Rhil 2

ATO-derived SP8 T cells were isolated as described above and expanded in vitro using irradiated antigen-expressing K562 aAPCs (K562 CD80-CD83-CD137L and HLA-A*02:01-B2M-NYESO_157–165 SCT) in a 1:3 aAPC:T cell ratio in AIM V (Thermo Fisher Scientific) supplemented with 5% human AB serum (Gemini Bio-Products), 20 ng ml⁻¹ rhIL-2 (Peprotech) and 5 ng ml⁻¹ rhIL-7 (R&D Systems). Fresh medium was added every 2–3 days, and cells were replated into larger wells as necessary. Restimulations for proliferation assays were performed every 5 days. Before functional and proliferation assays, expanded SP8 T cells were rested for an additional 2 days after the previous expansion cycle (7 days from aAPC stimulation) with reduced cytokines, 5 ng ml⁻¹ rhIL-2 (Peprotech) only, on the night before the assays.

For Th0 conditions, FACS-sorted naive CD4⁺ T cells were stimulated (day 0) with plate-bound anti-CD3ε (1 μg/mL; AB_394590; BD Biosciences) and anti-CD28 (3 μg/mL; AB_394763; BD Biosciences) on 48-well plates (0.2 × 10⁶ cells/well) in 1 mL T cell medium/well (RPMI1640 supplemented with 10% FCS [MilliporeSigma/Biowest], antibiotics, 50 mM β-ME) supplemented with 20 U/mL recombinant human IL-2 (rhIL-2, PeproTech) for 3 days, unless otherwise stated. Th1 cells were generated from sorted naive CD4⁺ T cells activated with anti-CD3ε/anti-CD28 in the presence of 20 U/mL rhIL-2 (PeproTech), 5 ng/mL IL-12 (R&D Systems), and 3 μg/mL anti–IL-4 (BioXcell) for 3 days. For assessment of cell proliferation, naive CD4⁺ T cells were labeled using Cell Proliferation Dye eFluor 450 (Thermo Fisher Scientific) according to the manufacturer’s protocol before activation. Cells were harvested and analyzed on day 3 unless otherwise indicated. For cytokine detection, activated cells were restimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA, 25 ng/mL) and ionomycin (Iono, 750 ng/mL), both from MilliporeSigma, in the presence of GolgiStop (BD Biosciences). CD8⁺ T cells were activated and cultured with anti-CD3ε/anti-CD28 as described above in the presence of 40 U/mL rhIL-2.

Peripheral blood mononuclear cells (PBMCs) were obtained from freshly heparinized venous blood from healthy adult peripheral blood by density gradient centrifugation using human lymphocyte isolation solution (TBD science, Tianjin, China). PBMCs (1 × 10⁶/mL) were seeded in 24-well culture plates (Labselect, Hefei, China) in complete RPMI-1640 medium (1 mL/well) (Gibco, USA), supplemented with 10% (v/v) heat-inactivated foetal bovine serum (FBS, EVERY GREEN, Hangzhou, China), 100 U/mL penicillin and 100 µg/mL streptomycin and cultured at 37 °C in 5% CO₂. rhIL-2 (50 U/mL) (PeproTech, USA), HDMAPP (5 μg/mL), Mtb-HAg (5 μg/mL), GroES (5 μg/mL), GroEL1 (5 μg/mL), HspX (5 μg/mL), GroEL2 (5 μg/mL), HtpG (5 μg/mL), DnaK (5 μg/mL), HbhA (5 μg/mL), Mpt63 (5 μg/mL), EsxB (5 μg/mL), and EsxN (5 μg/mL) were added to each well, and rhIL-2 (50 U/mL) was added to each well every 3–4 days.

To test the proliferating ability of Tregs, Tregs were isolated from the PBMCs by flow cytometry and then labelled with CFSE at room temperature for 1 hour. The anti‐CD3/28 antibodies were pre‐coated in the U‐bottom plate, followed by addition of rhIL‐2 (50 ng/mL, Peprotech, Rocky Hill, NJ). After incubation for 7 days, the cells were harvested and the proliferation rates of Tregs were analysed by flow cytometry through the signalling of CFSE. To test the regulatory activity of Tregs, 3.5 × 10⁴ CFSE‐labelled (2 μmol/L, Invitrogen, Life Technologies) CD8⁺ T cells were firstly seeded in 96‐well plates in 200 μL RPMI medium containing rhIL‐2, and then, the unlabelled CD4⁺CD25^high regulatory T cells isolated from patients or healthy controls were added into each well at a mixing ratio of 1:1. After co‐culturing for 7 days, the proliferation rate of CD8⁺ T cells was analysed by FACS through signalling of CFSE. The results were calculated by FlowJo software version 7.6.1 (Tree Star, Ashland, OR).

Purified CD19⁺ lymphocytes (1 × 10⁵ cells/well) were cultured in 96‐well plates in 50 μL medium (RPMI, 5%FBS) supplemented with 50 ng/mL rhIL‐10 (Miltenyi Biotec), 20 U rhIL‐2 (PeproTech), 0.1 μg/mL CD40L (Enzo Life Science) and 25 μg/mL anti‐human IgM F(ab’)₂ (Jackson ImmunoResearch). Purified CD4⁺ lymphocytes (2.5 × 10⁵‐3.5 × 10⁵ cells/well) were cultured for 36 h with rhIL‐2 (PeproTech, 50U/mL) at 37°C, 5% CO₂ in 48‐well plates that had been coated overnight with anti‐CD3 (2 μg/mL) (BioLegend, clone OKT3) +/‐anti‐CD46 (2 μg/mL) (R&D Systems, clone 344519). To assess B‐cell activation, 100 μL of T‐cell supernatant was added to B cells and cultured for 5–10 days at 37°C, 5%CO₂. To block the activity of IL‐10, a neutralizing mAb to IL‐10 (2.5 μg/mL) was used. To restore B‐cell proliferation in mixtures containing T‐cell supernatants rhIL‐10 (1 μg/mL) was added.

CD4⁺CD44^loFoxp3⁻ naive T cells from Foxp3-Tocky mice were isolated by cell sorting, and 10⁵ cells were cultured on anti-CD3 (clone 1452C11; 2 µg/ml; eBioscience) and anti-CD28 (clone 37.51; 10 µg/ml; eBioscience)–coated 96-well plates (Corning) in the presence of 100 U/ml rhIL-2 (Roche) and 2 ng/ml rhTGFβ (R&D) for 0–48 h in a final volume of 200 µl RPMI 1640 (Sigma-Aldrich) containing 10% FCS and penicillin/streptomycin (Thermo Fisher Scientific).
Mature Foxp3⁺ Treg cells from Foxp3-Tocky mice were isolated by cell sorting, and 10⁵ cells were cultured on anti-CD3 (clone 145.2C11; 2 µg/ml) and anti-CD28 (clone 37.51; 10 µg/ml) –coated 96-well plates in the presence of 100 U/ml rhIL-2 for 0–48 h in a final volume of 200 µl RPMI 1640 containing 10% FCS and penicillin/streptomycin.