Multiparametric Immune Cell Profiling

We analyzed and sorted all samples for single-cell and NGS analyses by MoFlo XDP (Beckman Coulter)60 (link)61 (link). Cell staining was performed by CXCR5-FITC (clone: RF8B2, BD Pharmingen), CXCR3-PE (clone: 1C6, BD Pharmingen), CD4-PerCP-Cy5.5 (clone: OKT4, Biolegend), CD3-PE-Cy7 (clone: VCHT1, Biolegend), CCR6-APC (clone: 11A9, BD Pharmingen), and CD45RO-APC-Cy7 (clone: UCHL1, BD Pharmingen). The definitions of each subset in this study were as follows: CD3⁺CD4⁺CD45RO⁺ for memory CD4⁺ T cells, CD3⁺CD4⁺CD45RO^- for naive CD4⁺ T cells, CD3⁺CD4⁺CD45RO⁺CXCR5⁺ for follicular helper T cells (Tfh), CD3⁺CD4⁺CD45RO⁺CXCR5⁻CXCR3⁺CCR6⁻ for Th1, CD3⁺CD4⁺CD45RO⁺CXCR5⁻CXCR3⁻CCR6⁺ for Th17, and CD3⁺CD4⁺CD45RO⁺CXCR5⁻CXCR3⁻CCR6⁻ for non-Th1/Th17/Tfh. We followed standard immunophenotyping approach of human immune cells based on chemokine receptors, which is now becoming widely accepted47 (link). The total count of sorted cells for the NGS analysis was listed in Table S2.