Characterization of Tfh, Tfr, and Treg cell populations. (A) Sorting strategy used for the transcriptome analysis of Tfh, Tfr, and Treg cell subsets. (B) Principal component analysis showing the distribution of Tfh, PD-1⁺ Tfr, PD-1^Neg Tfr, and Treg cells for the first two principal components. PC1 accounts for just over 50% of the variance whereas PC2 accounts for 20%. (C) Relative mRNA expression of BCL6, FOXP3, ICOS, and CTLA4 determined by RT-PCR for the sorted populations in A. Data are from nine subjects, but cells were not sufficient for analysis in all subsets for every subject. (D) Histograms showing the MFI for BCL6, FOXP3, ICOS, and CTLA-4; naive CXCR5⁻CD45RA⁺CD4⁺ T cells (gray histograms). (E) MFI for BCL6, FOXP3, ICOS, and CTLA-4 obtained by flow cytometry (n = 8 subjects). Naive CXCR5⁻CD45RA⁺CD4⁺ T cells were included as a baseline but were not included in the statistical analyses. Paired t tests; mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001.