Hil 2

All cell lines were obtained from ATCC. Peripheral blood mononuclear cells were isolated from buffy coats and T cells were purified by a negative selection using a human T-cell enrichment kit (R&D systems). Freshly isolated T cells were maintained for 4 days in precoated flask with OKT3 (10 μg ml⁻¹; Abcam ab86883) and anti CD28 (10 μg ml⁻¹; Abcam ab25234) in complete RPMI 1640 medium supplemented with 30 U ml⁻¹ of h-IL2 (R&D systems). The day before the test, T cells were transferred in an uncoated flask. The purity of activated T cells was confirmed by FACS analysis. MCF-7 (EpCAM-positive) were treated with serial dilutions of CD3 × EpCAM BiAb or control antibodies together with activated T cells (E:T=5:1) overnight at 37 °C in a round-bottom 96-well plate. Released lactate dehydrogenase was detected with a cytotoxicity detection kit (Roche). Data were processed and percentages of specific lysis were calculated and analysed using GraphPad Prism6 software.

At day 19, splenocytes were isolated from mouse and stimulated by hIL‐2 (R and D systems, Minneapolis, MN) plus the inactivated MCSCs for 5 days. MCSCs served as target cells, and splenocytes served as effector cells. MCSCs and splenocytes were seeded and incubated together; the supernatant was collected after 4 h. The lactate dehydrogenase activity was analyzed by cytotox 96 non‐radioactive cytotoxicity assay (Promega, Madison, WI). Calculation of cytotoxic T lymphocytes assay (CTL) percentage was described in early study 8.

CD25^low-intCD127^highTh cells and CD25^int-highCD127^lowTregs were isolated from PBMCs using the BD FACSAria Cell Sorter (BD Biosciences) and cultured with 20 ng/ml recombinant human IL-2 (hIL-2; R&D systems). Survival was determined with DAPI Nucleic Acid Stain (Life Technologies)-negative cells. To measure Treg survival and apoptosis in co-culture with Th cells, CD4⁺ T cells were isolated from PBMCs using the human CD4⁺ T cell isolation kit (MACS, Miltenyi Biotec), cultured with 20 ng/ml hIL-2 and examined using the FITC Annexin V Apoptosis Detection Kit I (BD biosciences). To assess Treg apoptotic susceptibility, CD4⁺ T cells were cultured either with 20 ng/ml hIL-2 or with hIL-2 and 500 ng/ml soluble CD95L (recombinant human soluble FasL, Enzo Life Sciences).

Naive CD4⁺ T (CD44^lowCD62L^highCD25⁻) cells were purified using a CD4⁺ naive T cell negative isolation kit according to the manufacturer's protocol (STEMCELL Technologies). The in vitro differentiation experiments were performed as previously described. Naive CD4⁺ T cells were stimulated with immobilized anti-CD3 (5 μg/ml; 145-2C11; eBioscience) and anti-CD28 (5 μg/ml; 37.51; eBioscience) for 2 days. Then, the cells were washed and transferred to a new plate and further expanded in medium with hIL-2 (50 U/ml, R&D Systems) for 2 days. For Tfh-like cell differentiation, naive CD4⁺ T cells were activated with anti-CD3 and anti-CD28 as above and treated with 20 ng/ml IL-6 (R&D Systems), 20 ng/ml IL-21 (R&D Systems), 10 μg/ml anti-IL-4 (11B11, eBioscience), 10 μg/ml anti-IFN-γ (XMG1.2, eBioscience), and 20 μg/ml anti-TGF-β (1D11, R&D Systems) for 4 days.

Splenic CD4+ T cells from 4.1-NOD.Rag2^–/– or NOD mice were purified with CD4 (LT34) micro-beads (Miltenyi) and activated for 48 h with Dynabeads™ Mouse T-Activator CD3/CD28 (Gibco) in complete T-cell medium (RPMI-1640) (Hyclone) supplemented with 50 µM 2-mercaptoethanol (Sigma), 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin, and 30 IU/ml hIL-2 (R&D). After stimulation, dynabeads were removed and expansion was maintained for 6–7 days with 100 IU/ml hIL-2 in complete T-cell medium.
Expanded cells were harvested and cocultured, in a U-bottom 96-well plate, with freshly isolated and red blood cell-lysed 10⁵ NOD splenocytes and 0.0001–10 µg/ml peptide in complete T-cell medium supplemented with 30 IU/ml of hIL-2. Cell culture supernatants were collected after 48 h and IFNγ concentrations were determined by ELISA using ELISA MAX™ Deluxe Set Mouse IFN-γ (BioLegend) according to the manufacturer’s instructions. Synthetic soluble peptides (purity >80%) were obtained from GenScript^®.