Cytometric Profiling of T Cell Subsets

Peripheral blood mononuclear cells (PBMC) were isolated from fresh blood by density-gradient centrifugation on Ficoll-Paque (GE Healthcare), with single cell suspensions stained with the following antibodies: APC-H7-conjugated anti-CD3 (clone SK7), PE-Cy5-conjugated anti-CD45RA (clone HI100), PE-conjugated anti-CD25 (clone M-A251), PE-Cy7-conjugated anti-PD-1 (clone EH12.1), Alexa Fluor 488-conjugated anti-CXCR5 (clone RF8B2) and V450-conjugated anti-CCR7 (clone 150503) (all from BD Bioscience), and Alexa Fluor 700-conjugated anti-CD4 (clone OKT4; from eBioscience). Stained cells were sorted into naive (CD3⁺CD4⁺CD45RA⁺CCR7^hi PD-1^lo), CXCR5⁺ central memory (Tcm: CD3⁺CD4⁺CD45RA⁻ CXCR5⁺ CD25^lo CCR7^highPD-1^low), and circulating Tfh (cTfh: CD3⁺CD4⁺CD45RA⁻ CXCR5^hi CD25^lo CCR7^low PD-1^high) by FACS Aria (BD biosciences), with exclusion of doublets by forward and side scatter.
For sequencing experiments, cells were loaded onto microwell arrays and sequenced as described above at an average depth of ∼20 000–40 000 reads per cell. Cells were filtered based on >10 000 reads, >1000 transcripts and >400 genes, and analyzed using Seurat package (25 ,26 (link)).