Magnitude and Kinetics of CD8+ T Cell Activation during Hyperacute HIV Infection Impact Viral Set Point

Abstract

CD8(+) T cells contribute to the control of HIV, but it is not clear whether initial immune responses modulate the viral set point. We screened high-risk uninfected women twice a week for plasma HIV RNA and identified 12 hyperacute infections. Onset of viremia elicited a massive HIV-specific CD8(+) T cell response, with limited bystander activation of non-HIV memory CD8(+) T cells. HIV-specific CD8(+) T cells secreted little interferon-γ, underwent rapid apoptosis, and failed to upregulate the interleukin-7 receptor, known to be important for T cell survival. The rapidity to peak CD8(+) T cell activation and the absolute magnitude of activation induced by the exponential rise in viremia were inversely correlated with set point viremia. These data indicate that rapid, high magnitude HIV-induced CD8(+) T cell responses are crucial for subsequent immune control of acute infection, which has important implications for HIV vaccine design.

Figure 1. Dynamics of absolute CD4 count and plasma viral loads during hyperacute HIV infection

Plasma HIV-1 RNA magnitude (red) and absolute CD4 counts (blue) before HIV infection and following onset of detectable plasma viremia in 12 subjects with hyperacute HIV infection that were not receiving antiretroviral treatment. See also table S1

Figure 2. CD8⁺ T cell activation following onset of plasma viremia

Longitudinal analysis of CD38 and HLA-DR expression on CD8⁺ cells after HIV infection. (a) Representative FACS plots gated on CD8⁺ cells expressing CD38 and HLA-DR. Days following onset of plasma viremia (DFOPV) are indicated (b) Longitudinal dynamics of activated (CD38⁺HLA-DR⁺) CD8⁺ cells for 11 donors in whom samples were available for analysis. (c) Representative relationship between HIV-induced CD8⁺ cell activation kinetics and contemporaneous viral load. (d) Correlation between time to peak activation and viral load setpoint. (e) Correlation between frequency of activated CD8⁺ cells at peak activation and viral load set point. Spearman’s rank correlation was used in (d) and (e).

Figure 3. CD8+ T cell proliferation and apoptosis following onset of viremia

(a) Representative FACS plots gated on CD8⁺ cells expressing Ki-67 and Bcl-2. (b) Summary data for the longitudinal analysis of proliferating and pro-apoptotic CD8⁺ cells for 11 donors evaluated. (c) Representative relationship between Ki67^negBcl-2^lowCD8⁺ T cells and contemporaneous viral load. (d) CD8⁺T cell co-expression of CD38, HLA-DR, Ki67 and Bcl-2. (e) Correlation between peak Ki67^negBcl-2^lowCD8⁺ T cells and viral load set point. Spearman’s rank correlation was used in (e).

Figure 4. Hyperacute HIV infection induces strong CD8⁺ T cell responses with degranulation potential

HIV-specific CD8⁺ T cell responses were measured by ICS after overnight incubation with autologous HIV-infected CD4⁺ cells. Uninfected autologous CD4⁺ cells co-cultured with CD8⁺ cells were used as a negative control. (a) Representative flow plots demonstrating CD107a expression on CD8⁺ cells. (b) Upper panel shows CD107a expression on ex vivo CD8+ cells from 14 DFOPV incubated for 12 hours with HIV infected autologous CD4+ T cells. The lower panel shows CD107a expression on CD8⁺ cells from 14 DFOPV incubated in IL-2 containing media for 5 days prior to co-culture with infected autologous CD4⁺ T cells. (c) Proportion of CD8+ cells secreting cytokines following stimulation with HIV infected autologous CD4⁺ cells. Background responses in the uninfected co-culture condition are subtracted. Statistical significance was determined using Mann-Whitney and Kruskal Wallis tests. (d) Comparison between proliferating CD8⁺ T cells and CD107a positive cells at peak activation time points (also see supplementary figures). Statistical significance was determined using Mann-Whitney test. See also Figure S1, S2 and S3

Figure 5. Analysis of HIV-specific CD8⁺ T cell responses during hyperacute HIV infection using MHC class I tetramers

PBMCs were stained with MHC class I tetramers specific for HIV and antibodies against CD38 and HLA-DR. All plots are gated on CD8⁺ T cells. Upper panels show frequencies of HIV-specific CD8⁺ T cells for each HIV tetramer tested. Blue dots depict the frequency of tetramer⁺ cells co-expressing CD38 and HLA-DR overlaid on total CD8⁺ T cells (red dots). Data for subjects 208, 079 and 093 are shown in panels (a), (b) and (c), respectively. (d) Flow plots show combined tetramer staining and cytokine secretion measured by ICS. Representative data for one of the four donors tested are shown. (e) Proportion of HIV-specific and CMV-specific tetramer⁺ cells secreting IFN-γ following optimal peptide stimulation of a peak activation sample. Statistical significance was determined using Mann-Whitney test.

Figure 6. Assessment of bystander CD8⁺ T cell activation during hyperacute HIV infection

Intra-donor activation (CD8⁺ CD38⁺ HLA-DR⁺ cells) profiles of HIV, CMV, EBV and influenza virus specific (tetramer⁺) CD8⁺ T cells. Data for two donors with detectable tetramer⁺ cells specific for three different pathogens are shown in panels (a) and (b). The first columns show flow plots gated on tetramer⁺ cells, the second column shows flow plots gated on tetramer⁺ cells that are double positive for CD38 and HLA-DR (blue dots) overlaid on total CD8⁺ T cells (red dots). (c) Activation data for 13 HIV, 5 CMV, 3 EBV and 3 influenza specific tetramer⁺ cells are graphed. Statistical significance was determined using Kruskal Wallis test.

Figure 7. Activation profiles of CD8⁺ T cells in hyperacute HIV infection

(a) Phenotypic analysis of HIV specific tetramer⁺ CD8⁺ cells. The first flow plot is gated on tetramer⁺ cells, the second, third and fourth plots depict tetramer⁺ cells (blue dots) overlaid on total CD8+ cells. Data showing frequencies of tetramer⁺ cells that are (b) Ki67^high, BCL2^low and (c) CD127^high are graphed. Statistical significance was determined using Mann-Whitney and Kruskal Wallis tests. (d) Frequencies of dead CD8⁺ T cells (annexin-V⁺, PI⁺ CD8⁺ cells). Statistical significance was determined using Mann-Whitney test. (e) Frequencies of dead CD4⁺ T cells (annexin-V⁺, PI⁺ CD4⁺ cells) after overnight incubation in R10. Statistical significance was determined using Mann-Whitney test. (f) Flow cytometric data for two donors showing ex vivo expression of annexin V by HIV tetramer⁺ and CMV tetramer⁺ cells at peak activation.