Spleens were collected from NP-CGG–immunized Prdm1-CKO or control mice 7 days after immunization. (A) Intracellular IL-17A was measured in CD4⁺ Tfh cells by flow cytometry. Representative flow images are on the left panel and the graph is the quantitation of IL-17A⁺ cells within Tfh cells (n = 7 for CTL and n = 5 for KO). Three independent experiments were performed. (B and C) Isolated Tfh cells were cultured overnight, and the supernatant was collected for cytokine assay, the cells were lysed, and total RNA was isolated. IL-17, IL-6, and IL-21 levels were quantified by MSD and IL-21 mRNA was measured by qPCR. The bar represents the mean from 5 independent experiments and each dot represents an individual mouse (n = 15 for CTL and n = 11 for KO) (n = 9 for both CTL and KO, only for the 4C Il21 qPCR experiment). (D) Intracellular staining of transcription factors (BCL6, c-MAF, and RORγt) was performed and measured within Tfh cells. Positive signal of BCL6 was obtained from Tfh cells and coexpression of c-MAF or RORγt was identified as in flow data (upper row). The percentage of c-MAF/BCL6 or RORγt/BCL6 was calculated from the total BCL6⁺ Tfh cells and the graph was made (bottom row). Each dot represents an individual animal and the bar represents the mean from 3 independent experiments (n = 7 for CTL and n = 9 for KO). An unpaired 2-tailed Student’s t test (Mann-Whitney) was used.