Background:  The long-term steady state established in long-term non-progressors (LTNP) between host and virus is characterized by low HIV RNA and HIV DNA levels and high specific immune responses. It represents a model to investigate distribution and stability of the HIV reservoir and the impact of CD4^–T cell differentiation on the long-term control of those HIV reservoirs.

Methods:  We included:  12 untreated LTNP HIV-seropositive for >8 years, CD4 T cells >600/µL, selected for HIV RNA <200 copies/mL in 7 cases and <3000 copies/mL in 5 cases defining elite and viremic controllers, respectively. Live resting CD4⁺ peripheral blood mononuclear cells (PBMC) were sorted according to anti-CD45RA, CD27, and CCR7 combinations in various memory subsets from central (TCM) to effector memory (TEM), effector, and naïve T cells. Ultrasensitive HIV DNA quantification was performed on the different sorted subsets and their ability to produce virions was tested by 6 days in vitro activation with anti-CD3⁺CD28 and interleukin-2 (IL-2). Comparisons were performed with the Mann-Whitney test.

Results:  The median HIV DNA in the 12 LTNP was 1.74_log copies/million PBMC and was distributed among resting CD4 T cells according to a constant hierarchy:  an intermediate TEM subset contained median values of 3.02_log copies/million intermediate TEM, followed by TCM (2.85_log), TEM (2.27_log), effector (1.98_log), and naive CD4 T cells (1.71_log). This distribution was highly stable and maintained over 10 years. The intermediate TEM subset contributed significantly more to this reservoir than the other subsets (median 47% and p <0.001 compared to all subsets). Elite differed from viremic controllers by a significantly lower infection level of this intermediate population (p = 0.035), independently of the genetic HLA or CCR5 background. Activation in vitro of these sorted subsets induced HIV replication from the 3 different memory CD4 T cell subsets, independently of their replicative capacity, but neither from naïve nor from effector T cells.

Conclusions:  In LTNP with stable long-term control of HIV, TCM are not the predominant reservoir of HIV but instead an intermediate TEM subset. The concentration of the HIV reservoir can vary by 10- to 100-fold depending on the subset maturation. Altogether our results demonstrate that the memory peripheral blood CD4 T cells in these LTNP contain a stable inducible reservoir with low production rate and distinct mechanisms of control of HIV dictated by T cell maturation.