Clone 53 6

Manufactured by BioLegend

Sourced in Germany, United States

Clone 53-6.7 is a rat monoclonal antibody that recognizes mouse CD8a. It is suitable for use in flow cytometry and other immunoassays to identify and isolate CD8a-positive cells.

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Anti-CD8 (clone 53-6.7, APC/Cy7-anti-CD8 mAb (clone 53–6.7, Anti-CD8-PerCP/Cy5.5 (clone 53–6.7) CD8 (clone 53-6.7, Anti-mouse CD8α APC-Cy7 (clone 53-6.7, FITC anti-CD8 (clone 53-6.7, Anti-CD8-FITC (clone 53.6.7; CD8a-APC (clone 53-6.7; Anti-CD8-PE/Cy5 (clone 53–6.7; Anti-mouse CD8-PE (clone 53-6,7,

21 protocols using clone 53 6

Cytotoxicity Assay of T Cell-Mediated Cancer Cell Killing

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Co-culture of cancer cells and T cells was performed as previously described (66 (link)). Briefly, B16F10 cells were maintained in complete DMEM media (10% FBS and 50U/ml of Penicillin-Streptomycin). CD8 T cells isolated from spleen and lymph nodes from Pmel-1 or OT-I mice were stimulated with anti-CD3/CD28 beads (ThermoFisher, 11452D) and then cultured in complete RPMI 1640 media (10%FBS, 20mM HEPES, 1mM sodium pyruvate, 0.05mM 2-mercaptoethanol, 2mM L-glutamine, and 50U/ml streptomycin and penicillin, 20ng/ml recombinant mouse IL-2).
To test the sensitivity of cancer cells to T cell-driven cytotoxicity (OT-I or Pmel-1 model), we plated B16F10 cells (sgRosa26, sgTraf3, pEF1a-Empty, or pEF1a-Traf3) at equal density in all wells, and added T cells at ratios to cancer cells. With the OT-I model, we first incubated the B16F10 cells with 1nM SIINFEKL peptide for 2 hours prior to co-culture with T cells. With the Pmel-1 model, B16F10 cells were either pre-treated with 1ng/ml IFNγ overnight or untreated prior to the co-culture. There are 2–4 cell-culture replicates for each condition. After a one-day or three-day co-culture with T cells, we counted the remaining cancer cells by FACS using the precision count beads (BioLegend, 424902). T cells present in these cultures were gated out based on antibodies specific for CD45 (Biolegend, clone 30-F11) or CD8 (BioLegend, clone 53-6.7).

Gu S.S., Zhang W., Wang X., Jiang P., Traugh N., Li Z., Meyer C., Stewig B., Xie Y., Bu X., Manos M.P., Font-Tello A., Gjini E., Lako A., Lim K., Conway J., Tewari A.K., Zeng Z., Sahu A.D., Tokheim C., Weirather J.L., Fu J., Zhang Y., Kroger B., Liang J.H., Cejas P., Freeman G.J., Rodig S., Long H.W., Gewurz B.E., Hodi F.S., Brown M, & Liu X.S. (2021). Therapeutically increasing MHC-I expression potentiates immune checkpoint blockade. Cancer discovery, 11(6), 1524-1541.

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Macrophage-mediated regulation of T cell activation

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Primary splenocytes were obtained from spleens of naïve C57BL/6 mice. Dissected spleens were dissociated in MAC buffer and passed through a 70 µm cell strainer to obtain a single cell suspension. Cells were centrifuged (400 x g) and red blood cells were lysed using 1x Red blood lysis buffer (Biolegend). Obtained splenocytes were cultured in RPMI supplemented with 10% FBS. For T cell activation assays, splenocytes were stimulated using Dynabeads Mouse T- Activator CD3/CD28 (Life Technology). Activated splenocytes (S) were then co-cultured with bone marrow derived macrophages (BMMs) from WT and Grn^-/- mice or with macrophages (M) magnetically isolated cells from day 6 and day 14 metastasis bearing livers (4:1 ratio, S: M). Cells were plated in 96 well plates and incubated at 37°C for 24h. Subsequently, Brefeldin A (eBioscience, 1:100) was added to the cells for 5h. Cells were then harvested and stained with CD8 (Biolegend, clone 53-6.7) and IFNγ (Biolegend, Clone XMG1.2) antibodies and analyzed by flow cytometry. For some experiments, recombinant mouse Progranulin protein (recGrn) (R&D systems; [1 µg/ml]) and mouse Periostin neutralizing antibody (R&D systems; [1 µg/ml]) were used.

Quaranta V., Rainer C., Nielsen S.R., Raymant M.L., Ahmed M.S., Engle D.D., Taylor A., Murray T., Campbell F., Palmer D.H., Tuveson D.A., Mielgo A, & Schmid M.C. (2018). Macrophage-derived granulin drives resistance to immune checkpoint inhibition in metastatic pancreatic cancer. Cancer research, 78(15), 4253-4269.

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Murine Leukocyte Functional Assay

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Leukocytes were extracted from murine spleen, salivary glands and lungs as described previously [13 (link),60 (link)]. For CD4⁺ functional responses, isolated leukocytes were stimulated for 2 hours with 3 μg/ml m09, (GYLYIYPSAGNSFDL), M25 (NHLYETPISATAMVI), m139 (TRPYRYPRVCDASLS), and m142 (RSRYLTAAAVTAVLQ) MCMV MHCII peptides (Genscript). Brefeldin A (Sigma) was then added and cells incubated for a further 4 hours. For CD8⁺ functional responses, leukocytes were stimulated with 2 μg/ml m139, IE3, M38, and M45 MCMV MHCI peptides in the presence of anti-mouse CD107a-FITC (Biolegend) with monensin (BD Biosciences) and Brefeldin A for 6 hours. Cells were subsequently stained with Zombie Aqua fixable viability dye (Biolegend) or LIVE/DEAD-Aqua (Life-Technologies), stained with anti-CD16/CD32 Fc-block (Biolegend) and then with either anti-CD4 Pacific-Blue or PercP, (clone RM4-5, Biolegend) or with anti-CD8 PercP (clone 53–6.7, Biolegend) and anti-CD262 (TRAILR DR5, clone MD5.1, eBioscience). Cells were fixed, saponin permeabilised and stained for anti-IFNγ FITC or Pacific-Blue (clone XMG1.2, Biolegend) and anti-IL-10 APC (clone JES5-16E3, eBioscience). Data was acquired using a BD FACSCantoII or a BD LSR II fortessa flow cytometer (BD Biosciences) and analysed with FlowJo software (TreeStar).

Clement M., Marsden M., Stacey M.A., Abdul-Karim J., Gimeno Brias S., Costa Bento D., Scurr M.J., Ghazal P., Weaver C.T., Carlesso G., Clare S., Jones S.A., Godkin A., Jones G.W, & Humphreys I.R. (2016). Cytomegalovirus-Specific IL-10-Producing CD4+ T Cells Are Governed by Type-I IFN-Induced IL-27 and Promote Virus Persistence. PLoS Pathogens, 12(12), e1006050.

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Immunofluorescent Staining of Tumor Tissues

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Excised tumor and spleen tissues were fixed in 4% formaldehyde overnight and then cryopreserved in 30% sucrose for 24 h. Tissue samples were embedded in Tissue-Tek OCT (Sakura Finetek, Alphen aan den Rijn, NL) and sectioned at 8 μm using a cryostat (Leica Microsystems, Diegem, BE). Sections were stained with antibodies directed against murine CD4, CD8, and FoxP3. Briefly, sections were first blocked with 0.2% (v/v) Triton X-100 (Sigma), 10% (w/v) goat serum, 5% rat serum, and 2% BSA in PBS for 1 h at room temperature. Subsequently, the primary antibodies (rat CD8a-FITC 1:500 [clone 53–6.7, BioLegend] and CD4-FITC 1:500 [clone GK1.5, BioLegend] and mFoxP3-APC 1:500 [clone FJK-16s, eBioscience] were applied to the slides for 1 h at room temperature protected from the light. After washing with PBS, the sections were mounted using Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI to visualize the cell nuclei. The slides were imaged using a structured illumination AxioImager microscope (Zeiss, Oberkochen, GE). The results are presented in Supplemental Information.

Kos S., Lopes A., Preat V., Cemazar M., Lampreht Tratar U., Ucakar B., Vanvarenberg K., Sersa G, & Vandermeulen G. (2019). Intradermal DNA vaccination combined with dual CTLA-4 and PD-1 blockade provides robust tumor immunity in murine melanoma. PLoS ONE, 14(5), e0217762.

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Tumor-Infiltrating CD8+ T Cell Isolation

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Single-cell tumour suspensions were kept on ice during the staining and sorting procedure. Cell suspensions from 3–5 tumours of the same treatment group were stained with the following antibodies: AF700 anti-CD45 (1:100; clone 30-F11, BioLegend), BV711 anti-CD8 (1:100; clone 53-6.7, BioLegend) and bin channel BV605 anti-CD4 (1:100; clone GK1.5, BioLegend), and BV605 anti-CD11c (1:100; clone N418, BioLegend). Discrimination of living cells from dead cells was performed using Live/Dead APC-Cy7 (eBioscience, 65-0865-14; 1:500 for non-fixed samples and cells with incubation for 20 min). Cells were washed twice, filtered through a 40-µm cell strainer, sorted on a FACSAria III instrument and acquired with FACSDiva (to enrich viable single CD45⁺CD8⁺CD11c⁻CD4⁻ cells.

Codarri Deak L., Nicolini V., Hashimoto M., Karagianni M., Schwalie P.C., Lauener L., Varypataki E.M., Richard M., Bommer E., Sam J., Joller S., Perro M., Cremasco F., Kunz L., Yanguez E., Hüsser T., Schlenker R., Mariani M., Tosevski V., Herter S., Bacac M., Waldhauer I., Colombetti S., Gueripel X., Wullschleger S., Tichet M., Hanahan D., Kissick H.T., Leclair S., Freimoser-Grundschober A., Seeber S., Teichgräber V., Ahmed R., Klein C, & Umaña P. (2022). PD-1-cis IL-2R agonism yields better effectors from stem-like CD8+ T cells. Nature, 610(7930), 161-172.

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Tumor Dissociation and Immune Cell Profiling

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Resected B78 tumors were mechanically dissociated for 45 min using a gentleMACS dissociator (Miltenyi Biotec) in HBSS supplemented with 1 mg/mL collagenase type D and 100 µg/mL DNAse I (Sigma-Aldrich) to obtain single cell suspensions.22 (link) Ghost Dye Red 780 (Tonbo Biosciences) was used for viability staining. For cell surface staining, cells were preincubated with mouse FC Block anti-mouse CD16/32 (clone 2.4G2, BD Biosciences). After blocking, the cells were labeled with CD3-PE-Cy5 (clone 145–2 C11, Biolegend), CD4-PE-Dazzle594 (clone GK1.5, Biolegend), CD8a-APC-R700 (clone 53–6.7, Biolegend), CD25-BB515 (clone PC61, BD Biosciences), CD45-BV510 (clone 30-F11, Biolegend), GD2-APC (clone 14G2a, Biolegend), and NK1.1-BV421 (clone PK136, Biolegend). Cells were then fixed and permeabilized overnight using Foxp3/Transcription Factor Staining Buffer Set (eBioscience). FoxP3 intracellular staining was then performed prior to flow cytometry. Flow cytometry data were acquired using an Attune NxT Flow Cytometer and analyzed using FlowJo V.10.7.1. The flow cytometry gating strategy is shown in online supplemental figure 1.

Aiken T.J., Komjathy D., Rodriguez M., Stuckwisch A., Feils A., Subbotin V., Birstler J., Gillies S.D., Rakhmilevich A.L., Erbe A.K, & Sondel P.M. (2022). Short-course neoadjuvant in situ vaccination for murine melanoma. Journal for Immunotherapy of Cancer, 10(1), e003586.

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Characterizing Islet Immune Infiltrates

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Assessment of islet infiltrate composition was performed by immunofluorescent staining of pancreatic sections for CD4 and CD8 T cell surface markers (using an average 15 sections/mouse). Slides were first stained for CD4 using primary rat anti-mouse CD4 (BioLegend, Clone GK1.5) antibody followed by a secondary donkey anti-rat Cy5-labeled detection antibody (Jackson Immunoresearch). After extensive washing, the slides were incubated for 1 hr with FITC-labeled rat anti-mouse CD8 antibody (BioLegend, Clone 53-6.7), washed and mounted in DAPI-containing Vectashield. As absolute numbers of islet-infiltrating T cells can vary widely between individual islets depending on the size of infiltrate, attempts to normalize this variance involved calculating the CD4⁺/CD8⁺ T cells ratios for each islet.
To count the frequency of T regulatory cells within islet infiltrates, indirect Foxp3 staining was performed with rat anti-mouse anti-Foxp3 primary antibody (eBioscience, San Diego, CA, Clone NRRF-30) followed by DyLight594 labeled donkey anti-rat-Ig (Jackson Immunoresearch). Anti-mouse CD4-FITC (eBioscience, Clone RM4-5) was then applied. All antibodies were diluted at 1∶100 for immunostaining.

Maneva-Radicheva L., Amatya C., Parker C., Ellefson J., Radichev I., Raghavan A., Charles M.L., Williams M.S., Robbins M.S, & Savinov A.Y. (2014). Autoimmune Diabetes Is Suppressed by Treatment with Recombinant Human Tissue Kallikrein-1. PLoS ONE, 9(9), e107213.

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Purification and Transfer of T-cell Subsets

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One week after the third recall, lymphocytes were collected from spleen of immunized or control group mice. Pelleted cells were resuspended in RobosepTM buffer (Stemcell technologies) at a final concentration of 10⁸ cells/mL. 5*10⁸ cells per group were purified by CD3+, CD8+ or CD4+ negative selection using an EasySep isolation kit (Stemcell Technologies). Cells were then injected intravenously (IV) in 4–5 week old C57BL/6 mice (10⁷ cell in 300μl). The remaining cells were marked with α-CD3-FITC (1:1000, clone 145-2C11, eBioscience), α-CD4-BrilliantViolet421 (1:500, clone M1/70, Biolegend) or α-CD8-PerCP (1:500, clone 53–6.7, Biolegend) stain and analyzed by FACS to assess the purity of the cell suspension, purity was always superior to 95%.

Castiglioni P., Hartley M.A., Rossi M., Prevel F., Desponds C., Utzschneider D.T., Eren R.O., Zangger H., Brunner L., Collin N., Zehn D., Kuhlmann F.M., Beverley S.M., Fasel N, & Ronet C. (2017). Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid. PLoS Neglected Tropical Diseases, 11(1), e0005240.

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Establishing Syngeneic Tumor Models for Immunotherapy Research

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To establish syngeneic tumor models, B16F10 (1 × 10⁶ cells to C57BL/6), CT26 (5 × 10⁵ cells to BALB/c), and EG7-OVA (1 × 10⁶ cells to C57BL/6) were injected subcutaneously to the right flank of mice. For lung metastasis, B16F10 (1 × 10⁵ cells) were injected intravenously to B16F10 tumor bearing mice, 4 days after subcutaneous injection. For dual flank model, 1 × 10⁶ and 5 × 10⁵ B16F10 cells were injected subcutaneously to the right and left flank of mice, respectively. When tumor reaches an average volume of 50–100 mm³, the mice were randomized into several groups according to the experimental protocol. Tumor volume (mm³) was calculated as (width)² × (length) × 0.5. IT dosing on the right tumors was performed three times with 3 days interval. To establish a mouse orthotopic hepatocarcinoma model, Hepa1-6 cells (1.5×10⁶) in HBSS medium were injected into spleen of C57BL/6 mice and the spleen was excised subsequently. IV dosing was performed three times with 3 days interval from Day 4 after cell inoculation. Isotype control antibody (10 mg/kg, BioLegend), Anti-CD8 (10 mg/kg, BioLegend, Clone 53–6.7), Anti-NK1.1 (10 mg/kg, BioLegend, Clone PK136), Anti-CSF1R (10 mg/kg, BioXcell, Clone AFS98), Anti-PD1 (10 mg/kg, BioLegend, Clone RMP1–14), and Anti-CTLA4 (5 mg/kg, BioLegend, Clone 9H10) were administered intraperitoneally.

Jang S.C., Economides K.D., Moniz R.J., Sia C.L., Lewis N., McCoy C., Zi T., Zhang K., Harrison R.A., Lim J., Dey J., Grenley M., Kirwin K., Ross N.L., Bourdeau R., Villiger-Oberbek A., Estes S., Xu K., Sanchez-Salazar J., Dooley K., Dahlberg W.K., Williams D.E, & Sathyanarayanan S. (2021). ExoSTING, an extracellular vesicle loaded with STING agonists, promotes tumor immune surveillance. Communications Biology, 4, 497.

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Bone Marrow Cell Enumeration by Flow Cytometry

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In order to determine cell numbers in the bone marrow, around 100.000 bone marrow cells were freshly isolated. Aliquots of bone marrows were washed once in H/S (132 mM NaCl, 20 mM HEPES [pH 7.4], 5 mM KCl, 1 mM CaCl₂, 0.7 mM MgCl₂, 0.8 mM MgSO₄), resuspended in 100 μL H/S, Fc-receptors were blocked by incubation for 30 min with Fc-block (anti-mouse CD16/CD32, Biolegend #101302), samples were washed once and aliquots were stained with FITC-coupled anti-mouse CD45 antibodies (1:200, clone RA3-6B2; Biolegend, #103228) and APC-coupled antibodies against mouse CD4 (1:200, clone GK1.5; Biolegend, #17-0041-81) or APC-coupled antibodies against mouse CD8 (1:200, clone 53-6.7; Biolegend, #17-0081-81) or PE-coupled antibodies against mouse CD19 (1:200, clone GD5; Biolegend, #115508) or APC-coupled antibodies against mouse Ly6G (Gr1) (1:200, clone RB6-BL5; Biolegend, #103107). In addition, cells were stained with Alexa 647-coupled anti-mouse CD45 antibodies (1:200, clone 30-F11; Biolegend, #103228) and FITC-coupled antibodies against mouse Sca-1 (1:200, clone D7; Biolegend, #108105). Cells were stained for 1 h at 4°C, washed once in H/S and analyzed on a FACS-Calibur. We analyzed the expression of the above described markers on 100 000 cells by flow cytometry.

Gulbins A., Horstmann M., Keitsch S., Soddemann M., Wilker B., Wilson G.C., Zeidan R., Hammer G.D., Daser A., Bechrakis N.E., Görtz G.E, & Eckstein A. (2023). Potential involvement of the bone marrow in experimental Graves’ disease and thyroid eye disease. Frontiers in Endocrinology, 14, 1252727.

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