Extended Data Fig. 9: Novel markers identify distinct human T_FH subpopulations across tonsil and PBMC.

a–c, scCITEseq surface protein expression of sorted CD3⁺CD4⁺CD45RA⁻CD45RO⁺CXCR5⁺CD27⁺ human tonsillar T_FH cells from three healthy adult donors (as in Fig. 7). scCITEseq surface protein expression (log counts) of (a) T cell identity markers, (b) chemokine receptors, and (c) cluster-identifying markers, overlaid onto scRNA-seq UMAP. d-h, CD3⁺CD4⁺CD45RA⁻CD45RO⁺CXCR5⁺ human T_FH cells from five PBMC samples and six tonsil samples from healthy adult donors. (d), Gating strategy to identify T_FH cells in human tonsil, adenoid, and PBMCs with PD-1⁺ expression in tonsil and PBMCs displayed in dot plots. (e) T_FH surface marker frequency in tonsil CD57⁺ or CD57⁻ T_FH cells (n = 6 samples per group). (f) CD151/CD99 T_FH populations (Q1-Q4) in T_FH cells distinguished by CD57 and PD-1 from tonsil (n = 6 tonsils per group) and PBMCs (n = 5 PBMCs per group). (g) CXCR3, CCR6, and CCR4 expression in CD57/PD-1 and CD151/CD99 T_FH populations with key to identify T_FH populations distinguished by CD57 and PD-1, and T_FH populations (Q1-Q4) distinguished by CD151 and CD99. (h) gMFI expression of T_FH markers (PD-1, ICOS, OX40), chemokine receptor markers (CXCR3, CCR6, CCR4), and novel markers (CD127, CD99, CD71, CD151, CD43, CD69) with (+) and without (-) 4 hr PMA and ionomycin stimulation. Data show experiments of 5 PBMC samples per group and 6 tonsil samples per group and mean ± SEM. Statistical tests: one-way ANOVA of multiple comparisons. **** P values are <0.0001 or otherwise indicated.

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