Determination of functionally distinct subsets of human tonsillar and blood T follicular helper (Tfh) cells.

T follicular helper (Tfh) cells represent a CD4+ T cell subset specialized for B-cell help. While our knowledge on the biology of Tfh cells has dramatically increased during the past decade, a vast majority of observations were made in mouse models. Our knowledge on the biology of human Tfh cells has been limited compared to mouse Tfh cells. Therefore, we aimed to determine the biology of Tfh cells in human tonsils. We hypothesized that human Tfh cells are composed of functionally distinct subsets. We also determined the type of Tfh responses induced by influenza vaccination in healthy adults. First, we identified a Tfh subset localized outside germinal centers (GCs) in human tonsils. This subset expressed low-levels of CXCR5 and ICOS (CXCR5loICOSlo) and helped naïve B cells more efficiently than GC-Tfh cells through high IL-21 production. Notably, CXCR5loICOSlo Tfh cells lacked the capacity to help GC-B cells due to FAS-L expression. This study shows that CXCR5loICOSlo Tfh cells are specialized for providing help to naïve and memory B cells outside GCs. Second, we found that influenza vaccination induces exclusive activation of CXCR3+ blood Tfh subset at day 7. The emergence of activated CXCR3+ blood Tfh cells at day 7 correlated with the generation of protective antibody responses. Mechanistically, we found that activated CXCR3+ blood Tfh cells efficiently help memory B cells, but not naïve B cells, to differentiate into plasma cells. Thus, our study has identified a Tfh subset responsible for the induction of antibody responses in influenza vaccinations. Last, we found that CXCR3+ Tfh cells localized in tonsillar GCs suppress the activity of GC-B cells and GC-Tfh cells. CXCR3+ GC-Tfh cells displayed three features distinct from GC-Tfh cells: expression of surface FAS-L, secretion of large amount of IFN- and the lack of CD40L expression. These three features contributed the suppression of survival, growth, and activation of GC-B cells and GC-Tfh cells. CXCR3+ GC-Tfh cells are clearly distinct from Foxp3+ Tregs, and therefore likely represent a previously undefined regulatory T cell subset in human GCs.