CD8 T-cell proliferative capacity is compromised in primary HIV-1 infection

Abstract

Understanding the correlates of immunity that control HIV-1 infection is imperative to our understanding of HIV-1 disease and vaccine development. HIV-1-specific cytotoxic T lymphocytes are fundamental to the control of viremia; however, which T-cell repertoire components enact this control remains unclear. We hypothesize that polyfunctional HIV-1-specific CD8 T cells capable of viral control are present in most patients early in infection and these cells are distinguished by their ability to secrete interleukin (IL)-2 and proliferate. We examined HIV-1-specific CD8 T-cell proliferation and cytokine secretion in primary HIV-1 infection (PHI) using known HIV-1 cytotoxic T-cell epitopes to exclude CD4 bystander effect. We found that only a subset of patients with PHI demonstrated "CD4-independent" CD8 proliferation ex vivo. The remainder of the patients lacked HIV-1-specific CD8 T cells with proliferative capacity, even after the addition of exogenous IL-2. Among the proliferators, IL-2 production from the total HIV-specific CD8 T-cell population correlated with proliferation. Surprisingly, though, we did not routinely detect both IL-2 secretion and proliferative capacity from the same antigen-specific CD8 T cells. Thus, there are distinct and heterogeneous populations of CD8 T cells, phenotypically characterized by either proliferation or IL-2 secretion and few with dual capacity. Generation of these responses may be an important measure of HIV-1 control but are not universal after PHI. Furthermore, the heterogeneity of this population suggests that a simple measure of an effective vaccine response remains elusive.

Figure 1. HIV-1-specific CTL proliferative capacity in PHI

PBMC were labeled with CFSE and stimulated with HIV-1 peptides, Non-HIV-1 peptides (CEF), or SEB for 7 days. CD3+ T cells were analyzed by flow cytometry for proliferation. Proliferation was determined to be 2X unstimulated (media) controls. Strong proliferative responses were seen to all peptides tested in this individual. Peptide key-CEF-(cytomegalovirus, Epstein Barr Virus, Influenza);SEB-(staphylococcal enterotoxin B); A*3 RK9 (RLRPGGKKK); B*35 VY10(VPLDEDFRKY); A*2 SL9- (SLYNTVATL); IG15- (IPLGDAKLVITTYWG)(B*51); B*35 VY8- VPLRPMTY; B*35 TY9-(TVLDVGDAY).

Figure 2. CD4 Independent CD8 T cell proliferation

A) CD4 Independent CD8 T cell proliferation. PBMC were depleted of CD4 T cells (> 96% purity) prior to labeling with CFSE and peptide stimulation for 7 days. CD3+ T cells were analyzed for proliferation by flow cytometry. Representative proliferative responses after CD4 depletion are shown. B) CD4 Independent CD8 T cell proliferation. The bar graph demonstrates CD8 T cell proliferation in the presence of CD4 T cells (shaded bars) and the maintenance of CD8 T cell proliferation in the absence of CD4 T cells (open bars) to multiple epitopes in two subjects(PHI 1 and PHI2). Peptide key- see Figure 1 legend and B*27 KK10-(KRWIILGLNK); vif 70 (QVDPDLADQLIHLYY); vpr31 (EAVRHFPRIWLHSLG). C) Induction of cytokines from CD8 T cells. PBMC from PHI 1 & 2 were stimulated with optimized HIV-1 epitopes and SEB. After gating on CD3+CD8+ T cells, the production of IFN-γ, IL-2 and co-expression of both cytokines was determined. A positive response was determined to be 2X media background.

Figure 3. Induction of cytokine secretion from CD4 and CD8 T cells in Acute HIV-1 infection

A/B) PBMC were stimulated with HIV-1 peptide pools encompassing the entirety of HIV-1. Representative Intracellular Cytokine Staining data demonstrates the induction of Gag specific secretion of IFN-γ, IL-2, and TNF-α from CD4 (panel A) and CD8 (panel B) T cells. C/D) Frequency of cytokine secreting cells from stimulation of CD4 (panel C) or CD8 T cells (panel D). PBMC from PHI subjects were stimulated in vitro with HIV-1 peptide pools (20 mers) encompassing the entirety of HIV (Gag, Pol, Env, Nef, and the rest of the accessory proteins). The cumulative responses for all patients are depicted for each cytokine, IFN-γ (black box), IL–2 (white box), and TNF–α (hatched box).

Figure 4. CD8 T cells with preserved proliferative capacity have increased IL-2 secretion from CD8 T cells compared with non-proliferators

A/B) Subjects were divided into 2 groups, proliferators and non-proliferators based on their proliferative capacity to optimized HIV-1 CTL epitopes. For each subject the portion of the total CD4 (panel A) or CD8 (panel B) T cells that secreted either IL-2 or IFN-γ was calculated and summed for the groups (proliferators vs. non-proliferators). C/D) The frequency of IL- 2 secreting CD4 (panel C) or CD8 (panel D) cells are depicted. Each dot represents one patient and peptide pool combination. The bar represents the median.