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. 2013 Jul 1:10:68.
doi: 10.1186/1742-4690-10-68.

Primary CD8+ T cells from elite suppressors effectively eliminate non-productively HIV-1 infected resting and activated CD4+ T cells

Robert W Buckheit 3rd  1 , Robert F Siliciano, Joel N Blankson
Affiliations

Primary CD8+ T cells from elite suppressors effectively eliminate non-productively HIV-1 infected resting and activated CD4+ T cells

Robert W Buckheit 3rd et al. Retrovirology. .

Abstract

Background: Elite controllers or suppressors have the remarkable capacity to maintain HIV-1 plasma RNA levels below the limit of detection of clinical assays (<50 copies/mL) without therapy and have a lower frequency of latently infected cells compared to chronic progressors. While it is unclear how this reduced seeding of the reservoir is achieved, it is possible that effective CTL responses play an in important role in limiting the size of the latent reservoir.

Results: Herein, we demonstrate that primary CD8+ T cells from HLA-B*57/5801 elite suppressors were able to efficiently eliminate resting and activated primary CD4+ T cells shortly after viral entry and prior to productive infection. CD8+ T cells from elite suppressors were significantly more effective at eliminating these cells than CD8+ T cells from chronic progressors.

Conclusions: Nonproductively infected CD4+ T cells may represent a subpopulation of cells that are precursors to latently infected cells; therefore, the effective elimination of these cells may partially explain why elite suppressors have a much lower frequency of latently infected cells compared to chronic progressors. Thus, a vaccine strategy that elicits early and potent CD8+ T cell responses may have the capacity to limit the seeding of the latent reservoir in HIV-1 infection.

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Figures

Figure 1
Figure 1
Early Gag positivity in spinoculated cells is CD4 and co-receptor dependant. CD4+ T cells were either untreated or treated with anti-CD4 antibody, MVC, ADM, or T20 for the duration of spinoculation. Percent Gag+ cells were analyzed immediately after spinoculation with R5 (A) or X4 (B) pseuotype virus. (C) X4 virus infection was analyzed at 0, 6 and 18 hours post spinoculation after treatment with anti-CD4 antibody, MVC, ADM, or T20 for the duration of the culture period. (D) Comparision of the percent infection 24 hours after spinoculation with and without T20 treatment (n=3). Standard error of the mean are indicated by black bars.
Figure 2
Figure 2
Elimination of non-productively infected CD4+ cells. (A) Representative FACS plots demonstrating the gating scheme employed for the calculation of normalized percent elimination. Cells in culture were stained with anti-CD3 and anti-CD8 antibodies to distinguish targets (CD3+/CD8-) and effector (CD3+/CD8+) populations. Target cells were then gated to determine the percent of gag positive cells, as determined by intracellular staining with an anti-Gag antibody. Uninfected target cells were used as a negative control. (B) An elimination assay was performed to determine the ability of CD8 T cells from B*57/5801+ ES (n=10; blue squares), B*57/5801+CP (n=9; orange squares), B*57/5801- CP (n=8; red squares) and healthy donors (HD, n=6; purple squares) to reduce the frequency of Gag positive target cells. Unstimulated CD8+ T cells or Gag Stimulated CD8+ T cells were co-cultured with untreated or EFV treated, autologous CD4+ T cell targets at various effector to target ratios. Elimination was analyzed after 6, 18 and 72 hours post infection. Data points where the level of elimination mediated by the ES CD8 T cells was significantly higher than all other experimental groups are indicated (black asterisks, p<.05). (C) The normalized percent elimination for ES Gag-stimulated and unstimulated effectors, for both untreated and 10 μM EFV treated were analyzed at 72 hours post infection for a 1:1 and 1:8 effector to target ratio. For all treatment groups, no statistical difference in the levels of elimination was observed. Median elimination levels are indicated.
Figure 3
Figure 3
Elimination of cells infected with inactivated virus. CD4+ T cells from two ES (top panels) and two HD (bottom panels) were infected with AT-2 inactivated virus, and the level of Gag positivity was analyzed for target alone wells, and targets with effectors at a 1:1 effector to target ratio. No elimination was mediated by CD8 T cells from HD, in contrast to high level of elimination observed for the two ES assayed.
Figure 4
Figure 4
Viral elimination is constant over a range of inoculum sizes. (A) Representative FACS plots showing the titration of gag positivity from a viral inoculum size of 1000 ng to a viral inoculum size of 62.5 ng per one million cells. (B) The average percent infection as a result of viral titration was calculated over a range of viral inoculum sizes in a subset of our ES cohort (n=6; top panel). Unstimulated CD8+ T cells were added to either untreated (Blue squares) or 10 μM EFV treated ( Red squares) CD4+ T cells targets at a 1:1 effector to target ratio and the level of elimination was calculated 72 hours after infection (bottom panel). Statistically significant differences in elimination between the viral inoculum size for the untreated CD4+ T cell target group is indicated (blue bracket and asterisks). Differences in elimination between the EFV treated and untreated treatment groups are indicated (black asterisks).
Figure 5
Figure 5
Equal elimination of resting and activated CD4+ T cells. In a subset of our ES cohort, target CD4+ T cells analyzed in the suppression assay were also stained with anti-HLA-DR, anti-CD25, and anti-CD69 antibodies and gated using the FACSDiva software to delineate resting and activated CD4+ cell populations. The level of elimination of untreated and EFV treated resting (blue diamonds), activated (red squares), and unfractionated (all CD3+/CD8- targets; orange circles) target cells was calculated at various effector to target ratios. This analysis was performed at 18 (top panels) and 72 hours (bottom panels) after infection. No statistically significant differences in the levels of elimination between each of the populations were observed.
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