anti tgf β, by BioXCell - Product details

1

Generation and Validation of Tr1 Cells

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For the generation of time-course microarray data of Tr1 cells and validation of Il10 regulators in Tr1 cells in vitro, CD4⁺CD44⁻CD62L⁺CD25⁻ naive cells were sorted from WT B6 or indicated KO and their corresponding control mice with BD FACSAria sorter, and then activated with plate-bound anti-CD3 and anti-CD28 (both at 1ug/ml) in the presence of 25ng/ml rmIL-27 (R & D systems). 10ug/ml anti-TGFβ (Bioxcell, Clone# 1D11.16.8) was also added for the microarray experiment.
For RNA-seq and qPCR analysis of IL-10 producing and non-producing T helper cells, naive CD4⁺CD44⁻CD62L⁺GFP⁻ cells were sorted from Foxp3-GFP; Il10-Thy1.1 double reporter mice using BD FACSAria sorter and were activated with irradiated splenocytes depleted of CD4 T cells (at the T: APC ratio of 1:6) and 2.5ug/ml soluble anti-CD3 in the presence of polarizing cytokines. Concentration of cytokines are as follows: 20ng/ml rmIL-12 (R & D systems) for Th1; 20ng/ml rmIL-4 (Miltenyi Biotec) for Th2; 2ng/ml of rhTGFb1 and 25ng/ml rmIL-6(both from Miltenyi Biotec) for non-pathogenic Th17; 20ng/ml rmIL-1β(Miltenyi Biotec), 25ng/ml rmIL-6 (Miltenyi Biotec), and 20ng/ml rmIL-23 (R&D systems) for pathogenic Th17; 25ng/ml of rmIL-27 (R & D systems) for Tr1. IL-10 positive (Thy1.1⁺) and negative (Thy1.1⁺) 7-AAD⁻TCRβ⁺CD4⁺GFP⁻ cells were re-sorted at 72 hours.

Zhang H., Madi A., Yosef N., Chihara N., Awasthi A., Pot C., Lambden C., Srivastava A., Burkett P.R., Nyman J., Christian E., Etminan Y., Lee A., Stroh H., Xia J., Karwacz K., Thakore P.I., Acharya N., Schnell A., Wang C., Apetoh L., Rozenblatt-Rosen O., Anderson A.C., Regev A, & Kuchroo V.K. (2020). An IL-27-Driven Transcriptional Network Identifies Regulators of IL-10 Expression across T Helper Cell Subsets. Cell reports, 33(8), 108433.

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2

Adoptive Transfer of Tolerant B Cells in Islet Transplantation

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Recipient mice received 100 μg anti-mouse CD45RB (clone: MB23G2, BioXCell, West Lebanon, NH) ip on days 0, 1, 3, 5, and 7 following transplantation. Islet recipient mice also received 500 μg anti-mouse TIM1 (clone RMT1–10, BioXCell, West Lebanon, NH) ip on day 0, and 300 μg on days 1 and 5 following transplantation. For the anti-TGF-β-treatment experiment, recipient mice were injected ip with 200 μg anti-TGF-β or isotype control (1D11.16.8 and MOPC-21, respectively, BioXCell, West Lebanon, NH) on days 0, 2, 4, 6, and 8 posttransplant.
For adoptive transfer experiments: after 21 days of skin graft survival, the tolerant animal is sacrificed, and spleen is isolated in sterile fashion. Spleens are disaggregated using a 70-μm nylon mesh and washed with sterile PBS. B cells are enriched by anti-CD19 magnetic bead isolation, following manufacturer protocol (Miltenyi, Auburn, CA). Purity of B cells is routinely >95%. Yield from an individual animal is typically 15–25 × 10⁶ B cells. If a greater number of cells is needed, B cells isolated from multiple tolerant mice are pooled. 5 × 10⁶ B cells from tolerant WT C57BL/6 or OBI mice are injected into B6.129S2-Ighmtm1Cgn/J (μMT) mice via tail or penile vein.

Kimura S., Rickert C.G., Kojima L., Aburawi M., Tanimine N., Fontan F., Deng K., Tector H., Mi Lee K., Yeh H, & Markmann J.F. (2019). Regulatory B cells require antigen recognition for effective allograft tolerance induction. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 20(4), 977-987.

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3

T Cell Differentiation and Expansion Protocols

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For iT_reg and T_activated cell differentiation, naive T cells were isolated and 24 well sterile tissue culture plates (Corning, Corning, NY) were coated anti-CD3ε (145–2C11, Biolegend) and anti-CD28 (37.51, Biolegend) as described ^{36 (link)}. Naive CD4⁺ T-cells were resuspended in complete RPMI media supplemented with 100 IU/ml recombinant human IL-2 for both iT_reg and T_activated, with additional 5 ng/ml recombinant human TGF-β for iT_reg and 10 μg/ml anti-TGF-β (1D11.16.8, BioXcell, West Lebanon, NH) for T_activated cultures. 3 × 10⁵ cells were added to the wells at a volume of 1 ml/well. Cells were cultured at 37°C 5% CO₂ for 3 days. Prior to experiments, live T_activated and iT_reg were FACS sorted based on their Foxp3-GFP expression status.
IL-2/anti–IL-2 mAb (JES6-1-A12, Bioxcell) complexes were prepared and injected as in ^{37 (link)} to expand antigen-specific tT_regs in vivo. Antigen-specific T_regs were isolated from spleens of the TCR transgenic animals by FACS sorting based on Foxp3 reporter expression. Alternatively, antigen-specific tT_regs were expanded in vitro. For in vitro proliferation, T_regs were cultured for three days in the presence of plate bound anti-CD3ε (145–2C11; 2 ug/mL), anti-CD28 (37.51; 2 ug/mL) and IL-2 (100 U/mL). On day 3, cells were split 1:2 and cultured with only IL-2. T_regs were harvested on day 5 and FACS sorted for Foxp3 reporter.

Akkaya B., Oya Y., Akkaya M., Souz J.A., Holstein A.H., Kamenyeva O., Kabat J., Matsumura R., Dorward D.W., Glass D.D, & Shevach E.M. (2019). T regulatory cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells. Nature immunology, 20(2), 218-231.

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Generation and Validation of Tr1 Cells

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For the generation of time-course microarray data of Tr1 cells and validation of Il10 regulators in Tr1 cells in vitro, CD4⁺CD44⁻CD62L⁺CD25⁻ naive cells were sorted from WT B6 or indicated KO and their corresponding control mice with BD FACSAria sorter, and then activated with plate-bound anti-CD3 and anti-CD28 (both at 1ug/ml) in the presence of 25ng/ml rmIL-27 (R & D systems). 10ug/ml anti-TGFβ (Bioxcell, Clone# 1D11.16.8) was also added for the microarray experiment.
For RNA-seq and qPCR analysis of IL-10 producing and non-producing T helper cells, naive CD4⁺CD44⁻CD62L⁺GFP⁻ cells were sorted from Foxp3-GFP; Il10-Thy1.1 double reporter mice using BD FACSAria sorter and were activated with irradiated splenocytes depleted of CD4 T cells (at the T: APC ratio of 1:6) and 2.5ug/ml soluble anti-CD3 in the presence of polarizing cytokines. Concentration of cytokines are as follows: 20ng/ml rmIL-12 (R & D systems) for Th1; 20ng/ml rmIL-4 (Miltenyi Biotec) for Th2; 2ng/ml of rhTGFb1 and 25ng/ml rmIL-6(both from Miltenyi Biotec) for non-pathogenic Th17; 20ng/ml rmIL-1β(Miltenyi Biotec), 25ng/ml rmIL-6 (Miltenyi Biotec), and 20ng/ml rmIL-23 (R&D systems) for pathogenic Th17; 25ng/ml of rmIL-27 (R & D systems) for Tr1. IL-10 positive (Thy1.1⁺) and negative (Thy1.1⁺) 7-AAD⁻TCRβ⁺CD4⁺GFP⁻ cells were re-sorted at 72 hours.

Zhang H., Madi A., Yosef N., Chihara N., Awasthi A., Pot C., Lambden C., Srivastava A., Burkett P.R., Nyman J., Christian E., Etminan Y., Lee A., Stroh H., Xia J., Karwacz K., Thakore P.I., Acharya N., Schnell A., Wang C., Apetoh L., Rozenblatt-Rosen O., Anderson A.C., Regev A, & Kuchroo V.K. (2020). An IL-27-Driven Transcriptional Network Identifies Regulators of IL-10 Expression across T Helper Cell Subsets. Cell reports, 33(8), 108433.

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5

T Cell Differentiation and Expansion Protocols

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For iT_reg and T_activated cell differentiation, naive T cells were isolated and 24 well sterile tissue culture plates (Corning, Corning, NY) were coated anti-CD3ε (145–2C11, Biolegend) and anti-CD28 (37.51, Biolegend) as described ^{36 (link)}. Naive CD4⁺ T-cells were resuspended in complete RPMI media supplemented with 100 IU/ml recombinant human IL-2 for both iT_reg and T_activated, with additional 5 ng/ml recombinant human TGF-β for iT_reg and 10 μg/ml anti-TGF-β (1D11.16.8, BioXcell, West Lebanon, NH) for T_activated cultures. 3 × 10⁵ cells were added to the wells at a volume of 1 ml/well. Cells were cultured at 37°C 5% CO₂ for 3 days. Prior to experiments, live T_activated and iT_reg were FACS sorted based on their Foxp3-GFP expression status.
IL-2/anti–IL-2 mAb (JES6-1-A12, Bioxcell) complexes were prepared and injected as in ^{37 (link)} to expand antigen-specific tT_regs in vivo. Antigen-specific T_regs were isolated from spleens of the TCR transgenic animals by FACS sorting based on Foxp3 reporter expression. Alternatively, antigen-specific tT_regs were expanded in vitro. For in vitro proliferation, T_regs were cultured for three days in the presence of plate bound anti-CD3ε (145–2C11; 2 ug/mL), anti-CD28 (37.51; 2 ug/mL) and IL-2 (100 U/mL). On day 3, cells were split 1:2 and cultured with only IL-2. T_regs were harvested on day 5 and FACS sorted for Foxp3 reporter.

Akkaya B., Oya Y., Akkaya M., Souz J.A., Holstein A.H., Kamenyeva O., Kabat J., Matsumura R., Dorward D.W., Glass D.D, & Shevach E.M. (2019). T regulatory cells mediate specific suppression by depleting peptide-MHC class II from dendritic cells. Nature immunology, 20(2), 218-231.

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In Vitro Differentiation of Tfh Subsets

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The method to differentiate iTfh1, iTfh2, and iTfh17-polarized cells in vitro was developed based on our previous paper (Gao et al., 2020 (link)). In brief, red blood cell lysed splenocytes from WT mice were left untreated (for differentiating iTh0/1/2/17) or pre-treated by 1 μg/mL lipopolysaccharide (LPS) for 24 hr in the complete RPMI media. 5×10⁵ per well LPS pre-treated splenocytes were co-cultured with FACS purified OT-II cells at the ratio of 50:1 in the presence of 1 μg/mL OVA_323-339 peptide and indicated Cytokines (Table 3) for 72 hr to differentiate iTfh1/2/17 cells. No Cytokines were added for differentiating Th0 cells. Neutralizing antibodies anti-IL-4, anti-IFN-γ, and anti-TGF-β (BioxCell) were used at 10 μg/mL. Cytokines were purchased from PeproTech.

Gao X., Luo K., Wang D., Wei Y., Yao Y., Deng J., Yang Y., Zeng Q., Dong X., Xiong L., Gong D., Lin L., Pohl K., Liu S., Liu Y., Liu L., Nguyen T.H., Allen L.F., Kedzierska K., Jin Y., Du M.R., Chen W., Lu L., Shen N., Liu Z., Cockburn I.A., Luo W, & Yu D. (2023). T follicular helper 17 (Tfh17) cells are superior for immunological memory maintenance. eLife, 12, e82217.

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Nonpolarizing CD4+ T Cell Activation

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For nonpolarizing CD4⁺ T cell (Th₀) activation, a protocol previously described elsewhere was used (18 (link)). Briefly, aseptically purified naïve CD4⁺ T cells were resuspended in complete media supplemented with 100 U/ml IL-2 (Peprotech; Rocky Hill, NJ, USA) and 10 µg/ml anti-TGF-β (Bioxcell; West Lebanon, NH, USA) and plated on sterile 24 well tissue culture plates (Corning/Life Sciences; Tewksbury, MA, USA) previously coated with 4 µg/ml anti-mouse CD3ε (Clone 145–2C11, Biolegend; San Diego, CA, USA) and 4 µg/ml anti-mouse CD28 (Clone 37.12, Biolegend). Cells were incubated for up to 96 h in a 5% CO₂ humidified tissue culture incubator at 37°C.

Akkaya B., Roesler A.S., Miozzo P., Theall B.P., Souz J.A., Smelkinson M.G., Kabat J., Traba J., Sack M.N., Brzostowski J.A., Pena M., Dorward D.W., Pierce S.K, & Akkaya M. (2018). Increased mitochondrial biogenesis and ROS production accompany prolonged CD4+ T cell activation. Journal of immunology (Baltimore, Md. : 1950), 201(11), 3294-3306.

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8

In Vitro Activation and Adoptive Transfer of Naïve T Cells

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24 well plates were coated with 10 μg/ml anti-CD3ε (BioXCell BE0001–1) overnight at 4°C. Plates were washed out with PBS, and plated with 5×10⁵ to 1.5×10⁶ naïve (CD4⁺Foxp3⁻CD25⁻CD44^loCD62L^hi) T cells FACS sorted from TCli TCRαβ Foxp3^IRES-GFPRag1^+/− Tg or TCli TCRβ Foxp3^Thy1.1 IFNγ^YFP IL-17A^GFPTcra^+/− mice in D10 in Th0 conditions (1 μg/ml anti-CD28 (BioXcell #BE0015–1), 10 μg/ml anti-TGFβ (BioXcell #BE0057), 5 μg/ml anti-IL-12 (BioXCell #BE0052), 5 μg/ml anti-IFNγ (BioXCell #BE0054), 5 mg/ml anti-IL4 (BioXCell #BE0045)). TCRα chain IRES-Thy1.1 retroviral transduction was performed as described (Hsieh et al., 2004 (link)) using TransIT-293 (Thermo Fisher #MIR2700). Transduced cultures were left to rest in Th0 media for ~66 hours. Foxp3⁻ TCRα-transduced Thy1.1+ cells were sorted by FACS and 2×10⁴ sorted cells were retro-orbitally injected into each host for in vivo analysis unless otherwise specified. Cells were stained with CTV (Thermo Fisher #C34571) in some experiments. Transferred cells were identified as CD4⁺ Vβ6⁺ Thy1.1⁺. For transcription factor staining experiments, 10⁵ cells were injected per mouse to increase recovery. Transcription factors were stained using the Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher #00–5523-00).

Russler-Germain E.V., Jung J., Miller A.T., Young S., Yi J., Wehmeier A., Fox L.E., Monte K.J., Chai J.N., Kulkarni D.H., Funkhouser-Jones L.J., Wilke G., Durai V., Zinselmeyer B.H., Czepielewski R.S., Greco S., Murphy K.M., Newberry R.D., Sibley L.D, & Hsieh C.S. (2021). Commensal Cryptosporidium colonization elicits a cDC1-dependent Th1 response that promotes intestinal homeostasis and restricts other infections. Immunity, 54(11), 2547-2564.e7.

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Co-culture of Tumor Cells and T Cells

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CD4⁺ T cells were isolated as described above. MC38 or Hepa1-6 tumor cells were harvested from culture, along with the conditioned medium. The tumor cells were treated with 50 µg/mL mitomycin-c for 30 min at 37 °C. Tumor cells were then washed twice with medium, and co-cultured with T cells at a 1:2 ratio (50 K tumor cells to 100 K T cells) and 50 K anti-CD3/CD28 Dynabeads. The tumor conditioned medium was transferred back to the coculture, supplemented with HEPES, L-glutamine, MEM, b-mercaptoethanol, and IL-2. T cells were analyzed by flow cytometry after 4 days. In experiments with anti-TGFβ blockade, anti-TGFβ (Bioxcell) or isotype control was added into culture at 20 µg/mL.

John P., Pulanco M.C., Galbo PM J.r., Wei Y., Ohaegbulam K.C., Zheng D, & Zang X. (2022). The immune checkpoint B7x expands tumor-infiltrating Tregs and promotes resistance to anti-CTLA-4 therapy. Nature Communications, 13, 2506.

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Anti tgf β

Lab products found in correlation

9 protocols using anti tgf β

Generation and Validation of Tr1 Cells

Adoptive Transfer of Tolerant B Cells in Islet Transplantation

T Cell Differentiation and Expansion Protocols

Generation and Validation of Tr1 Cells

T Cell Differentiation and Expansion Protocols

In Vitro Differentiation of Tfh Subsets

Nonpolarizing CD4+ T Cell Activation

In Vitro Activation and Adoptive Transfer of Naïve T Cells

Co-culture of Tumor Cells and T Cells

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