Fig. 3.

HIV-specific cTfh produce higher levels of Tfh cytokines than CMV-specific cTfh. Ag-specific cTfh responses were analyzed by flow cytometry for expression of CXCL13 and IL-21 using a delayed ICS assay. (a) Schematic representation of the delayed ICS assay. (b) Example plot showing CXCL13 expression in unstimulated or HIV-stimulated CD4⁺ cells after gating on either total CD4⁺ or CD69⁺CD40L⁺ Ag-specific CD4⁺. Plots shown are from the same sample. (c) Example plot showing CXCL13 or IL-21 expression in CD69⁺CD40L⁺ cTfh. (d) Comparison of cytokine⁺ HIV-specific vs CMV-specific cTfh. (e) Example plot showing phenotype of total cTfh cells (black) or HIV-specific CXCL13+ or IL-21+ cTfh (shown in red) based on PD-1 and TIGIT expression. (f) Frequency of cytokine⁺ cells in cTfh subsets based on TIGIT/PD-1 expression. n = 9. Significant p-values shown were calculated by Wilcoxon test (d) or Friedman test with Dunn's post test (f). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)