pTfh and MBC differentiation phenotypes in cocultures.

PBMCs at T2 were thawed and rested overnight. pTfh (Aqua−CD19−CD3+CD8−CD4+CD45RO+CXCR5+), MBCs (Aqua−CD3−CD19+CD27+), and APC (Aqua−CD3−CD19−CD8−) populations were purified by cell sorting. B cells were cocultured with pTfh and APC at a 1:1:1 ratio in medium alone or in the presence of 5 μg/mL of H1N1 vaccine Ag +/− a cytokine cocktail containing IL-21 (50 ng/ml), anti-IL-2 (10 μg/ml), and anti-TNF-α (2 μg/ml) for 7 d. (A–D) Harvested pTfh were analyzed for T helper differentiation based on CCR4, CCR6, and CXCR3 expression into (A) Th1 (CCR4−CCR6−CXCR3+), (B) Th1/Th17(CCR4−CCR6+CXCR3+), (C) Th17 (CCR4−CCR6+CXCR3−), and (D) Th2 (CCR4+CCR6−CXCR3−) cells. For unpaired data, “Mann-Whitney” U test and for paired data Wilcoxon Signed Rank Test were performed. p p p p S1 Data. Ag, antigen; APC, antigen presenting cell; CCR, CC-chemokine receptor; CXCR, CXC chemokine receptor; IL-2, interleukin 2; MBC, memory B cell; PBMC, peripheral blood mononuclear cell; pTfh, peripheral T follicular helper; Th17, T helper 17; TNFα, tumor necrosis factor alpha.