T cell culturing, in vitro activation, and TCR titration

For T cell activation, sorted naïve CD4⁺ T cells were cultured at a concentration of 1.5 × 10⁶ cells/ml in T-helper 1 (T_H1) medium and activated with Dynabeads Human T-activator anti-CD3/anti-CD28 beads (Gibco, catalog no. 1131D). T_H1 medium is composed of complete RPMI [RPMI with GlutaMAX-I (Gibco), 10% (v/v) fetal bovine serum (Gibco), 1% (v/v) nonessential amino acids, 1 mM sodium pyruvate, penicillin (50 U/ml), and streptomycin (50 μg/ml)] supplemented with T_H1-specific cytokines [recombinant IL-2 (20 IU/ml; Miltenyi Biotec; catalog no. 130-097-744), recombinant IL-12 (10 ng/ml; Miltenyi Biotec; catalog no. 130-096-704), and neutralizing anti-IL-4 (2 μg/ml; Miltenyi Biotec; catalog no. 130-095-753)]. For the TCR titration assay, neonatal and adult naïve CD4⁺ T cells were cultured for 16 hours at a concentration of 1.5 × 10⁶ cells/ml in complete RPMI supplemented with IL-2 (20 IU/ml) in MaxiSorp Nunc-Immuno 96-well plates precoated with 2 μg of anti-CD28 (BioLegend, catalog no. 302933, RRID:AB_11150591) and different concentrations of anti-CD3 [i.e., 0.75, 1.5, or 3 μg of anti-CD3 (BioLegend, catalog no. 317325, RRID:AB_11147370)]. Cells were then evaluated by FACS (fluorescence-activated cell sorting) analysis for the expression of CD69, CD44, and IL2RA activation markers. For the assay involving naïve CD4⁺ T cells maintained in culture with autologous APC, with or without an antibody against MHC-II molecules, the procedures were as follows: (i) naïve T cells alone: FACS-sorted naïve T cells were used for subsequent analysis as freshly isolated naïve CD4⁺ T cells. (ii) Naïve T cells cultured with autologous APC: FACS-sorted naïve CD4⁺ T cells were cultured at a 1:3 ratio with FACS-sorted APC at a final concentration of 5.5 × 10⁶ cells/ml in complete RPMI. (iii) Naïve T cells cultured with autologous APCs plus anti-MHC-II antibodies: 2.5 × 10⁶ APC were incubated with 30 μg of α-MHC-II antibody in 300 μl of complete RPMI for 40 min at 37°C. After incubation, the excess antibody was washed out, and the APC were incubated with naïve T cells as described in point (ii). After coculture, naïve T cells were magnetic FACS-isolated to be subjected to subsequent analysis: for intracellular staining of phospho-Zap70 and phospho-ERK1/2, cells were cultured for 1 hour; for puromycin and cell cycle analysis, cells were cocultured for 1 hour, FACS isolated, and activated for 24 hours; last, for the RT-qPCR assay, cells were cocultured for 24 hours. T cells were maintained at 37°C in a 5% CO₂ humidified incubator.