T follicular helper cells (Tfhs) are a phenotypically heterogeneous cell population generally defined by the expression of CXCR5 and PD-1. Tfhs serve as a major reservoir for HIV transcription and production in both viremic and long-term ART treated subjects. In the presents study, we have dissected the phenotypic and functional heterogeneity of Tfhs and the role of the different Tfhs populations in serving as HIV reservoir and their relationship with HIV-specific B cell responses.
Lymph nodes (LN) biopsies were obtained from 17 HIV uninfected, 27 viremic untreated and 23 aviremic ART treated subjects. Definition of B and T cell populations and cytokines was performed by mass cytometry using a panel of 40 metal-conjugated antibodies. To dissect the heterogeneity of Tfh cells, we performed self-organizing map (FlowSOM) and consensus clustering. Cell-associated HIV-RNA was assessed in Tfh cells sorted on the basis of CD38 and CXCR3 expression (CD38+CXCR3+, CD38+CXCR3-, CD38-CXCR3+ and CD38-CXCR3-).
Unsupervised clustering identified 20 different populations of Tfh cells within CXCR5highPD-1high Tfh cells. CD38+CXCR3+ Tfhs significantly increased in viremics (41.18%) as compared to ART treated (17.8%) and HIV uninfected (6.9%) subjects (p<0.0001). Frequencies of CD38+CXCR3+ Tfh cells positively correlated with the percentage of total germinal center B cells (r=0.58, p<0.0001) and GC gp140+ B cells (r = 0.65, p<0.0003). CD38+CXCR3+ Tfhs expressed higher levels of expression of BCL-6, ICOS, CD57, CD40L, CCR5, HLA-DR, T-bet as compared to the CD38+CXCR3-, CD38-CXCR3+ and the CD38-CXCR3- and of production of the Tfh signature cytokine IL-21 (averages of 33%) as compared to the other three Tfh cell populations (p<0.0002). Of note, only the percentage of CD38+CXCR3+ Tfhs positively correlated with viremia (r=0.5, p=0.01) in untreated subjects. More importantly, CD38+CXCR3+ Tfhs were greatly enriched in cell-associated HIV-RNA as compared to CD38+CXCR3- (average 3.3 fold), CD38-CXCR3+ (average 6.24 fold) and CD38-CXCR3- (average 8.24 fold) Tfh cell populations (p<0.05).
CD38+CXCR3+ Tfhs correspond to a population of phenotypically and functionally active Tfh cells. The higher levels of expression of CCR5 may render these cells more susceptible to HIV infection and it explains why CD38+CXCR3+ Tfh cells serve as the major active HIV reservoir within the total Tfh cell population.