Anti cd28 37

Antibodies against the following proteins were purchased from BD Biosciences: CD4 (RM4-5), CD69 (H1.2F3), CD90.1 (OX-7), phospho-STAT5^Y694 (C71E5), and CD90.2 (53-2.1). Antibodies against the following proteins were purchased from eBioscience: CD44 (IM7), CD98 (RL388), ICOS (7E.17G9), IRF4 (3E4), Ki-67 (SolA15), CD39 (24DMS1), KLRG1 (2F1), and FoxP3 (FJK-16s). Antibodies against the following proteins were purchased from BioLegend: CD4 (RM4-5), CD45 (30-F11), CD62L (MEL-14), CTLA-4 (UC10-4F10-11), PD-1 (29F.1A12), CD25 (PC61), and Bcl2 (BCL/10C4). Normal rabbit IgG (2729) and anti-phospho-S6^S240/244 (5364) were purchased from Cell Signaling Technology. Goat anti-rabbit-Alexa Fluor 647 secondary antibody was purchased from Invitrogen. Fc Block (2.4G2) and anti-CD28 (37.51) were purchased from Bio X Cell. Stimulatory anti-CD3 (2C11) was purified from hybridoma supernatants prepared in-house. Fixable viability dye eFluor780 was purchased from eBioscience. MitoTracker Deep Red dye was purchased from Invitrogen. Flow cytometry experiments were performed on a FACSCalibur, LSR II, or FACSCelesta (BD Biosciences), and analyzed using FlowJo software (Treestar, v.10.3) or FCS Express (De Novo Software, v.6). Cell sorting was performed on a FACSAria II or FACSAria Fusion (BD Biosciences).

Naïve (CD4⁺CD25⁻CD44^lowCD62L^high) T cells were sorted from the peripheral lymph nodes and spleens of mice. Cells were then activated either in plates coated with 10μg/ml anti-CD3 (145-2C11, BioXCell) and 10μg/ml anti-CD28 (37.51, BioXCell) or by soluble 1μg/ml anti-CD3 and irradiated (3000 cGy) T-cell-depleted splenocytes. Cells were cultured in RPMI medium with 10% FBS and 1% antibiotics unless specifically indicated. Cells were cultured in the presence of 20μg/ml anti-IFN-γ (XMG1.2, BioXcell) and 20μg/ml anti-IL-4 (11B11, BioXcell). For IL-6+TGF-β condition, cells were cultured in the presence of 40ng/ml recombinant IL-6, 1ng/ml TGF-β1 (R&D systems), 20μg/ml anti-IFN-γ and 20μg/ml anti-IL-4. For IL-6 condition, cells were cultured in the presence of 40ng/ml recombinant IL-6, 20μg/ml anti-IFN-γ, 20μg/ml anti-IL-4. 100ng/ml Recombinant Human Activin A (Biolegend) was used in indicated conditions. 10μM TGFβR inhibitor SB525334 (Selleckchem) was added into culture medium with indication of “i”. For retroviral transduction, CD4⁺ T cells were isolated and cultured under various conditions on day 0 and then spin inoculated with indicated retroviruses at 1500g at 30°C for 1.5 hours on day 1. Cells were harvested and analyzed by flow-cytometry on day 4 unless stated otherwise in the figure legends.

Naïve (CD62L^highCD44^low) T cells were sorted from the peripheral lymph-nodes and/or spleens of mice. Cells were then activation by stimulation via the TCR by anti-CD3 (145-2C11; BioXCell) and anti-CD28 (37.51; BioXCell). For proliferation assays, cells were labeled with CFSE (carboxyfluorescein diacetate succinimidyl ester) or CellTrace Violet (BD Biosciences) and then cultured under the appropriate conditions. Proliferation was assessed by the dilution of live dye with flow-cytometry 72–96 hours after T cell activation. Th1 cells were differentiated in the presence of 20ng/ml rIL-12 (R&D systems) and 20μg/ml anti-IL-4 (11B11, BioXcell). Th2 cells were differentiated in the presence of 20ng/ml rIL-4 (R&D systems) and 20μg/ml anti-IFN-γ (XMG1.2, BioXcell). Treg cells were differentiated in the presence of 2ng/ml rTGF-β1 (R&D systems). To assess the efficacy of Treg-mediated immune suppression in vitro, 2x10⁴ sorted CD4⁺CD25⁻CD45RB^hi responder T cells were labeled with CFSE and mixed with varying amounts (as indicated) of CD4⁺CD25⁺ Treg suppressor cells. Cell mixtures were stimulated with soluble anti-CD3 antibody (1μg/ml) in the presence of 1x10⁵ irradiated (3000 cGy) T-cell depleted splenocytes as APC. The proliferation of responder cells was assessed by CFSE dilution detected by flow-cytometry 72 hours post stimulation.