.9

Tnfaip3nuclear receptor subfamily 4, group A,6.8387.82639.0343.7220.7

member 1

Map1bsurfeit gene 22.1399.8852.2696.3204.0

Cdv3N(alpha)-acetyltransferase 25, NatB auxiliary2.1405.7847.3669.5194.1

subunit

Plac8yrdC domain containing (E, coli)2.0406.7830.8635.3267.0

Mrpl3La ribonucleoprotein domain family, member2.2408.8887.9586.6358.3

Surf2SDA1 domain containing 12.2419.8939.9631.4284.7

Ubxn2aimportin 42.8420.31183.6777.8173.5

Utp18inducible T cell co-stimulator2.2423.9920.9818.8796.9

Isg20solute carrier family 7 (cationic amino acid2.1439.4934.4842.6344.6

transporter, y+ system), member 1

Dnajc2arsA arsenite transporter, ATP-binding,2.6446.61165.0717.9963.9

homolog 1 (bacterial)

Jak2polymerase (RNA) I polypeptide C2.7447.81208.4854.0295.9

Slc7a1spermatogenesis associated 52.0450.8920.2516.0361.6

Syde2ubiquitin specific peptidase 182.7451.81240.5296.0250.7

Slc5a6placenta-specific 82.1452.4967.3888.6590.8

Dnttip2general transcription factor IIF, polypeptide 12.3454.81063.9890.0680.8

Idi2nuclear factor of kappa light polypeptide3.4456.41535.5679.1502.7

gene enhancer in B cells inhibitor, beta

Dus2PHD finger protein 62.5462.01159.5775.8510.4

Pitrm1RRN3 RNA polymerase I transcription factor2.1462.2948.4913.2388.9

homolog (yeast)

Plxna1cytotoxic and regulatory T cell molecule2.5473.71177.8586.8431.8

Cdk5r1COP9 (constitutive photomorphogenic)

homolog, subunit 6 (Arabidopsis thaliana)2.3483.61101.9947.8560.3

Ube2cbpasparagine-linked glycosylation 3 (alpha-1,3-

mannosyltransferase)2.1485.91006.3758.7339.4

Tnfsf11tryptophanyl-tRNA synthetase2.0486.1987.1897.1504.7

Pop7hypoxia up-regulated 12.0494.3996.6802.4690.3

Psme3family with sequence similarity 60, member A2.0500.81002.1834.7417.6

Mir17hgbone marrow stromal cell antigen 23.8502.51922.9925.5246.0

Tsr1nuclear factor of kappa light polypeptide2.4503.21231.8494.0341.8

gene enhancer in B cells 2, p49/p100

Rbpms2UTP20, small subunit (SSU) processome2.4510.51240.2696.4245.8

component, homolog (yeast)

Mrpl47CD274 antigen2.2516.61128.7246.9220.2

Rab8bproviral integration site 13.4518.41766.4676.9970.0

Plagl2signal transducer and activator of

transcription 5A2.3530.01210.4496.6507.8

GrhI1CD69 antigen3.2535.71725.8289.5153.9

Zeb2pitrilysin metallepetidase 12.1544.91153.8968.4349.3

sept-02cyclin-dependent kinase 62.7550.31476.51064.0642.1

Slc5a3DEAD (Asp-Glu-Ala-Asp) box polypeptide 272.3556.21286.9987.2480.4

Naa25polymerase (RNA) I polypeptide B2.8556.21536.01070.4201.3

Plaurtumor necrosis factor, alpha-induced protein2.2560.61212.2255.5446.0

Metap1nodal modulator 12.1563.01161.0988.9439.8

Alg3NOP14 nucleolar protein2.5570.91418.9925.3398.0

Mrpl15ribosomal protein L7-like 12.5586.71448.71030.2687.2

Oasl1methionyl aminopeptidase 12.1597.51244.11139.3433.4

Rorchypoxia inducible factor 1, alpha subunit3.0624.21854.6809.4838.4

Nomo1Janus kinase 22.1624.51328.7390.6917.8

Tgif1nuclear factor of kappa light polypeptide2.9661.51913.3713.9720.5

gene enhancer in B cells 1, p105

Lipgreticuloendotheliosis oncogene2.5678.91686.4409.8580.5

Rrn3septin 22.1687.31436.01354.11181.3

Dnajc21nucleolar protein interacting with the FHA2.3733.41658.21280.0407.2

domain of MKI67

Yrdcelongation factor Tu GTP binding domain2.0739.31483.51439.0904.3

containing 2

Acsl6myelocytomatosis oncogene4.0761.03022.81064.0211.5

Spata5dyskeratosis congenita 1, dyskerin2.7778.22112.01549.5484.2

Urb2carnitine deficiency-associated gene2.1801.61718.21274.71010.3

expressed in ventricle 3

NlelGTP binding protein 42.4824.21942.61578.7567.3

WarsHEAT repeat containing 12.4830.32020.61235.5495.4

Cremproteaseome (prosome, macropain) activator2.1838.41763.51471.1936.1

subunit 3 (PA28 gamma, Ki)

Larp1La ribonucleoprotein domain family, member2.0861.71742.11250.9854.3

Eif2ak2DNA segment, Chr 19, Brigham & Women's2.3868.61978.41218.0653.4

Genetics 1357 expressed

Hyou1eukaryotic translation initiation factor 3,2.2909.11971.61641.9920.6

subunit D

Senp3TSR1 20S rRNA accumulation2.1913.91915.91474.6477.2

Tmtc2MYB binding protein (P160) 1a2.61140.02962.92200.7459.8

FosbT cell activation Rho GTPase activating2.41176.72794.4489.3704.2

protein

Pdcd11RAB8B, member RAS oncogene family2.11189.52492.21671.32512.5

Usp31DEAD (Asp-Glu-Ala-Asp) box polypeptide 212.41210.22928.02221.11098.2

Cdk8chaperonin containing Tcp1, subunit 4 (delta)2.31321.42989.72462.51294.8

Eftud2coiled-coil-helix-coiled-coil-helix domain2.31374.23171.22636.91008.9

containing 2

Fam60aWD repeat domain 432.31727.63912.62927.51014.9

TABLE 7

Selection of preferred endogenous genes that are constantly active during

immune cell activation (dependent or independent from T-cell activation).

SymbolGene description

CD3GCD3 gamma

Rn28s128S ribosomal RNA

Rn18s18S ribosomal RNA

Rn7skRNA, 7SK, nuclear

Actg1actin, gamma, cytoplasmic 1

B2mbeta-2 microglobulin

Rpl18aribosomal protein L18A

Pabpc1poly(A) binding protein, cytoplasmic 1

Gapdhglyceraldehyde-3-phosphate dehydrogenase

Rpl19ribosomal protein L19

Rpl17ribosomal protein L17

Rplp0ribosomal protein, large, P0

Cfl1cofilin 1, non-muscle

Pfn1profilin 1

TABLE 8

Selection of genes that are transiently

upregulated upon T-cell activation.

SymbolGene description

II3interleukin 3

II2interleukin 2

Ccl4chemokine (C-C motif) ligand 4

II21interleukin 21

Gp49aglycoprotein 49 A

Nr4a3nuclear receptor subfamily 4, group A, member 3

Lilrb4leukocyte immunoglobulin-like receptor, subfamily B,

member 4

Cd200CD200 antigen

Cdknacyclin-dependent kinase inhibitor 1A (P21)

Gzmcgranzyme C

Nr4a2nuclear receptor subfamily 4, group A, member 2

Cishcytokine inducible SH2-containing protein

Ccr8chemokine (C-C motif) receptor 8

Lad1ladinin

Crabp2cellular retinoic acid binding protein 11

TABLE 9

Selection of genes that are upregulated

over more than 24 hours upon T-cell activation.

SymbolDescription

Gzmbgranzyme B

Tbx21T-box 21

Pdcd1programmed cell death 1

Plekpleckstrin

Chek1checkpoint kinase 1

Slamf7SLAM family member 7

Zbtb32zinc finger and BTB domain containing 32

TigitT cell immunoreceptor with Ig and ITIM domains

Lag3lymphocyte-activation gene 3

Gzmagranzyme A

Wee1WEE 1 homolog 1 (S. pombe)

II12rb2interleukin 12 receptor, beta 2

Ccr5chemokine (C-C motif) receptor 5

Eea1early endosome antigen 1

Dtldenticleless homolog (Drosophila)

TABLE 10

Selection of genes that are down-regulated

upon immune cell activation.

SymbolGene description

Apata6spermatogenesis associated 6

Itga6integrin alpha 6

Rcbtb2regulator of chromosome condensation (RCC1) and

BTB (POZ) domain containing protein 2

Cd1d1CD1d1 antigen

St8sia45T8 alpha-N-acetyl-neuraminide alpha-2,8-

sialyltransferase 4

Itgaeintegrin alpha E, epithelial-associated

Fam214afamily with sequence similarity 214, member A

Slc6a19solute carrier family 6 (neurotransmitter transporter),

member 19

Cd55CD55 antigen

XkrxX Kell blood group precursor related X linked

Mturnmaturin, neural progenitor differentiation regulator

homolog (Xenopus)

H2-Obhistocompatibility 2, O region beta locus

Cnr2cannabinoid receptor 2 (macrophage)

Itgaeintegrin alpha E, epithelial-associated

Raver2ribonucleoprotein, PTB-binding 2

Zbtb20zinc finger and BTB domain containing 20

Arrb1arrestin, beta 1

Abca1ATP-binding cassette, sub-family A (ABC1), member 1

Tet1tet methylcytosine dioxygenase 1

Slc16a5solute carrier family 16 (monocarboxylic acid

transporters), member 5

Trav14-1T cell receptor alpha variable 14-1

Ampd3adenosine monophosphate deaminase 3

TABLE 11

Selection of human genes that are silent upon T-cell activation

(safe harbor gene targeted integration loci).

SymbolGene description

Zfp640zinc finger protein 640

LOC100038422uncharacterized LOC100038422

Zfp600zinc finger protein 600

Serpinb3aserine (or cysteine) peptidase inhibitor,

clade B (ovalbumin), member 3A

Tas2r106taste receptor, type 2, member 106

Magea3melanoma antigen, family A, 3

Omt2aoocyte maturation, alpha

Cpxcr1CPX chromosome region, candidate 1

Hsf3heat shock transcription factor 3

PbsnProbasin

Sbpspermine binding protein

Wfdc6bWAP four-disulfide core domain 6B

Meiobmeiosis specific with OB domains

Dnm3osdynamin 3, opposite strand

Skint11selection and upkeep of intraepithelial T cells 11

TABLE 12

List of gene loci upregulated in tumor exhausted infiltrating

lymphocytes from multiple tumors) useful for gene integration

of exogenous (compiled coding sequences as per the present invention

Gene namesUniprot ID (human)

CXCL13O43927

TNFRSF1BP20333

RGS2P41220

TIGITQ495A1

CD27P26842

TNFRSF9Q12933

SLAQ13239

INPP5FQ01968

XCL2Q9UBD3

HLA-DMAP28067

FAM3CQ92520

WARSP23381

EIF3LQ9Y262

KCN K5095279

TMBIM6P55061

CD200P41217

C3H7AO60880

SH2D1AO60880

ATP1B3P54709

THADAQ6YHU6

PARK7Q99497

EGR2P11161

FDFT1P37268

CRTAMO95727

IFO16Q16666

TABLE 13

List of gene loci upregulated in hypoxic tumor conditions useful for gene

integration of exogenous coding sequences as per the present invention

Gene namesStrategy

CTLA-4KO/KITarget shown to be upregulated

LAG-3 (CD223)KO/KIin T-cells upon hypoxia exposure

PD1KO/KIand T cell exhaustion

4-1BB (CD137)KI

GITRKI

OX40KI

IL10KO/KI

ABCB1KIHIF target

ABCG2KI

ADMKI

ADRA1BKI

AK3KI

ALDOAKI

BHLHB2KI

BHLHB3KI

BNIP3KI

BNIP3LKI

CA9KI

CCNG2KI

CD99KI

CDKN1AKI

CITED2KI

COL5A1KI

CPKI

CTGFKI

CTSDKI

CXCL12KI

CXCR4KI

CYP2S1KI

DDIT4KI

DEC1KI

EDN1KI

EGLN1KI

EGLN3KI

ENGKI

EN01KI

EPOKI

ETS1KI

FECHKI

FN1KI

FURINKI

GAPDHKI

GPIKI

GPX3KI

HK1KI

HK2KI

HMOX1KI

HSP90B1KI

ID2KI

IGF2KI

IGFBP1KI

IGFBP2KI

IGFBP3KI

ITGB2KI

KRTI4KI

KRTI8KI

KRTI9KI

LDHAKI

LEPKI

LOXKI

LRPIKI

MCLIKI

METKI

MMPI4KI

MMP2KI

MXI1KI

NOS2AKI

NOS3KI

NPMIKI

NR4A1KI

NT5EKI

PDGFAKI

PDKIKI

PFKFB3KI

PFKLKI

PGKIKI

PH-4KI

PKM2KI

PLAURKI

PMAIPIKI

PPP5CKI

PROKI12

SERPINE1KI

SLC2A1KI

TERTKI

TFKI

TFF3KI

TFRCKI

TGFAKI

TGFB3KI

TGM2KI

TPI1KI

VEGFAKI

VIMKI

TMEM45AKI

AKAP12KI

SEC24AKI

ANKRD37KI

RSBN1KI

GOPCKI

SAMD12KI

CRKLKI

EDEM3KI

TRIM9KI

GOSR2KI

MIFKI

ASPHKI

WDR33KI

DHX40KI

KLF10KI

R3HDM1KI

RARAKI

L0C162073KI

PGRMC2KI

ZWILCHKI

TPCN1KI

WSB1KI

SPAG4KI

GYS1KI

RRP9KI

SLC25A28KI

NTRK2KI

NARFKI

ASCC1KI

UFM1KI

TXN I PKI

MGAT2KI

VDAC1KI

SEC61GKI

SRP19KI

JMJD2CKI

SNRPD1KI

RASSF4KI

US11873511B2. Targeted gene insertion for improved immune cells therapy (2024-01-16). CELLECTIS [FR]. Inventors: Brian Busser [US], Philippe Duchateau [FR], Alexandre Juillerat [US], Laurent Poirot [FR], Julien Valton [US].

Full text: Click here

Patent 2024

Top products related to «Activating transcription factor 6, human»

Rneasy plus mini kit by Qiagen

Sourced in Germany, United States, United Kingdom, Netherlands, Canada, Japan, France, Spain, China, Australia, Italy, Switzerland, Belgium, Denmark, Sweden, Norway, Singapore, Jamaica, Hong Kong

The RNeasy Plus Mini Kit is a product from Qiagen designed for the purification of total RNA from a variety of sample types. It utilizes a silica-membrane-based technology to effectively capture and purify RNA molecules.

High capacity rna to cdna kit by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Spain, Japan, Lithuania, Canada, Australia, Sweden, France, Netherlands, Switzerland, Ireland, China, Belgium, Italy

The High Capacity RNA-to-cDNA Kit is a laboratory product designed to efficiently convert extracted RNA into complementary DNA (cDNA). It provides a streamlined workflow for the reverse transcription process, allowing for the generation of high-quality cDNA from a wide range of RNA input amounts.

Beas 2b by Merck Group

Sourced in United States, Germany

BEAS-2B is a human bronchial epithelial cell line that is commonly used in cell culture experiments. It is derived from normal human bronchial epithelial cells and exhibits characteristics of the bronchial epithelium. The BEAS-2B cell line is widely used for research purposes in the fields of respiratory biology and disease.

High capacity cdna reverse transcription kit by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Japan, Lithuania, France, Italy, China, Spain, Canada, Switzerland, Poland, Australia, Belgium, Denmark, Sweden, Hungary, Austria, Ireland, Netherlands, Brazil, Macao, Israel, Singapore, Egypt, Morocco, Palestine, State of, Slovakia

The High-Capacity cDNA Reverse Transcription Kit is a laboratory tool used to convert RNA into complementary DNA (cDNA) molecules. It provides a reliable and efficient method for performing reverse transcription, a fundamental step in various molecular biology applications.

Spss statistical software by IBM

Sourced in United States, Japan, United Kingdom, Belgium, Austria, Australia, Spain

SPSS is a comprehensive statistical software package that enables data analysis, data management, and data visualization. It provides a wide range of statistical techniques, including descriptive statistics, bivariate analysis, predictive analytics, and advanced modeling. SPSS is designed to help users efficiently manage and analyze large datasets, making it a valuable tool for researchers, statisticians, and data analysts.

Spss statistical package by IBM

Sourced in United States, United Kingdom, Japan, Spain, Italy

SPSS is a software package developed by IBM that provides statistical analysis capabilities. It allows users to perform a wide range of statistical analyses, including data management, descriptive statistics, and advanced analytics. SPSS is designed to help users explore, visualize, and interpret data, though its specific use cases may vary depending on the needs of the user or organization.

Qrt pcr by Bio-Rad

Sourced in United States, Germany

QRT-PCR is a laboratory equipment used for Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) analysis. It is designed to detect and quantify specific nucleic acid sequences in a sample.

Dimethyl sulfoxide (dmso) by Merck Group

Sourced in United States, Germany, United Kingdom, China, Italy, Sao Tome and Principe, France, Macao, India, Canada, Switzerland, Japan, Australia, Spain, Poland, Belgium, Brazil, Czechia, Portugal, Austria, Denmark, Israel, Sweden, Ireland, Hungary, Mexico, Netherlands, Singapore, Indonesia, Slovakia, Cameroon, Norway, Thailand, Chile, Finland, Malaysia, Latvia, New Zealand, Hong Kong, Pakistan, Uruguay, Bangladesh

DMSO is a versatile organic solvent commonly used in laboratory settings. It has a high boiling point, low viscosity, and the ability to dissolve a wide range of polar and non-polar compounds. DMSO's core function is as a solvent, allowing for the effective dissolution and handling of various chemical substances during research and experimentation.

Ripa buffer by Thermo Fisher Scientific

Sourced in United States, Germany, China, Switzerland, Japan, United Kingdom, Italy, Australia, Sweden, Belgium, Canada, Denmark, France, Sao Tome and Principe, Macao, Austria, Morocco, Norway, Israel

RIPA buffer is a commonly used lysis buffer for the extraction and solubilization of proteins from cells and tissues. It contains a mixture of ionic and non-ionic detergents that help disrupt cell membranes and release cellular contents, including proteins. The buffer also includes salts and buffers to maintain the pH and ionic strength of the solution.

Secondary anti mouse and anti rabbit antibodies by Santa Cruz Biotechnology

Sourced in United States

Secondary anti-mouse and anti-rabbit antibodies are laboratory reagents used to detect the presence of primary antibodies that have been raised against mouse or rabbit antigens. These secondary antibodies are conjugated with enzymes or fluorescent dyes, which allow for the visualization and quantification of the primary antibodies in various experimental techniques, such as Western blotting, immunohistochemistry, and ELISA.

What is the role of Activating transcription factor 6 (ATF6) in human cells?

Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response in humans. It serves as a sensor for endoplasmic reticulum stress and triggers adaptive signaling pathways that promote cell survival and homeostasis. ATF6 plays a central role in maintaining proteostasis and modulating various cellular processes, including metabolism, inflammation, and apoptosis. Disregulation of ATF6 activity has been implicated in the pathogenesis of diverse human diseases, such as neurodegenerative disorders, cardiovascular disease, and metabolic syndromes.

How can PubCompare.ai help in research related to Activating transcription factor 6 (ATF6)?

PubCompare.ai allows you to screen protocol literature more efficietly and leverage AI to pinpoint critical insights. The platform can help researchers identify the most effective protocols related to Activating transcription factor 6 (ATF6) for their specific research goals. PubCompare.ai's AI-driven analysis can highlight key differences in protocol effectiveness, enabling you to choose the best option for reproducibility and accuracy.

What are some common applications of Activating transcription factor 6 (ATF6) in human research?

Activating transcription factor 6 (ATF6) is widely studied for its role in maintaining cellular proteostasis and modulating key processes like metabolism, inflammation, and apoptosis. Researchers often investigate how ATF6 dysregulation contributes to the pathogenesis of various human diseases, such as neurodegenerative disorders, cardiovascular disease, and metabolic syndromes. Understanding the power of ATF6 activation can provide valuable insights into disease mechanisms and identify potential therapeutic targets.

Are there different variants or types of Activating transcription factor 6 (ATF6) in humans?

Yes, there are two main isoforms of Activating transcription factor 6 (ATF6) found in humans: ATF6α and ATF6β. Both isoforms serve as sensors for endoplasmic reticulum stress and trigger adaptive signaling pathways, but they may have distinct roles and regulation in the unfolded protein response and other cellular processes. Researchers often study the individual and combined effects of these ATF6 isoforms in various human cell types and disease models.

More about "Activating transcription factor 6, human"

Activating transcription factor 6 (ATF6) is a key regulator of the unfolded protein response in humans.
It serves as a sensor for endoplasmic reticulum (ER) stress, triggering adaptive signaling pathways that promote cell survival and homeostasis.
ATF6 plays a central role in maintaining proteostasis and modulating various cellular processes, including metabolism, inflammation, and apoptosis.
Disregulation of ATF6 activity has been implicated in the pathogenesis of diverse human diseases, such as neurodegenerative disorders, cardiovascular disease, and metabolic syndromes.
Exploring the power of ATF6 activation in humans can provide valuable insights into disease mechanisms and potential therapeutic targets.
Researchers can utilize AI-driven platforms like PubCompare.ai to discover protocols from literature, preprints, and patents, while receiving AI-powered comparisons to enhance research reproducibility and accuracy.
This single, typo-free platform can help uncover the best protocols and products for ATF6-related studies.
When conducting ATF6 research, scientists may employ techniques such as real-time quantitative PCR (QRT-PCR) to measure gene expression levels, and utilize cell lines like BEAS-2B for in vitro experiments.
The RNeasy Plus Mini Kit and High Capacity RNA-to-cDNA Kit can be used for RNA extraction and cDNA synthesis, respectively.
Downstream analyses may involve the use of SPSS statistical software for data analysis.
Cellular lysis and protein extraction can be performed using RIPA buffer, and Western blotting can be carried out with secondary anti-mouse and anti-rabbit antibodies to detect ATF6 and related proteins.
Dimethyl sulfoxide (DMSO) may be used as a solvent for various experimental compounds.
By leveraging these tools and techniques, researchers can delve deeper into the role of ATF6 in human physiology and pathology, ultimately contributing to the understanding of disease mechanisms and the development of potential therapeutic interventions.