T follicular helper cells (Tfh) are expanded in lymphoid tissue during HIV-1 infection, but are also highly infected with HIV DNA, despite reportedly being CCR5-. We studied how Tfh cells are infected, but still increase relative to other CD4+ T-cells.

Tfh were studied from fine needle biopsies of lymph nodes (LN FNB) and by splenectomy and tonsillectomy from HIV-uninfected controls and HIV+ subjects. Tfh and germinal centre B cells were measured by multiparameter flow cytometry, and HIV DNA by qPCR. From SIV-infected macaque LN cells, and from human tonsil cells, CCR5+ and CCR5- subsets of PD-1^intermediate pre-Tfh and PD-1^high CXCR5+ Tfh were purified by cell sorting.

In LN FNB samples, Tfh were elevated 4.3-fold, and GC B cells 110-fold in untreated HIV+ subjects, compared to controls. Proviral HIV env DNA sequences from splenic Tfh were CCR5-using. In the macaque model, SIV-infected CCR5+ PD-1^intermediate pre-Tfh could differentiate in vitro into SIV-infected PD-1^high Tfh, but many Tfh also came from CCR5-negative pre-Tfh. Human tonsillar Tfh are largely CCR5-, and could not be infected in vitro with CCR5-using HIV, but surprisingly, median 39% of PD-1^high Tfh expressed CCR5 at a low level, detected using optimized staining, and could be infected.

Altogether, the expansion of Tfh (leading to the huge increase in GC B cells) comes from both CCR5- and CCR5+ pre-Tfh. The latter can be infected, in addition to the small subset of CCR5^low Tfh, leading to the significant HIV-1 DNA reservoir in Tfh in germinal centres, which is resistant to antiretroviral therapy.