HIV-1 and SIV Predominantly Use CCR5 Expressed on a Precursor Population to Establish Infection in T Follicular Helper Cells

Yin Xu, Chansavath Phetsouphanh, Kazuo Suzuki, Anu Aggrawal, Stephanie Graff-Dubois, Michael Roche, Michelle Bailey, Sheilajen Alcantara, Kieran Cashin, Rahuram Sivasubramaniam, Kersten K. Koelsch, Brigitte Autran, Richard Harvey, Paul R. Gorry, Arnaud Moris, David A. Cooper, Stuart Turville, Stephen J. Kent, Anthony D. Kelleher, John Zaunders

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.