HIV controller CD4+ T cells respond to minimal amounts of Gag antigen due to high TCR avidity

Abstract

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral treatment. Emerging evidence indicates that HIV control is mediated through very active cellular immune responses, though how such responses can persist over time without immune exhaustion is not yet understood. To investigate the nature of memory CD4+ T cells responsible for long-term anti-HIV responses, we characterized the growth kinetics, Vbeta repertoire, and avidity for antigen of patient-derived primary CD4+ T cell lines. Specific cell lines were obtained at a high rate for both HIV controllers (16/17) and efficiently treated patients (19/20) in response to the immunodominant Gag293 peptide. However, lines from controllers showed faster growth kinetics than those of treated patients. After normalizing for growth rates, IFN-gamma responses directed against the immunodominant Gag293 peptide showed higher functional avidity in HIV controllers, indicating differentiation into highly efficient effector cells. In contrast, responses to Gag161, Gag263, or CMV peptides did not differ between groups. Gag293-specific CD4+ T cells were characterized by a diverse Vbeta repertoire, suggesting that multiple clones contributed to the high avidity CD4+ T cell population in controllers. The high functional avidity of the Gag293-specific response could be explained by a high avidity interaction between the TCR and the peptide-MHC complex, as demonstrated by MHC class II tetramer binding. Thus, HIV controllers harbor a pool of memory CD4+ T cells with the intrinsic ability to recognize minimal amounts of Gag antigen, which may explain how they maintain an active antiviral response in the face of very low viremia.

Figure 1. Rapid Growth of CD4+ T cell lines from HIV controllers.

Growth of viable CD4+ T cell lines specific for Gag293, Gag263, Gag161 and CMV peptides was measured in groups of HIV controllers (HIC), viremic patients (VIR), and treated patients (HAART). The growth ratio, corresponding to the number of observed cells divided by the number of input cells, was evaluated at day 7 (A) and day 14 (B) of culture. Each symbol corresponds to one subject. Horizontal bars indicate medians. All statistically significant differences (P<0.05) evaluated by the Mann-Whitney U test are reported on graphs.

Figure 2. Increased IFN-γ production by CD4+ T cell lines from HIV controllers.

Production of IFN-γ by CD4+ T cell lines specific for Gag293, Gag263, Gag161 and CMV peptides were compared between groups of HIV controllers (HIC), viremic patients (VIR), and treated patients (HAART). IFN-γ production was measured by ELISPOT assay on CD4+ T cell lines restimulated with peptide, and was expressed by the number of spot forming cells (SFC) per million cells. All measurements were made at CD4+ T cell line doubling time. Horizontal bars indicate medians. All statistically significant differences (P<0.05) evaluated by the Mann-Whitney U test are reported on graphs.

Figure 3. Increased functional avidity of memory CD4+ T cells from HIV controllers.

The functional avidity was evaluated by IFN-γ ELISPOT assay in the presence of decreasing peptide concentrations (serial dilutions from 4×10⁻⁶ M to 10⁻¹¹ M). For each peptide dilution, IFN-γ production was expressed as the number of spot forming cells (SFC) per million cells. The functional avidity was defined as the last peptide dilution that gave a positive IFN-γ response at least 2 fold above background level. (A) Representative examples of functional avidity measurement in response to Gag293 for one HIV controller (HIC) and one treated patient (HAART). The peptide dilution at half response (EC₅₀) and the last positive peptide dilution are indicated. (B) Functional avidity of CD4+ T cell lines specific for Gag293, Gag263, Gag161 and CMV peptides were compared between groups of HIV controllers (HIC), viremic patients (VIR), and treated patients (HAART). All measurements were made at CD4+ T cell line doubling time. Horizontal bars indicate medians. All statistically significant differences (P<0.05) evaluated by the Mann-Whitney U test are reported on graphs.

Figure 4. Characterization of HIV specific CD4+ T cells by MHC class II tetramer staining.

(A) Representative examples of class II tetramer staining for Gag293-specific CD4+ T cells in cell lines at doubling time. Four distinct tetramers corresponding to alleles DRB1*0101, DRB1*0401, DRB1*0701 and DRB5*0101 were loaded with the Gag293 peptide and used to stain CD4+ T cell lines from HIV controllers (HIC) and treated patients (HAART) with matching HLA DR alleles. Negative controls corresponding to cell lines labeled with a DR-matched CLIP-loaded tetramer are shown for the 4 HAART cell lines. (B) Class II tetramer staining for Gag293-specific CD4+ T cells in cell lines from two HIV controllers (HIC) after 18 days of culture, showing the amplification of the tetramer+ population. (C) The percentage of Gag293-specific tetramer+ cells in CD4+ T cell lines at doubling time did not differ significantly between the HIC and HAART groups, as evaluated by the Mann-Whitney U test.

Figure 5. Gag293-specific CD4+ T cells have a diverse Vβ repertoire.

(A) Example of TCR Vβ staining: comparison of three Vβ specificities in the tetramer-negative (left panel) and tetramer-positive (right panel) CD4+ T cell populations obtained for a Gag293-specific cell line from an HIV controller. In this case, Vb13.6 expression is amplified in the tetramer-positive population. (B) Example of Vβ repertoire analysis in an HIV controller CD4+ T cell line at doubling time. The clonogram indicates the percentage of CD4+ T cells labeled with a given Vβ-specific antibody within the tetramer-negative (black bars) and tetramer-positive (red bars) populations. The presence of multiple Vβ specificities within tetramer-positive population indicates that Gag293-specific CD4+ T cells have a diverse Vβ repertoire.

Figure 6. Increased TCR avidity of memory CD4+ T cells in HIV controllers.

(A) The TCR avidity of Gag293-specific CD4+ T cells was determined by MHC class II tetramer staining in the presence of decreasing tetramer concentrations. An example shows the progressive decrease of Gag293-DRB1*0101 tetramer staining as a function of tetramer concentration in an HIV controller CD4+ T cell line at doubling time. (B) Representative examples of TCR avidity measurement in Gag293-specific CD4+ T cell lines from one HIV controller (HIC) and one treated patient (HAART). The tetramer dilution at half response (EC₅₀) and the last positive tetramer dilution are indicated. (C) The TCR avidity was defined as the inverse of the last tetramer dilution that gave a positive staining at least 2 fold above control CLIP-tetramer staining. The TCR avidity of Gag293-specific CD4+ T cell lines was significantly higher in the controller group (HIC) than in the treated patient group (HAART) as evaluated by the Mann-Whitney U test. Horizontal bars indicate medians.