Cd45ro apc cy7

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.

All flow cytometry reagents were from Biolegend unless otherwise stated. Data was acquired using a LSR-II flow cytometer (BD Biosciences) and analysed using FACS DIVA software (BD Biosciences); Fluorescence Minus One (FMO) controls established gating strategies. Figures were generated using FlowJo software (Tree Star Inc.). Immune cells were further purified by separation by density gradient centrifugation over lymphoprep (Axis Shield Diagnostics) then cells were washed twice with PBS/2% FCS and consecutive centrifugation of 800 × g and 200 × g for 10 minutes.
For flow cytometry, cells were incubated with antibodies for 20 minutes at 4 °C, washed ×2 in PBS/2% FCS and fixed in Fluorofix prior to data acquisition. Antibodies used were CD4-FITC(OKT4), CD8a-PE(HIT8a), CD3-PeCy5(UCHT1), CD56-PeCy7(BD Biosciences; B159), CD16-APCCy7(BD Biosciences; 3G8), CD45-APC(HI30), CD103-FITC(BER-ACT8), CD69-PE(FN90), CD8a-PECy7(RPA-T8), CD45RO-APC-Cy7(UCHL1), CD127-AlexaFluor647(A019D5), PD-1(CD279)-APC(EH12.2H7).

PB containing the anticoagulant sodium heparin was centrifuged at 1,500 rpm for 5 min at room temperature. Red blood cells were lysed by Red Blood Cell Lysis Buffer (Sigma), and the white blood cells were pelleted at 300g for 3 min. Flow cytometric analysis was performed using the LSR FORTESSA device (BD Biosciences, San Jose, CA, USA). The samples were incubated with the following antibodies to identify T and regulatory T (Treg) cells: anti-human CD3 FITC (BD Biosciences), anti-human CD4 Pacific Blue (BD Biosciences), anti-human CD8 APC (BD Biosciences), anti-human CD45 PE (BD Biosciences), IFNγ-APC (BioLegend), CD45RA- PE (BD Biosciences), CD45RO-APC-cy7 (BD Biosciences), anti-human FOXP3 PE (BD Biosciences), CFSE FITC (BD Biosciences), CD25 PE (BD Biosciences), CD127 PE-cy7 (BD Biosciences), CD56 PE (BD Biosciences), and HLA-DR PE-cy7 (BD Biosciences). The following controls were used: unstained cells and single-stained cells; and dead cells, which in conjunction with AutoComp software were used to set accurate compensation and data analysis. Cells were counted per sample, and the data were analyzed with FlowJo V10.