T cell subsets.

The following mAbs were added to 100 μL of whole blood: anti-CD45 (catalog 652803), anti-CD3 (catalog 663490), anti-CD4 (catalog 560345), anti-CD8 (catalog 335822), anti-CD28 (catalog 662797), anti–HLA-DR (catalog 652809), anti-CD45RA (catalog 662840), anti-CD45RO (catalog 663530), anti-CD25 (catalog 662525), anti-CD127 (catalog 560822), anti–PD-1 (catalog 557860), and anti–Tim-3 (catalog 565558) (all from BD Biosciences). Different T cell subsets were defined as following: costimulatory molecule (CD28⁺CD4⁺ or CD28⁺CD8⁺ T cells), activated T cells (HLA-DR⁺CD4⁺ or HLA-DR⁺CD8⁺ T cells), naive/memory CD4⁺ T cells (CD45RA⁺/CD45RO⁺ CD4⁺ T cells), Tregs (CD25^hiCD127^loCD4⁺ T cells), and natural Tregs (CD45RA⁺ Tregs). PD-1 and Tim-3 were also used as activation markers of T cells. Gating strategies of T cell subsets are shown in Supplemental Figure 1, A, B, and F.