T follicular helper (Tfh) cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and the mechanism by which they are infected is unclear. We have previously shown that macaque Tfh lack CCR5, but are infected in vivo with CCR5-using SIV at levels comparable to other memory CD4⁺ T cells. Similarly, human splenic Tfh cells are highly infected with HIV-1 DNA. Therefore, we set out to examine the mechanism of infection of Tfh cells.

Tfh and other CD4⁺ T cell subsets from macaque lymph nodes and spleens, splenic Tfh from HIV⁺ subjects, and tonsillar Tfh from HIV-uninfected subjects were isolated by cell sorting prior to cell surface and molecular characterization. HIV proviral gp120 sequences were submitted to genotypic and phenotypic tropism assays. Entry of CCR5- and CXCR4-using viruses into Tfh from uninfected tonsillar tissue was measured using a fusion assay.

Phylogenetic analysis, genotypic, and phenotypic analysis showed that splenic Tfh cells from chronic HIV⁺ subjects were predominantly infected with CCR5-using viruses. In macaques, purified CCR5⁺PD-1^{intermediate(int)+} memory CD4⁺ T cells were shown to include pre-Tfh cells capable of differentiating in vitro to Tfh by upregulation of PD-1 and Bcl6, confirmed by qRT-PCR and single-cell multiplex PCR. Infected PD-1^int cells survive, carry SIV provirus, and differentiate into PD-1^hi Tfh after T cell receptor stimulation, suggesting a pathway for SIV infection of Tfh. In addition, a small subset of macaque and human PD-1^hi Tfh can express low levels of CCR5, which makes them susceptible to infection. Fusion assays demonstrated CCR5-using HIV-1 entry into CCR5⁺ Tfh and pre-Tfh cells from human tonsils.

The major route of infection of Tfh in macaques and humans appears to be via a CCR5-expressing pre-Tfh population. As the generation of Tfh are important for establishing effective immune responses during primary infections, Tfh are likely to be an early target of HIV-1 following transmission, creating an important component of the reservoir that has the potential to expand over time.