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. 2013 Aug 7;8(8):e71879.
doi: 10.1371/journal.pone.0071879. eCollection 2013.

Gag-positive reservoir cells are susceptible to HIV-specific cytotoxic T lymphocyte mediated clearance in vitro and can be detected in vivo [corrected]

Erin H Graf  1 , Matthew J Pace, Bennett A Peterson, Lindsay J Lynch, Steve B Chukwulebe, Angela M Mexas, Farida Shaheen, Jeffrey N Martin, Steven G Deeks, Mark Connors, Stephen A Migueles, Una O'Doherty
Affiliations

Gag-positive reservoir cells are susceptible to HIV-specific cytotoxic T lymphocyte mediated clearance in vitro and can be detected in vivo [corrected]

Erin H Graf et al. PLoS One. .

Erratum in

  • PLoS One. 2013;8(9). doi:10.1371/annotation/3aa92c3d-b6dd-4c6e-8cee-9587ce80a9c9

Abstract

Resting CD4+T cells infected with HIV persist in the presence of suppressive anti-viral therapy (ART) and are barriers to a cure. One potential curative approach, therapeutic vaccination, is fueled by recognition of the ability of a subset of elite controllers (EC) to control virus without therapy due to robust anti-HIV immune responses. Controllers have low levels of integrated HIV DNA and low levels of replication competent virus, suggesting a small reservoir. As our recent data indicates some reservoir cells can produce HIV proteins (termed GPR cells for Gag-positive reservoir cells), we hypothesized that a fraction of HIV-expressing resting CD4+T cells could be efficiently targeted and cleared in individuals who control HIV via anti-HIV cytotoxic T lymphocytes (CTL). To test this we examined if superinfected resting CD4+T cells from EC express HIV Gag without producing infectious virus and the susceptibility of these cells to CTL. We found that resting CD4+T cells expressed HIV Gag and were cleared by autologous CD8+T cells from EC. Importantly, we found the extent of CTL clearance in our in vitro assay correlates with in vivo reservoir size and that a population of Gag expressing resting CD4+T cells exists in vivo in patients well controlled on therapy.

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Conflict of interest statement

Competing Interests: The authors have received funding from Merck for some of the data reported. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Superinfected resting CD4+ T cells express HIV proteins without producing detectable viral spread.
(A) Resting CD4+ T cells were isolated from frozen PBMC by negative bead depletion and spinoculated with HIV-1NL4-3. Cells were cultured for 3 days without stimulation in the presence saquinavir (SQV). Resting conditions refer to culture in RPMI+ 10% FCS. After 3 days cells were collected, labeled with a viability dye and activation markers, then fixed and permeabilized and stained for intracellular HIV Gag. (B) Resting CD4+ T cells were gated by excluding HLA-DR, CD25 and CD69 and analyzed for the presence of intracellular Gag with or without addition of Raltegravir at the time of infection, using a reverse-transcriptase inhibitor-treated control also added at the time of infection to set the Gag positive gates. At time 0 and day 3 resting CD4+ T were determined to be 99% negative for activation markers (not shown) but consistent with [24]. (C) The frequency of cells expressing HIV Gag after in vitro infection and 3 day culture was compared among normal donors (ND, n=6) and elite controllers (EC, n=6). (D) Total HIV DNA was measured in superinfected resting or activated cells and compared between +SQV and -SQV fractions from one representative experiment in an EC. (E) Summary data from 4 different EC donors as in D, showing the ratio of total HIV DNA in the -SQV divided by the +SQV fraction. Lines represent the median and p values were generated using the Mann-Whitney test.
Figure 2
Figure 2. GPR cells from EC can be eliminated by autologous CD8+ T cells.
(A) Resting CD4+ T cells were isolated from frozen PBMC and infected as in Figure 1. Autologous CD8+ T cells were purified from a second vial of frozen PBMC by negative bead depletion on day 2 and rested for 24 hours. After labeling the resting CD4+ T cell targets with a viability dye and activation markers, CD8+ T cells and CD4+ T cells were cocultured at a ratio of 10:1 for 1 hour. Cells were then fixed, permeabilized and stained for intracellular Gag and gated on activation marker negative CD4+ T cells for analysis. (B) A representative plot of gated resting CD4+ T cells from an EC or a normal donor that were either cultured alone for 1 hour (no coculture) or cocultured with CD8+ T cells for 1 hour (1hr with CD8s). The same cocultures were treated with an anti-MHC antibody or Raltegravir. The numbers represent the percent of cells that were positive for HIV Gag as above. (C) The percent of GPR cells after 3 days of culture post-infection was compared among samples either cultured alone or cultured with autologous CD8+ T cells for 1 hour (n=10). Lines represent the median and p values were calculated using a Wilcoxon matched-pairs signed rank test. (D) A representative plot of either uninfected or infected EC resting CD4+ T cells after loading with a granzyme B fluorescent substrate and coculture with autologous CD8+ T cells for 1 hour. The values represent the percent of cells positive for cleavage of the granzyme B substrate. (E) Uninfected and infected cells positive for cleavage of the granzyme B substrate were compared (n=4). Lines represent the median and p values were calculated using a Wilcoxon matched-pairs signed rank test. (F) A representative plot of EC resting CD4+ T cells infected with HIV, cultured for 3 days, and either cultured alone or cocultured with autologous CD8+ T cells at a ratio of 10:1 or 1:10 effectors to targets. The values represent the percent of cells positive for HIV Gag. (G) Combination of 3 experiments showing a linear correlation between the percent of infected cells eliminated as calculated by the reduction in HIV Gag positive cells and the natural log of the ratio of effectors to targets at 5 E:T ratios.
Figure 3
Figure 3. Integrated HIV DNA is preferentially cleared over 2-LTR circles after coculture providing an explanation of the EC phenotype of high levels of unintegrated relative to integrated HIV DNA; this clearance of integrated HIV DNA is not an artifact spinoculation.
EC resting CD4+T cells were inoculated without spinoculation, cultured for 3 days and cocultured with autologous CD8+T cells or cultured alone for 16 hours. DNA was then isolated. The fold change is calculated by dividing the copies of the specific HIV DNA intermediate per resting CD4+T cell (i.e. integrated or 2-LTRs) cultured alone by the copies from the same samples cultured with autologous CD8+T cells. For cocultured samples the level measured was multiplied by 10, which is the dilution factor of effectors added to targets. The line represents the median and p value was calculated using the Mann-Whitney test.
Figure 4
Figure 4. GPR cells are more efficiently cleared by CTL in EC than in non-controllers
The percent of GPR cells eliminated after 1 hour coculture with autologous CD8+T cells was compared between EC (n=8) and chronic non-controllers (n=8). The line represents the median and the p value was calculated by a Mann-Whitney test.
Figure 5
Figure 5. CTL clearance of GPR cells correlates with reservoir size in vivo.
(A) Integrated copies per PBMC are plotted for EC with either B57 or B27 or EC without either allele. The line represents the median and the p value was calculated by a Mann-Whitney test. (B) A subset of the patients measured in A was assayed for killing ability as in Figure 4 (n=4 for either HLA group). The line represents the median and the p value was calculated by a Mann-Whitney test. (C) Percent of GPR cells eliminated (as in Figure 4B) after one-hour coculture with autologous CD8+T cells (at 1:1 E:T) is plotted on the x-axis. Integrated copies per million PBMC are plotted for the same patients. EC are represented by red squares and non-controllers by blue circles. The linear correlation was examined using the Pearson test with the p and r2 values reported (n=15).
Figure 6
Figure 6. GPR cells are detectable in vivo.
(A) Representative sort plots and gating strategies from a treated non-controller showing selection of resting CD4+T cells by lineage (except CD4) and activation markers stained in PE (left plot) and the selection of Gag positive and negative cells (right plot). The numbers inside the boxes represent proportion of resting CD4+T cells. Numbers outside of the boxes represent the copies of HIV DNA per cell. TP1 in Table 1. (B) Summary data from 3 sort experiments where HIV DNA was measured in Gag negative and Gag positive sorted populations from ART-suppressed non-controllers as in (A). A one-tailed paired Student’s t-test was used to determine statistical significance. We chose a one-tailed test because a priori we expected that sorted Gag positive cells would be enriched for HIV DNA. If for example, the staining were entirely nonspecific which is the alternative to our hypothesis, we would expect that there would be no enrichment of HIV DNA. There is no rationale for why the sorted Gag positive cells would be depleted for HIV DNA. Lines represent median values. (C) Sort data from one individual was gated more conservatively than the 3 previous sorts for Gag positive cells. The numbers inside the boxes represent proportion of resting CD4+T cells. Numbers outside of the boxes represent the copies of HIV DNA per cell. TP3 in Table 1.
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