Interleukin 2 (il 2)

Naive CD4⁺CD25⁻CD62L^hiCD44^lo T cells from spleens and lymph nodes were isolated using a FACSAria sorter (BD Biosciences). Purified naive T cells were stimulated with 2 μg/mL plate-bound anti-CD3 (2C11, Bio X Cell) and 2 μg/mL anti-CD28 (37.51, Bio X Cell) in the presence of 5 μg/mL anti–IFN-γ (XMG1.2, Bio X Cell), 5 μg/mL anti–IL-4 (11B11, Bio X Cell), and 40 U/mL IL-2 (Peprotech) for the generation of Th0 cells; 20 ng/mL IL-12 (Peprotech), 5 μg/mL anti–IL-4, and 40 U/mL IL-2 for the generation of Th1 cells; 10 ng/mL IL-4 (Peprotech), 10 μg/mL anti–IFN-γ and 40 U/mL IL-2 for the generation of Th2 cells; 1 ng/mL TGF-β1 (Peprotech), 10 ng/mL IL-6 (Peprotech), 5 μg/mL anti–IFN-γ, and 5 μg/mL anti–IL-4 or 10 ng/mL IL-23, 10 ng/mL IL-1β (Peprotech), 10 ng/mL IL-6, 5 μg/mL anti–IFN-γ, and 5 μg/mL anti–IL-4 for the generation of Th17 cells; and 1 ng/mL TGF-β1, 5 μg/mL anti–IFN-γ, 5 μg/mL anti–IL-4, and 40 U/mL IL-2 for the generation of iTregs. Cells were cultured in complete medium (RPMI medium containing 10% FBS, supplemented with penicillin-streptomycin, HEPES, l-glutamine, sodium pyruvate, and 2-mercaptoethanol) for 3–5 days, followed by intracellular staining and RNA preparation.

Naïve CD4 T cells were enriched from the spleen of WT mice using MACS beads (130-104-453, Miltenyi Biotech) and cultured with plated-bound anti-CD3 (5 μg/ml) and soluble anti-CD28 (2 μg/ml) (eBioscience). Cytokines and neutralizing antibodies were added at the following concentrations: (Th1) 5 ng/ml IL-2, 10 ng/ml IL-12 (Peprotech), 10 μg/ml anti-IL-4 (ebiosciences); (Th17) 5 ng/ml IL-2, 5 ng/ml TGF-β, 10 ng/ml IL-6 (Peprotech); for Tr1 conditions, only 5 ng/ml IL-2 was added. Enriched CD4 T cells (1×10⁵) were plated in the bottom compartment of the cell culture wells while sorted CD1d^hiCD5⁺ B cells (1×10⁵) were cultured in the trans-well inserts (0.4 μm pore size; Corning). After 3 days, CD4⁺ T cells were stimulated for 5 hours with PMA and ionomycin in the presence of monensin for intracellular cytokine staining.

Purified CD4⁺ T cells from Itpkb^+/+ and Itpkb^fl/fl mice were stimulated at a 1:1 ratio with anti-CD3/28 beads in Th1- or Th2-skewing conditions. Th1 skewing conditions were anti-IL-4 (eBioscience, 5μg/ml), IL-12 (R&D Systems, 10ng/ml) and IL-2 (Peprotech, at 5ng/ml); Th-2 skewing conditions were IL-4 (R&D Systems, 5ng/ml), anti-IFNγ (eBioscience, 5μg/ml), anti-IL-12 (eBioscience, 4.25μg/ml) and IL-2 (5ng/ml); Cell were cultured at 37°C, for 6 days in the presence of exogenous IL-2 (5ng/ml). On day 6, 100K cells restimulated with anti-CD3/28 beads (~1:1 ratio) for 6 h at 37°C. The final 2h included Golgi stop (BD) and Golgi plug (BD). Cells were then surface stained with anti-CD4- APC-Cy7 (BioLegend), fixed, permeabilized, stained intracellularly with anti-IFNγ-APC, anti-IL4-PE (BioLegend), and anti-IL2-PE-Cy7 (eBioscience), and then run on the flow cytometer.

Control T cells or PD-1 KO T-cells post transfection were cultured in AIM-V medium supplemented with IL-2 (300 U/ml, Peprotech) and half replaced by fresh complete medium containing IL-2 every 2 ~ 3 d until analyzes. For antigen stimulated T cells. Mature DCs were pulsed by peptide (25 μg/mL) for 2 ~ 3 h at 37°C, washed with pre-warmed PBS and then incubated with control T cells or PD-1 KO T-cells at a ratio of 1:10 in complete AIM-V medium supplemented with IL-2 (100 U/ml, Peprotech) in 6-well-plates (5 × 10⁶cells/well) on day 7 post electroporation. IL-7 and IL-15 (5 ng/mL, Peprotech) were added with fresh medium. For re-stimulation, autologous DCs were pulsed with peptide (25 μg/mL) for 2 h and added to the cultured cells for another 7 d. Fresh complete medium was added containing cytokines every 2 to 3 d until use for experiments.

Human naïve CD4⁺ T cells were activated and differentiated under certain conditions. Briefly, cells were cultured under the stimulation of precoated anti-CD3 antibody (2 μg/ml, Sigma-Aldrich, USA) and anti-CD28 antibody (1 μg/ml, Sigma-Aldrich) for the desired time with the intention of activation. In addition to anti-CD3 and anti-CD28 antibodies, human naïve CD4⁺ T cells were cultured under the following polarization conditions for cell differentiation: anti-IL-4 antibody (10 μg/ml, Peprotech, USA), IL-12 (10 ng/ml, Peprotech), IL-2 (5 ng/ml, Peprotech) for Th1 polarization; anti-IFN-γ antibody (10 μg/ml, Peprotech), IL-2 (5 ng/ml), IL-4 (25 ng/ml, Peprotech) for Th2 polarization; anti-IFN-γ antibody (10 μg/ml), anti-IL-4 antibody (10 μg/ml), IL-6 (25 ng/ml, Peprotech), TGF-β (5 ng/ml, R&D System, USA), IL-1β (12.5 ng/ml, Peprotech), IL-21 (20 ng/ml, R&D System), IL-23 (25 ng/ml, Peprotech) for Th17 polarization; IL-6 (20 ng/ml), IL-21 (20 ng/ml), IL-12 (10 ng/ml), TGF-β (5 ng/ml) for Tfh polarization, IL-2 (10 ng/ml), TGF-β (5 ng/ml) for Treg polarization.

Naïve CD4⁺ T cells (1 × 10⁶) were activated with soluble α-CD3 (clone 2C11, 2 μg/mL) in the presence of irradiated WT splenocytes (5 × 10⁶) and cultured for 5 days in lymphocyte culture media. In some experiments, whole-splenocyte suspensions were depleted of CD4⁺ and CD8⁺ T cells using α-mouse CD4 (clone L3T4) and α-mouse CD8 (clone Ly-2) microbeads (Miltenyi) followed by magnetic-activated cell sorting. For pathogenic Th17 differentiation, culture media was further supplemented with 20 ng/mL IL-6 (Miltneyi), 10 ng/mL IL-1β (Miltenyi), 10 ng/mL IL-23 (R&D Systems), 10 μg α-IFNγ mAbs (clone XMG1.2, BioXCell) and 10 μg/mL α-IL-4 mAbs (clone 11B11, BioXCell). For Th0 differentiation, culture media was further supplemented with 200 U/mL IL-2 (Peprotech). For Th1 differentiation, culture media was further supplemented with 200 U/mL IL-2, 10 ng/mL IL-12 (Peprotech) and 10 μg/mL α-IL-4 mAbs (clone 11B11, BioXCell). For Th2 differentiation, culture media was further supplemented with 200 U/mL IL-2, 10 ng/mL IL-4 (Peprotech) and 10 μg/mL α-IFNγ mAbs (clone XMG1.2, BioXCell). For iTreg differentiation, naïve CD4⁺ T cells were stimulated with 10 μg/mL soluble α-CD3 (clone 2C11, BD Pharmingen) and culture media was further supplemented with 100 U/mL IL-2 and 5 ng/mL TGF-β.