HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection

Abstract

Immune control of viral infections is heavily dependent on helper CD4⁺ T cell function. However, the understanding of the contribution of HIV-specific CD4⁺ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4⁺ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4⁺ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4⁺ T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4⁺ T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4⁺ T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4⁺ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4⁺ T cell responses in natural infections.IMPORTANCE Increasing evidence suggests that virus-specific CD4⁺ T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4⁺ T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4⁺ T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4⁺ T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4⁺ T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections.

Keywords: CD4 T helper cells; MHC class II tetramers; human immunodeficiency virus, HIV.

FIG 1

(A) Frequency of targeting of HIV-specific CD4⁺ T cell responses to overlapping peptides across the HIV-1 proteome. HIV-specific CD4⁺ T cell responses against a panel of 410 OLPs spanning the entire HIV proteome were screened. The labels on the x axis indicate the start of the relevant HIV protein or subprotein. The percentages of responders (30/72 individuals screened) with epitope-specific CD4⁺ T cell responses are shown. (B) Percentages of epitope-specific CD4⁺ T cell responses targeting the respective OLPs across the Gag and Nef proteins between controllers (n = 13) and progressors (n = 17) from a chronically infected cohort. No significant differences were observed between the two groups (P = 0.65, based on a nonparametric two-tailed t test). Further analysis of each individual response indicated a significant (*, P = 0.01, Fisher's exact test) more predominant targeting of Gag25 in the Gag p24 region by progressors than by controllers.

FIG 2

HLA class II restriction characteristics of HIV-specific CD4⁺ T cell responses in a cohort with chronic clade C infection. (A) Frequency of various HLA-DRB1 allele variants in CD4⁺ T cell responders (n = 30). The number of responders possessing each allele is indicated in parentheses above each bar. (B) HLA-DRB1 allele restriction characteristics of HIV-specific CD4⁺ T cell responses in individuals with clade C infection (promiscuous epitopes). The restricting HLA alleles for each overlapping peptide are indicated above each bar. Alleles that are highlighted in red were used to generate MHC class II tetramers.

FIG 3

Frequency of tetramer-positive CD4⁺ T cells in chronically infected controllers and progressors and HIV-negative subjects. (A, B) Representative flow plots indicating a dual PE and APC tetramer-staining strategy with gating on double-stained cells, which was used to increase the ability to detect genuine tetramer⁺ cells and minimize nonspecific background staining on CD8⁺ T cells, from an HIV-infected individual (A) and from an HIV-negative individual (B). (C, D) Differences in the percentage of CD4⁺ tetramer⁺ T cells between controllers (Cont) and progressors (Prog) for all class II DRB1*03:01, DRB1*11:01, and DRB1*13:01 tetramers utilized (P = 0.0001) (C) and for only the DRB1*11:01 tetramer (P = 0.0005) (D).

FIG 4

HIV-specific CD4⁺ T cell polyfunctional responses. (A) Graphical representation of intracellular cytokine staining of controller and progressor individuals on the basis of CD107a stimulation. (B to D) The expression of CD107a, IFN-γ, IL-2, and IL-21 in 14 chronically infected subjects divided into controllers and progressors in response to Gag pool stimulation (stim) (B), to Gag41 stimulation (C), and to SEB stimulation (D) was measured. (E, F) The bars depict the frequency of CD4⁺ T cells expressing a combination of the functions indicated below the x axis.

FIG 5

Gag-specific tetramer⁺ CD4⁺ T cell responses correlate with markers of HIV disease progression. (A) The frequency of HIV-specific CD4⁺ T cells measured by four class II tetramers negatively correlated with the contemporaneous viral load (VL) (Spearman r = −0.5, P = 0.005). (B) A negative correlation between the most dominant Gag41 response (DRB1*11:01) and the contemporaneous viral load was also observed. (C) IFN-γ secretion, measured by ICS, exhibited a similar negative correlation with the viral load (Spearman r = −0.7, P = 0.003). (D to G) In addition, the relationship between tetramer⁺ CD4⁺ T cells and cytokine⁺ cells following stimulation with HIV peptides was analyzed.

FIG 6

Frequency of tetramer-positive CD4⁺ T cells in chronically infected controller and progressor individuals following cell expansion. (A, C) Representative flow plots indicating a dual PE and APC tetramer-staining strategy with gating on double-stained cells at the baseline and 2 weeks following expansion of peptide-specific T cell lines from an HIV controller (A) and from an HIV progressor (C). (B, D) Differences in the percentage of CD4⁺ tetramer⁺ T cells in 3 controllers, indicating the responses at the baseline and 2 weeks after expansion in culture (red) for all class II DRB1*03:01, DRB1*11:01, and DRB1*13:01 tetramers utilized (P = 0.02) (B), and for 3 progressors, using the same principle (D). (E) Representative flow plots indicating the functional responses by IFN-γ-specific CD4⁺ T cells to granzyme A, granzyme B, and CD107a following 2 weeks of expansion. (F) Comparison of IFN-γ-specific CD4⁺ T cells to granzyme A (P = 0.03), granzyme B (P = 0.01), and CD107a (P = 0.9) between controllers and progressors.