ICOS expression in pTFH cells is correlated with IL-21 production and Ab response to H1N1/09 vaccine. (A) Representative dot plots showing the gating strategy for IL-21 and ICOS expression in pTFH cells, defined as CD4⁺CXCR5⁺ events, using appropriate isotype control Abs. Freshly isolated PBMCs at T2 were cultured in complete medium alone or in the presence of PMA, ionomycin, and brefeldin A for 5 hours and stained for intracellular IL-21. (B) Linear correlation of frequency of ICOS⁺ pTFH cells with IL-21–producing pTFH cells at T2 of 8 HCs, 7 HIV⁺ responders (R), and 7 HIV⁺ nonresponders (NR). (C) Linear correlation of frequency of ICOS⁺ pTFH cells with H1N1 serum Ab titer at T2 for 8 HCs, 7 HIV⁺ responders, and 7 HIV⁺ nonresponders. (D) Maturation subsets within ICOS⁺ pTFH cells. (E) Cryopreserved PBMCs were thawed, rested overnight, and cultured at 1 × 10⁶ cells/mL for 5 days at 37°C with H1N1 vaccine Ag or medium as described. Supernatants were harvested and CXCL13 levels were determined by ELISA. Bar graphs show CXCL13 levels at T0 and T2 after vaccination in 5 HCs, 6 HIV⁺ responders, and 6 HIV⁺ nonresponders. Box plots represent median with 25th and 75th percentile borders. Error bars represent the 10th and 90th percentiles.