Splenic TFH phenotype and localization. (A) To determine splenic TFH percentage by FCM, CD3⁺CD4⁺ T cells were first gated within lymphocytes and then discriminated for CXCR5 expression. Within CXCR5⁺ T cells, the percentage of ICOS⁺PD-1^hi cells was measured. (B) The expression of different markers was assessed on CD3⁺CD4⁺CXCR5⁺ICOS⁺PD-1^hi (gray shaded histogram) and compared with CD3⁺CD4⁺CXCR5⁻ (full line) and for some markers with B cells (dashed lined). Representative histograms of 1 ITP patient are depicted; the dotted line represents isotype control. Cells were stimulated for 5 hours with phorbol-12-myristate-23-acetate and ionomycin to determine CD154 expression, and in the presence of brefeldin A to measure IL-21 production. (C) CXCR4 expression was compared between CD3⁺CD4⁺CXCR5⁺ICOS⁺PD-1^hi (TFHs), CD4⁺CXCR5⁻, and B cells. (D) The expression of IL-21R on TFHs was compared with CD4⁺CXCR5⁻. (E) TFH frequency within tonsils (n = 4) was compared with the one in the spleens (6 controls and 13 ITP patients). Data are depicted in box-and-whisker graphs. P value derived by Mann-Whitney test. (F) TFHs were localized by immunohistochemistry. Follicles and GC (arrowheads) were identified within the spleen by using CD20 staining. Then TFHs were identified by the expression of PD-1 (black arrows) and CD4 (white arrows). Representative spleens of 1 control and 1 ITP patient (magnification ×200).