The gating strategies for memory and naïve T cell panels (panel 6), T cell subsets, and CXCR3 and Ca5R expression (panel 7).

(A) For panel 6, the strategies of the first to fourth gates were the same as panel 4 (Fig 4). After gating on CD3+CD45+ T cells (G5) and then gating on CD8 T cells (G6) or CD4+ T cells (G7), CD45RA vs CCR7 and CD45RA vs CD62L identified naïve (CD45RA+CCR7+/CD62L+), central memory (CD45RA-CC7+/CD62L+), effector memory (CD45RA-CC7-/CD62L-) and terminal differentiated effector memory (TEMRA) (CD45RA+CC7-/CD62L-) CD4+ or CD8+ T cells. CD8 vs CD25 and CD4 vs CD25 can be used to estimate the ratio of CD8+CD25+, CD8+CD25+bright, CD4+CD25+ and CD4+CD25+bright T cells respectively. To identify Treg cells, the initial gate is on CD4+ T cells (G7), then evaluation of CD25 vs CD127 expression with the CD25+CD127-/dim population defined as Treg cells. Further gating on these Tregs (G8), and gating on CD45RA vs CCR7 and CD45RA vs CD62L separated CD45RA+CCR7+/CD62L+ naïve-Tregs from CD45RA-CCR7+/CD62L+ and CD45RA-CCR7-/CD62L- activated Tregs. (B) For panel 7, the first part focuses on CD3+ T cells. The first to fourth gates (G1-G4) were the same as in panel 6. After gating on CD3+CD45+ T cells (G5a), TCRαβ vs TCRγδ showed the proportion of TCRαβ and TCRγδ in CD3+ T cells. Gating on TCRγδ (G6a) and TCRαβ (G7a), CD4 vs CD8 showed the proportion of CD4+, CD8+, CD4-CD8- and/or CD4+CD8+ T cells on these two TCR cell-populations. Additionally, after gating on CD3+CD45+ T cells (G5a), then gating on CD8+ (G8a) or CD4+ (G9a) T cells on CD4 vs CD8, expressions of CXCR3 and C5aR on CD45RO+ and CD45RO- of CD8+ and CD4+ T cells were assessed on CD45RO vs CXCR3 or CD45RO vs C5aR respectively. In the second portion of the panel, the first gate was the same as panel 2 (Fig 3). After gating for granulocytes (G2b) (CD45+dimSSC-Ahigh), the third gate (G3b) was on FCS-W vs FSC-H to exclude double-cells, the percentage of CD16+bright cells on the histogram of CD16 represents neutrophils, and the MFI of CD16+bright cells was measured. After gating on neutrophils (G4b), the histogram of CD88 (C5aR) expression was assessed by MFI.