Lsrii instrument

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient (Sigma) and were washed once with FACS buffer (PBS, with 1% bovine serum albumin). For staining, 1 × 10⁶ cells were incubated with conjugated antibodies APC-cy7-conjugated anti-CD3, PerCP-conjugated anti-CD8 (BD Biosciences Pharmingen), PE-conjugated anti-Tim-3, and FITC-conjugated anti-PD-1 (Biolegend) for 30 min at 4°C. Analysis was performed using an LSRII instrument (BD Biosciences), and at least 10,000 events were collected. Tim-3 and PD-1 expression on CD4+ (defined as CD3+CD8− cells) and CD8+ T cells were analyzed with FACSDiva software (BD Biosciences). For the detection of cytokine-induced Tim-3 and PD-1 expression, freshly isolated PBMCs were plated in round-bottom 96-microtiter plates at 0.5 million cells/well in 200 μL complete RPMI 1640 containing anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) antibodies (BD Biosciences) or recombinant IL-2, IL-7, IL-15, and IL-21 (25 ng/mL) (IL-2, IL-7, and IL-15: R&D Systems; IL-21: Biosource). Cells were cultured for five days and were incubated with conjugated antibodies against PerCP-conjugated anti-CD3, APC-cy7-conjugated anti-CD4 (BD Biosciences Pharmingen), PE-conjugated anti-Tim-3, and FITC-conjugated anti-PD-1 for 30 min at 4°C. Analysis was performed using an LSRII instrument and analyzed with FlowJo software.

Cells were stained with specific antibodies or isotype controls (Table S8) in 96-well plates on ice and analyzed by flow cytometry using a BD LSR II instrument with high-throughput sampler and evaluated using FlowJo V10 software.