HIV-1 persistence in CD4+ T cells with stem cell-like properties

Abstract

Cellular HIV-1 reservoirs that persist despite antiretroviral treatment are incompletely defined. We show that during suppressive antiretroviral therapy, CD4(+) T memory stem cells (TSCM cells) harbor high per-cell levels of HIV-1 DNA and make increasing contributions to the total viral CD4(+) T cell reservoir over time. Moreover, we conducted phylogenetic studies that suggested long-term persistence of viral quasispecies in CD4(+) TSCM cells. Thus, HIV-1 may exploit the stem cell characteristics of cellular immune memory to promote long-term viral persistence.

Figure 1. CD4⁺ T_SCM cells represent a long-term reservoir for HIV-1 in HAART-treated patients

(a): Proportion of HIV-1 infected cells after ex-vivo infection with GFP-encoding R5-tropic or VSV-G pseudotyped HIV-1 (R5: n=17, VSV-G: n=14). (b) Cell-associated HIV-1 DNA in sorted CD4⁺ T cell populations (left panel) and corresponding contributions to the total HIV-1 reservoir in CD4⁺ T cells from HAART-treated individuals (right panel). (c): Left panel: Representative pie charts reflecting the contribution of CD4⁺ T_SCM cells to the total viral CD4⁺ T cell reservoir in two persons with large and small HIV-1 reservoirs in total CD4⁺ T cells, respectively. Right panel: Spearman correlation between contributions of CD4⁺ T_SCM cells to the total HIV-1 CD4⁺ T cell reservoir, and corresponding size of the HIV-1 reservoir in total CD4⁺ T cells. (d): Reactivation of replication-competent HIV-1 from memory CD4⁺ T cell subsets. Orange bars reflect proportions of wells with detectable replication-competent HIV-1, blue bars indicate proportions of wells without detectable replication-competent HIV-1. Numbers above columns reflect total numbers of wells analyzed for each CD4⁺ T cell population; numbers below columns reflect estimated frequencies of cells with replication-competent HIV-1 per million cells (IUPM) based on limiting-dilution analysis. (e): Longitudinal evolution of HIV-1 DNA in CD4⁺ T cell subsets in n=8 study persons who initiated antiretroviral therapy in primary infection. (f): Pair-wise fold-differences in HIV-1 DNA measured after short-term and long-term antiretroviral therapy. Mean and standard error are shown. (g): Corresponding contribution of individual CD4⁺ T cell subsets to the total CD4⁺ T cell pool, and to the total HIV-1 CD4⁺ T cell HIV-1 reservoir after short-term and long-term antiretroviral therapy. Statistical significance was tested with Wilcoxon rank sum test. *,**, *** reflect p<0.05, p<0.01, p<0.001, respectively, after Bonferroni correction for multiple comparisons in panel a and b.

Figure 2. Phylogenetic analysis of HIV-1 sequences isolated from CD4⁺ T_SCM cells

(a): Longitudinal evolution of CD4⁺ T cell counts and viral loads in the three study patients. Shaded areas reflect periods of antiretroviral treatment exposure. Arrows indicate time of CD4⁺ T cell and plasma sampling. (b): Phylogenetic analysis of HIV-1 sequences longitudinally amplified from sorted CD4⁺ T_SCM and TCM cells at the beginning of antiretroviral treatment initiation, and after 4–8 years of continuous suppressive therapy in three study persons. Identical HIV-1 sequences in T_SCM cells (1) and TCM cells (2) are highlighted by gray circles. (c): Circular phylogenetic trees of HIV-1 sequences amplified from indicated CD4⁺ T cell subsets and from plasma collected at indicated timepoints. Gray circles reflect: Phylogenetic relationships between HIV-1 DNA sequences from CD4⁺ T_SCM cells and circulating HIV-1 viral RNA sequences isolated during early untreated disease (3) or during contemporaneous and ensuing timepoints (4). Identical HIV-1 sequences isolated from CD4⁺ T_SCM, and from CD4⁺ TCM, TEM, TTD cells isolated at later timepoints, are also highlighted (5).