Anti il 4

Spleens were harvested from adult mice and red blood cells were lysed with ammonium chloride. CD4⁺ T cells were enriched using magnetic-activated cell sorting (Miltenyi Biotec) and live (DAPI⁻) CD4⁺CD25⁻CD62L⁺CD44^lo/− naive T cells were subsequently FACS-sorted to a purity of >98%. Cells were then cultured during 3 days in 48-well plates (5 × 10⁵cells per well) coated with 5 μg/mL anti CD3ε (145-2C11, BD Biosciences) in complete DMEM medium with GlutaMAX (Invitrogen) with 2 μg/mL soluble anti CD28 (37.51, BD Biosciences). Cultures were supplemented as follows. Th1: IL-12 (2 ng/mL, PeproTech) + neutralising anti IL-4 (2 μg/mL, eBioscience); Th2: IL-4 (2 ng/mL, eBioscience) + neutralising anti IFNγ (2 μg/mL, eBioscience); Th17: IL-6 (20 ng/mL, Sigma) + TGF-β (0.3 ng/mL, R&D systems) + neutralising anti IFNγ and anti IL-4; iTreg: TGF-β (5 ng/mL) + neutralising anti IFNγ and anti IL-4. Gene expression in Th1, Th2, Th17 and iTregs was compared to immature bone marrow-derived DCs generated as previously described13 (link).

PBMCs were isolated from pSS patients and HCs using Ficoll–Paque gradient centrifugation. CD4⁺CD45RA⁺ naïve T cells were purified from PBMCs using Naïve CD4⁺ T-Cell Isolation Kit II (Miltenyi Biotec, Germany) according to the instructions of the manufacturer, with a purity of more than 95% by flow cytometry. Naïve CD4⁺ T cells were activated with anti-CD3 (5 µg/ml, BD Bioscience) and anti-CD28 (5 µg/ml, BD Bioscience). For the T-cell differentiation, IL-12 (10 ng/ml, R&D systems) plus anti-IL4 (10 µg/ml, PeproTech), IL-4 (2 ng/ml, PeproTech) plus anti-IFNγ(10 µg/ml, BD Bioscience), anti-IL4 (10 µg/ml) plus anti-IFNγ(10 µg/ml) plus TGF-β(5 ng/ml, R&D systems) plus IL-1β(12.5 ng/ml, PeproTech) plus IL-6 (25 ng/ml, PeproTech) plus IL-23 (25 ng/ml, PeproTech), TGF-β1 (5 ng/ml, R&D systems) plus IL-12 (1 ng/ml, R&D systems), or IL-2 (5 ng/ml, R&D systems) plus TGF-β (5 ng/ml, R&D systems) were supplemented for Th1, Th2, Th17, Tfh or Treg differentiation, respectively. T cells were incubated in RPMI-1640 medium (Thermo Fisher) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and at 37°C, 5% CO2 for 3–5 days.

Cells were stimulated via CD3/CD28 and cultured under Th17-polarizing conditions (IL-1β (10 ng/ml), IL-6 (50 ng/ml), and IL-23 (40 ng/ml), and anti-IL-4 (1 μg/ml) and anti-IFN-γ Abs (10 μg/ml) (R&D Systems)) or Th1-polarizing conditions (IL-12 (5 ng/ml) (R&D Systems) and anti-IL-4 Abs (1 μg/ml)) for 4 days. Cells were washed and plated in media containing the Th17- or Th1-polarization cocktails together with recombinant IL-2 (5 ng/ml) for 10 additional days. Polarizing cytokines were replenished every 2–3 days and cells were split to optimal density (<2 × 10⁶ cells/ml).

PBMCs were isolated from peripheral blood using Ficoll density-gradient centrifugation. Treg cells were obtained as CD4⁺CD25⁻T cell subsets, and naïve CD4⁺ T cells were isolated and purified using a naïve CD4⁺ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. Then, CD4⁺CD25⁻ T cells (1 × 10⁶/well) were cultured with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) antibodies, with the addition of recombinant human TGF-β1 (10 ng/ml; R&D Systems, USA) and IL-2 (100 U/ml; Peprotech, USA) to induce Treg cell conversion. After culturing for 5–6 days, the cells were collected for the measurement of CD4⁺CD25⁺ percentages by flow cytometry.
Tfh cells were obtained as naive CD4⁺ T cells (1 × 10⁶/well) and stimulated with soluble anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) antibodies, with the addition of recombinant human IL-2 (100 U/ml; Peprotech, USA), IL-6 (20 ng/ml; Peprotech, USA), anti-IL-4 (10 μg/ml; R&D Systems, USA), anti-IFN-γ (10 μg/ml; R&D Systems, USA) and anti-TGF-β (10 μg/ml; R&D Systems, USA). After 5–6 days, the cells were collected for the measurement of CXCR5⁺⁺PD-1⁺⁺CD4⁺ T cell or CXCR5^{+ +}PD-1⁺⁺ Foxp3⁺CD4⁺ T cell percentages by flow cytometry.

FACS-sorted Th-like Tregs (0.5–1 × 10⁵), total memory Tregs (0.5 × 10⁵) and Teffs (1 × 10⁵) were stimulated with anti-CD3/CD28 beads at a 4:1 (cell/bead) ratio in the absence or presence of the neutralizing antibodies anti-IL-2 (10 μg/mL, BioSource International), anti-IL-4, anti-IFN-γ, or anti-IL-17 (all 10 μg/mL, R&D Systems) or in the presence of different concentrations of exogenous IL-2 (Novartis). After 16 hr, STAT5 and p53 were evaluated using western blot. After 72 hr, viability and apoptosis were evaluated using the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit and Annexin V, and supernatants were used to detect human T cell cytokine production.

Mouse splenocytes were incubated in 24 well plates coated with 1 μg/mL anti-CD3 and 1 μg/mL soluble anti-CD28. Tr1 inducing conditions: 50 ng/mL of IL-27 (R&D systems, Minneapolis, MN) was added according to previously described protocols [30 (link)]. Th1 inducing conditions: 20 ng/mL IL-2, 20 ng/mL IL-12, and 10 μg/mL anti-IL-4 (R&D systems, Minneapolis, MN) was added. Th0 inducing conditions: no added cytokine. After 5 days, cells were counted and resuspended at 1x10⁶ cells/mL in 75 % fresh cell culture media (without cytokines) to rest for 2 days.
Restimulation of mouse CD4⁺ T cells: Rested cells were plated at 1 x 10⁶ cells/mL in fresh media in 96 or 24 well plates coated with 5 μg/mL anti-CD3 with a brief spin (200 x g for 2 min) to ensure contact between cells and plate bottom. Alternatively, 100 ng/ml PMA was added to wells. At indicated time points, cells were collected, put on ice for 3 min and centrifuged at 300 x g for 5 min at 4 C. Supernatants were stored at -80 C until cytokine analysis by ELISA. For production of ROS, cells were coated with anti-CD3 [10 μg/mL] (BD Biosciences, Franklin Lakes, NJ) on ice and restimulated by addition of anti-hamster IgG [10 μg/mL] in warm media according to previously described protocols [43 (link)].