Characterization of peripheral TFH cells in HCs and HIV-infected patients. (A) Representative dot plots show gating strategy for pTFH cells, defined as CD4⁺CXCR5⁺ cells in an HIV-infected patient. Isotype control Ab was used to set the gate for CXCR5. T-cell differentiation subsets in the CD4⁺CXCR5⁺ and CD4⁺CXCR5⁻ subsets were based on the surface expression of CD45RO, CD27, and CD57 as T_N (CD45RO⁻CD27⁺), T_CM: (CD45RO⁺CD27⁺), T_EM (CD45RO⁺CD27⁻), T_E (CD45RO⁺CD57⁺), and T_TE (CD45RO⁻CD57⁺). (B) Frequency of pTFH (CD4⁺CXCR5⁺) cells are indicated as a percentage of total CD4 T cells in HCs and HIV-infected patients. (C-D) Distribution of CD4⁺CXCR5⁺ (C) and CD4⁺CXCR5⁻ (D) cells within the differentiation subsets. Box plots represent median with 25th and 75th percentile borders. Error bars represent 10th and 90th percentile means from 17 HCs and 25 HIV-infected aviremic patients. (E) Phenotyping for differentiation subsets in IL-21⁺ pTFH cells obtained after 5 hours of stimulation with 50 ng/mL of PMA, 1 μg/mL of ionomycin, and 10 μg/mL of brefeldin A.