Peripheral T follicular helper cells are the major HIV reservoir within central memory CD4 T cells in peripheral blood from chronically HIV-infected individuals on combination antiretroviral therapy

In this study, we examined the peripheral blood (PB) central memory (T_CM) CD4⁺ T cell subsets designated peripheral T follicular helper cells (pTfh cells) and non-pTfh cells to assess HIV permissiveness and persistence. Purified pTfh and non-pTfh cells from healthy HIV-negative donors were tested for HIV permissiveness using green fluorescent protein (GFP)-expressing HIV- 1NL4-3/Ba-L, followed by viral reactivation using beads coated with anti-CD3/anti-CD28 monoclonal antibodies. The role of pTfh cells in HIV persistence was analyzed in 12 chronically HIV-1 infected patients before and 48 weeks after initiation of raltegravir- containing combination antiretroviral therapy (cART). Total cellular HIV-1 DNA and episomes containing two copies of the viral long terminal repeat (2LTR circles) were analyzed in using droplet digital PCR in the purified pTfh and non-pTfh cells. Activation-inducible HIV p24 expression was determined by flow cytometry. Results indicate that pTfh cells, in particular PD1⁺ pTfh cells, showed greater permissiveness for HIV infection than non-pTfh cells. At week 48 on cART, HIV DNA levels were unchanged from pre-cART levels, although a significant decrease in 2LTR circles was observed in both cell subsets. Inducible HIV p24 expression was higher in pTfh cells than in non-pTfh cells, with the highest frequencies in the PD1⁺ CXCR3^- pTfh cell subset. Frequencies of HLADR⁺ CD38⁺ activated CD4 T cells correlated with 2LTR circles in pTfh and non-pTfh cells at both time points and with p24⁺ cells at entry. In conclusion, among CD4 T_CM cells in PB of aviremic patients on cART, pTfh cells, in particular the PD1⁺ CXCR3^- subset, constitute a major HIV reservoir that is sustained by ongoing residual immune activation. The inducible HIV p24 assay is useful for monitoring HIV reservoirs in defined CD4 T cell subsets.