Increased HIV-specific CD8+ T-cell cytotoxic potential in HIV elite controllers is associated with T-bet expression

Abstract

Recent data suggest that CD8+ T-cell effector activity is an important component in the control of HIV replication in elite controllers (ECs). One critical element of CD8+ T-cell effector function and differentiation is the T-box transcription factor T-bet. In the present study, we assessed T-bet expression, together with the effector proteins perforin, granzyme A (Grz A), granzyme B (Grz B), and granulysin, in HIV-specific CD8+ T cells from ECs (n = 20), chronically infected progressors (CPs; n = 18), and highly active antiretroviral therapy (HAART)-suppressed individuals (n = 19). Compared with the other cohort groups, HIV-specific CD8+ T cells among ECs demonstrated a superior ability to express perforin and Grz B, but with no detectable difference in the levels of Grz A or granulysin. We also observed higher levels of T-bet in HIV-specific CD8+ T cells from ECs, with an ensuing positive correlation between T-bet and levels of both perforin and Grz B. Moreover, HIV-specific CD8+ T cells in ECs up-regulated T-bet to a greater extent than CPs after in vitro expansion, with concomitant up-regulation of perforin and Grz B. These results suggest that T-bet may play an important role in driving effector function, and its modulation may lead to enhanced effector activity against HIV.

Figure 1

HIV-specific CD8⁺ T cells from ECs express higher amounts of perforin and Grz B than CPs after short-term stimulation. (A) Representative flow cytometric plots showing perforin, Grz B, Grz A, and granulysin versus IFN-γ from ECs (top row) and CPs (bottom row). Percentages represent the proportion of IFN-γ–expressing cells that were either positive or negative for each cytolytic protein. The determination of gate placement was aided by consideration of cytolytic protein expression, which is often low or absent, within CD19⁺ B cells or CD4⁺ T cells. (B-C) Relative contribution of perforin, Grz B, Grz A, and granulysin to Gag-specific (B) and Nef-specific (C) CD8⁺ T-cell responses, as identified by staining for IFN-γ, TNFα, or CD107a, is shown among all ECs, CPs, and HAART subjects. For each cytolytic molecule, statistical analysis was carried out using 1-way ANOVA tests (nonparametric; Kruskal-Wallis) followed by a Dunn test for multiple comparisons. *P < .05; **P < .01. Bars represent the means and error bars indicate SDs.

Figure 2

Effector CD8⁺ T cells contain the highest levels of T-bet, which is associated with perforin and Grz B expression in resting cells. (A) Three populations of T-bet are typically observed in human CD8⁺ T cells. Shown is a representative flow cytometric plot of the T-bet staining profile within CD3⁺CD8⁺ T cells from an HIV-negative subject. (B) Representative flow cytometric plots of the memory distribution, as determined by CD27 and CD45RO, of the 3 populations of T-bet: negative (blue), dull (yellow), and bright (red). These various T-bet populations were overlaid onto density plots (black shading) of total CD8⁺ T cells. Percentages represent the fraction of overlaid cells that fell within each quadrant. (C) The memory distributions of T-bet^neg (blue), T-bet^dull (yellow), and T-bet^bright (red) populations, as determined by CD27, CD45RO, and CD57, were established for HIV-negative subjects (n = 8). Bars represent the means and error bars indicate SDs. (D-E) Representative flow cytometric plots showing T-bet against both perforin and Grz B among total CD8⁺ T cells from an HIV-negative (D) and an HIV-positive (E) subject. Percentages represent the fraction of perforin or Grz B that was negative, dull, or bright for T-bet expression.

Figure 3

HIV-specific CD8⁺ T cells from ECs express higher amounts of T-bet than CPs after short-term stimulation. (A) The fraction of Gag- or Nef-specific CD8⁺ T cells, as identified by staining for IFN-γ, TNFα, or CD107a, that fell within the T-bet^bright gate was determined for all ECs, CPs, and HAART subjects. (B) Representative flow cytometric plots from ECs and CPs showing the fraction of the Nef-specific response that fell within the 3 T-bet gates. Events shown have been gated on CD8⁺ T cells. (C) The fraction of CMV-specific CD8⁺ T cells that fell within the T-bet^bright gate was determined for all ECs, CPs, and HAART subjects as in panel A. (A-C) Statistical analysis was carried out using 1-way ANOVA (nonparametric; Kruskal-Wallis) followed by a Dunn test for multiple comparisons. *P < .05; **P < .01. Bars represent the means and error bars indicate SDs.

Figure 4

Positive correlation between T-bet and the expression of both perforin and Grz B within activated CD8⁺ T cells. (A-B) The proportion of Nef-specific perforin (A) and Grz B (B) expression was plotted against the fraction of the response that was T-bet^bright within all therapy-naive subjects (ECs, blue; CPs, red). Spearman correlation tests (nonparametric; 2-tailed) were performed to determine statistical significance as indicated. Only those subjects in whom a Nef-specific response was detected are plotted. (C) Relative levels of T-bet expression are shown within perforin⁺IFN-γ⁺ (gray) and perforin⁻IFN-γ⁺ (red) CD8⁺ T cells from an example Nef-specific response. (D) T-bet expression profile of all perforin⁺ and perforin⁻ Nef-specific CD8⁺ T cells (as identified by staining for IFN-γ, TNFα, or CD107a) was determined among ECs. (E) Relative levels of perforin expression are shown within T-bet^brightIFN-γ⁺ (blue) and T-bet^dull/negIFN-γ⁺ (orange) CD8⁺ T cells from an example Nef-specific response. (F) The degree of perforin and Grz B positivity was determined for all T-bet^bright and T-bet^dull/neg Nef-specific CD8⁺ T cells (as identified by staining for IFN-γ, TNFα, or CD107a) among ECs. (D and F) Mann-Whitney tests (nonparametric; 2-tailed) were performed to determine statistical significance. ***P < .001. Bars represent the means and error bars indicate SDs.

Figure 5

Kinetics of T-bet, perforin, and Grz B up-regulation are tightly linked during an antigen-specific recall response. (A) The degree of Grz B, perforin, and T-bet^bright positivity was determined at baseline for TPR- and RAK-specific CD8⁺ T cells (labeled by the peptide: MHC I tetramer) from 2 HIV-negative donors, ND172 and ND232, respectively. Percentages represent the fraction of tetramer⁺ cells that fell within each gate. (B-C) PBMCs from ND232 were incubated for either 2 (B) or 3 (C) days in the presence of RAK peptide. The levels of Grz B, perforin, and T-bet are shown within RAK-specific cells, which were identified either by peptide restimulation for 6 hours or by tetramer staining. Events shown have been gated on CD8⁺ T cells. (D) CFSE-labeled PBMCs from ND232 were incubated for either 3 or 5 days in the presence of RAK peptide, and the expression of Grz B, perforin, and T-bet was subsequently determined. All flow cytometric plots show only RAK-specific cells, as determined by tetramer staining. Percentages represent the fraction of tetramer⁺ cells that fell within each quadrant. (E) CFSE-labeled PBMCs from ND172 were incubated for 5 days in the presence of the TPR peptide. The MFI of Grz B, perforin, and T-bet within TPR-specific cells was plotted against each generation number, which was established with FlowJo software Version 8.8.4.

Figure 6

HIV-specific CD8⁺ T cells from ECs express higher amounts of T-bet than CPs after proliferation. (A) CFSE-labeled PMBCs from a subset of ECs and CPs were stimulated for 5 days in the presence of a pool of Nef, Gag, or optimal 9-11mer HIV peptides. Representative flow cytometric plots show HIV-specific CD8⁺ T cells, as determined by the production of IFN-γ after restimulation, that have proliferated. Percentages represent the fraction of total CD8⁺ T cells within each gate. (B) The proportion of IFN-γ⁺CFSE^low HIV-specific CD8⁺ T cells positive for perforin, Grz B, and Grz A was determined on day 5 for ECs and CPs. (C-D) T-bet expression was also determined on day 5 after HIV-specific stimulation, with levels reported as the percentage (C) or MFI (D) among IFN-γ⁺CFSE^low HIV-specific CD8⁺ T cells. The reported MFI values indicate the difference between the T-bet^neg population and the T-bet MFI of the IFN-γ⁺CFSE^low cells for each subject. (B-D) Mann-Whitney tests (nonparametric; 2-tailed) were performed to compare ECs with CPs for each functional parameter. **P < .01. Error bars indicate the means and SDs.