HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation

Abstract

HIV persists in a reservoir of latently infected CD4(+) T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory (T(CM)) and transitional memory (T(TM)) CD4(+) T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in T(CM) cells in subjects showing reconstitution of the CD4(+) compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in T(TM) cells from aviremic individuals with low CD4(+) counts and higher amounts of interleukin-7-mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells.

Figure 1

Integrated DNA is the major molecular form of HIV during HAART and is harbored by T_CM and T_TM cells in vivo. (a) Frequencies of total, integrated and 2-LTR circle DNA molecules in CD4⁺ T cells before and after HAART initiation. Molecular forms of HIV were quantified by real-time PCR in longitudinal samples from four individuals who initiated HAART during the follow-up period. Off and On refer to the therapy status. The time before (−) and after (+) HAART initiation (in months) and the viral load at each time point (in RNA copies per ml of plasma) are indicated. (b) Frequencies of total, integrated and 2-LTR circle HIV DNA in 1 × 10⁶ CD4⁺ T cells from ten subjects receiving HAART for more than 32 months. (c) Percentage of CD4⁺ T cell subsets among total CD4⁺ T cells in a cohort of 31 individuals with undetectable viral load. Percentages of cells were determined by flow cytometry. (d) Frequencies of cells harboring integrated HIV DNA in CD4⁺ T cell subsets from three representative aviremic subjects. Results are expressed as the HIV copy number in 1 × 10⁶ cells of a given subset. (e) Contribution of T_N, T_CM, T_TM+EM and T_TD cell subsets to the pool of HIV-infected cells calculated in 17 aviremic HIV-infected subjects. HIV integrated DNA copy number was determined in sorted subsets by highly sensitive real-time PCR. Each symbol represents a different individual. In ten subjects, CD45RA⁻CCR7⁻ CD4⁺ T cells were further sorted according to the expression of CD27 to distinguish between T_TM cells and T_EM cells. Horizontal lines indicate mean values.

Figure 2

CD4⁺ T cell depletion drives the size and the localization of the HIV reservoir. (a–d) Integrated HIV DNA copy number, as determined in the CD4⁺ T cells from 33 virally suppressed HIV-infected subjects by Alu real-time PCR. (a) Comparison of the size of the HIV reservoir in subjects with a CD4/CD8 ratio o1 or 41. (b) Correlation between the size of the HIV reservoir and the nadir CD4⁺ count. (c) Comparison of the size of the HIV reservoir in subjects who have started HAART before or after the first year of infection. (d) Correlation between the size of the HIV reservoir and absolute CD4⁺ counts. (e) Correlation between the absolute CD4⁺ count and the T_CM/T_TM ratio in 33 subjects receiving HAART. The frequencies of T_CM and T_TM cells within total CD4⁺ T cells were measured by flow cytometry after staining for the CD4, CD45RA, CCR7 and CD27 markers. (f) Correlation between the T_CM/T_TM+EM infection frequency ratio and absolute CD4⁺ counts in the 17 individuals on whom HIV quantifications in CD4⁺ T cell subsets were performed. HIV integrated DNA copy number was determined in sorted subsets by highly sensitive real-time PCR. The ratio was calculated by dividing the number of copies of integrated HIV DNA in 1 × 10⁶ T_CM by the number of copies of integrated HIV DNA in 1 × 10⁶ T_TM+EM cells. (g) Correlations between the contribution of T_CM cells and T_TM+EM cells to the HIV reservoir and the absolute CD4⁺ count in the same individuals. To calculate the contribution of each CD4⁺ T cell subset to the global pool of HIV-infected cells, the percentage of each subset within the CD4⁺ compartment as well as the frequency of the cells in the subset harboring HIV integrated DNA were taken into account. Horizontal lines indicate mean values.

Figure 3

Proliferation of CD4⁺ T cells drives the size and the localization of the HIV reservoir. (a) Correlation between the absolute CD4⁺ count and the level of cellular proliferation, as measured by intracellular Ki67 expression in CD4⁺ T cells from 33 individuals receiving potent HAART. (b) Correlation between the size of the HIV reservoir and the frequencies of Ki67⁺ cells within total CD4⁺ T cells. The frequency of cells harboring HIV integrated DNA in total CD4⁺ T cells from 33 individuals was determined by Alu real-time PCR. (c) Correlation between the T_CM/T_TM+EM infection frequency ratio and the expression of Ki67 on total CD4⁺ T cells in the 17 subjects on whom HIV quantifications in CD4⁺ T cell subsets were performed. (d) Correlations between contributions of T_CM and T_TM+EM cells to the HIV reservoir and the expression of Ki67 in the same individuals as in a–c. (e) Percentage of CD4⁺ T cells from each cellular subset expressing Ki67 and PD-1. (f) Correlations between the contribution of a given CD4⁺ T cell subset to the HIV reservoir and the expression of Ki67 and PD-1. (g) Correlation between PD-1 and Ki67 expression in total CD4⁺ T cells. (h) T_CM cells and T_TM cells from three and four virally suppressed subjects were sorted according to their relative expression of PD-1. The frequency of cells harboring HIV proviral DNA was determined in both fractions by Alu real-time PCR.

Figure 4

T_CM and T_TM cells define a distinct HIV reservoir. (a) Neighbor-joining trees derived from HIV sequences obtained from T_CM cells and T_TM cells of four representative HAART-treated HIV-infected subjects. T_CM and T_TM cells from ten aviremic subjects were sorted, and a minimum of 11 clones deriving from at least ten independent positive PCRs of the Env gene were sequenced in each case. The numbers near nodes indicate the percentage of bootstrap replicates (1,000 resampling). The scale refers to the distance between sequences. Vertical lines are for clarity only. (b) Correlation of the mean genetic distances of HIV Env sequences between T_CM and T_TM cells to the percentage of Ki67⁺ T_TM cells in ten aviremic subjects. Each diamond represents the mean genetic distance existing between the clones obtained from T_CM cells and T_TM cells. (c) Correlations between the HIV genetic diversity in T_CM cells and T_TM cells and the expression of Ki67 in these subsets. HIV diversity was estimated by calculating the mean genetic distance between the clones within a given subset.

Figure 5

Proliferation of T_TM cells is associated with a genetic stability of the HIV reservoir over time. (a) Neighbor-joining trees derived from HIV sequences obtained from T_CM cells and T_TM cells of two representative aviremic individuals at first and second time points (closed and filled symbols, respectively). Percentages of T_CM cells and T_TM cells expressing the Ki67 proliferation marker are indicated. (b) Genetic evolution of the HIV reservoir in T_CM cells and T_TM cells from five aviremic individuals followed longitudinally. Results are expressed as the mean genetic distance existing between HIV sequences from both time points, corresponding to the estimated genetic divergences of the Env region (left). Percentage of Ki67⁺T_TM cells (middle) and plasma IL-7 concentrations (right) are also shown. (c) Evolution of the HIV reservoir size in eight aviremic individuals. The integrated HIV DNA copy number was measured by Alu real-time PCR in purified CD4⁺ T cells at two time points. For each subject, the line between the two time points reflects the slope of the decrease in the frequency of CD4⁺ T cells harboring HIV proviral DNA. (d) Correlation between the slope decrease in the number of HIV-infected CD4⁺ T cells and the percentage of Ki67⁺ T_TM cells. The decrease in the HIV reservoir size is expressed as a decrease of the percentage of cells harboring HIV proviral DNA per day. (e) Correlation between the slope decrease in the number of HIV-infected CD4⁺ T cells and the plasma IL-7 level measured by ELISA.

Figure 6

IL-7 induces homeostatic proliferation of CD4⁺ T cells and ensures HIV reservoir stability through cellular proliferation at low levels. (a) Phenotype of CD4⁺ T cells after TCR-induced (CD3/CD28) or IL-7 proliferation. Cells were harvested at day 8, and their phenotype was determined by flow cytometry. (b) Percentage of CD4⁺ T cells expressing the proliferation marker Ki67 after 8 d of CD3/CD28 or IL-7 stimulation. (c) HIV genetic diversities at baseline and after 8 d of treatment. Each dot represents the mean genetic distance between one given clone and the entire population. The horizontal bar represents the mean of these values and is reflecting the genetic diversity of the viral population in the various conditions.