Alternative effector-function profiling identifies broad HIV-specific T-cell responses in highly HIV-exposed individuals who remain uninfected

Abstract

The characterization of host immune responses to human immunodeficiency virus (HIV) in HIV controllers and individuals with high exposure but seronegativity to HIV (HESN) is needed to guide the development of effective preventive and therapeutic vaccine candidates. However, several technical hurdles severely limit the definition of an effective virus-specific T-cell response. By using a toggle-peptide approach, which takes HIV sequence diversity into account, and a novel, boosted cytokine staining/flow cytometry strategy, we here describe new patterns of T-cell responses to HIV that would be missed by standard assays. Importantly, this approach also allows detection of broad and strong virus-specific T-cell responses in HESN individuals that are characterized by a T-helper type 1 cytokine-like effector profile and produce cytokines that have been associated with potential control of HIV infection, including interleukin 10, interleukin 13, and interleukin 22. These results establish a novel approach to improve the current understanding of HIV-specific T-cell immunity and identify cellular immune responses and individual cytokines as potential markers of relative HIV resistance. As such, the findings also help develop similar strategies for more-comprehensive assessments of host immune responses to other human infections and immune-mediated disorders.

Keywords: HIV infection; T-cell responses; Th1 cytokines; Th17 cytokines; Th2 cytokines; boosted flow cytometry; highly exposed seronegative; toggled peptides.

Figure 1.

Results of single-cytokine intracellular staining assays and boosted flow for human immunodeficiency virus (HIV)-uninfected subjects. Freshly isolated peripheral blood mononuclear cells were stimulated with either CD3/CD28 magnetic beads, PMA plus ionomycin, or Epstein-Barr virus peptide pools, and intracellular cytokine staining (ICS) and boosted-flow signals were compared. Representative histograms of mean fluorescence intensities after FITC (left) and PE-Cy7 (right) staining for individual cytokines measured by single ICS assays (colors) and by boosted flow (black) are shown after polyclonal stimulation with PMA plus ionomycin (A) or upon T-cell–specific activation through anti-CD3 and anti-CD28 (B). The magnitude of antigen-specific responses detected by boosted flow was compared with respective single-cytokine levels (C) or summed magnitudes (D). Assays are shown for 4 uninfected subjects upon 3 positive stimulations. Medians of magnitudes with ranges (C and D) are represented and compared by the Wilcoxon signed rank test. Abbreviations: IFN-γ, interferon γ; IL, interleukin; TNF-α, tumor necrosis factor α.

Figure 2.

Boosted flow and interferon γ (IFN-γ) enzyme-linked immunosorbent assay (ELISpot) responses against toggle peptides in human immunodeficiency virus (HIV)-infected subjects and individuals with high exposure but seronegativity to HIV (HESN). Freshly isolated peripheral blood mononuclear cells were stimulated with individual toggle peptides spanning the HIV Gag p24 consensus B sequence. Boosted flow and ELISpot analyses were conducted during 5-hour and 16-hour incubation periods, respectively. A, The median breadths of T-cell responses in 17 HIV-infected people, 8 HESN individuals, and 8 HIV-unexposed subjects after applying cutoffs based on positive responses exceeding T-cell responses by 0.03% (white), positive responses exceeding the background activity (Bck) by 3-fold (dark grey), and the threshold value, determined as described in Methods (black). B, The number of positive responses upon toggle-peptide stimulations detected using boosted flow (grey) or ELISpot (white) in 17 HIV-infected people (circles) and 8 HESN individuals (squares). Horizontal lines indicate the median of number of toggle proteins eliciting a positive response. C, The median magnitude of T-cell responses detected using boosted flow (left; grey) or ELISpot (right; white) in 17 HIV-infected patients, 8 HESN individuals, and 8 HIV-unexposed donors. Whiskers denote ranges, and tops and bottoms of boxes denote interquartile ranges. D, Median percentages of matches between the HLA types in subjects and the HLA restriction of the optimal epitope contained in the toggle peptide. Ranges are denoted by whiskers. Response rates between groups were compared by the Mann–Whitney test. Abbreviation: SFC, spot-forming cell.

Figure 3.

Increased detection of human immunodeficiency virus (HIV)–specific responses in individuals with high exposure but seronegativity to HIV (HESN), using boosted flow. Response rates associated with 30 toggle peptides covering HIV Gag p24 are shown for total T cells (A; dark grey) for 7 HIV noncontrollers (squares), 10 HIV controllers (triangles), and 8 HESN individuals (circles). Responses are further stratified into CD4⁺ T cells (B; dark grey) and CD8⁺ T cells (C; dark grey). D, CD8⁺ T-cell responses in either the T-helper type 1 (Th1)–like (light grey) or Th2/17-like (white) cytokine channel are shown. Horizontal bars indicate median numbers of toggle peptides eliciting a positive response. Response rates between groups were compared by the Mann–Whitney test.

Figure 4.

Detection of 13 released cytokines in culture supernatants of peripheral blood mononuclear cells stimulated with toggle peptides. Supernatants of 1-week cultures were assessed for T-helper type 1 (Th1)–like cytokines (interferon γ [IFN-γ], tumor necrosis factor α [TNF-α], interleukin 1β [IL-1β], interleukin 2 [IL-2], and interleukin 12p70 [IL-12p70]), Th2-like cytokines (interleukin 4 [IL-4], interleukin 5 [IL-5], interleukin 6 [IL-6], interleukin 10 [IL-10], and interleukin 13 [IL-13]), and interleukin 17A (IL-17A), interleukin 9 (IL-9), and interleukin 22 (IL-22). The latter 3 cytokines are grouped in panel A as “other Th.” The number of positive responses in each panel is given as the percentage of all stimulated wells. B, Distribution of cytokine signals among all positive responses in 8 individuals with high exposure but seronegativity to human immunodeficiency virus (HIV; HESN), 10 HIV controllers, and 7 noncontrollers. C, Individual cytokine levels in 17 HIV-infected individuals and 8 HESN individuals. P values are from comparisons by the Mann–Whitney test. Horizontal lines indicate median of cytokine levels (pg/ml) per patient and stimulation.