CD4+ T-cell help enhances NK cell function following therapeutic HIV-1 vaccination

Abstract

Increasing data suggest that NK cells can mediate antiviral activity in HIV-1-infected humans, and as such, novel approaches harnessing the anti-HIV-1 function of both T cells and NK cells represent attractive options to improve future HIV-1 immunotherapies. Chronic progressive HIV-1 infection has been associated with a loss of CD4(+) T helper cell function and with the accumulation of anergic NK cells. As several studies have suggested that cytokines produced by CD4(+) T cells are required to enhance NK cell function in various infection models, we hypothesized that reconstitution of HIV-1-specific CD4(+) T-cell responses by therapeutic immunization would restore NK cell activity in infected individuals. Using flow cytometry, we examined the function of CD4(+) T cells and NK cells in response to HIV-1 in subjects with treated chronic HIV-1 infection before and after immunization with an adjuvanted HIV-1 Gp120/NefTat subunit protein vaccine candidate provided by GlaxoSmithKline. Vaccination induced an increased expression of interleukin-2 (IL-2) by Gp120-specific CD4(+) T cells in response to HIV-1 peptides ex vivo, which was associated with enhanced production of gamma interferon (IFN-γ) by NK cells. Our data show that reconstitution of HIV-1-specific CD4(+) T-cell function by therapeutic immunization can enhance NK cell activity in HIV-1-infected individuals.

Importance: NK cells are effector cells of the innate immune system and are important in the control of viral infection. Recent studies have demonstrated the crucial role played by NK cells in controlling and/or limiting acquisition of HIV-1 infection. However, NK cell function is impaired during progressive HIV-1 infection. We recently showed that therapeutic immunization of treated HIV-1-infected individuals reconstituted strong T-cell responses, measured notably by their production of IL-2, a cytokine that can activate NK cells. The current study suggests that reconstitution of T-cell function by therapeutic vaccination can enhance NK cell activity in individuals with chronic HIV-1 infection. Our findings provide new insights into the interplay between adaptive and innate immune mechanisms involved in HIV-1 immunity and unveil opportunities to harness NK cell function in future therapeutic vaccine strategies to target HIV-1.

FIG 1

Increased production of IFN-γ by NK cells correlates with IL-2 production in response to HIV-1 Gag. (A) Quantification of IL-2 in the supernatant after 20 h of incubation of PBMCs in the presence of HIV-1 Gp120- or Gag-derived peptides in 17 HIV-infected individuals, including 6 with chronic ART-treated infection, 5 with untreated progressive infection, and 6 elite controllers. P < 0.05 (*) and P < 0.005 (**) compared to unstimulated PBMCs. (B) Each bar represents the mean ± SEM concentration of IL-2 in the supernatant for each cohort after stimulation with the indicated peptides. (C) Flow cytometry panels show representative examples of IL-2 production by HIV-1-specific CD4⁺ T cells (upper row) and IFN-γ production by NK cells (lower row) after intracellular staining. (D) Percentages of IFN-γ⁺ NK cells after 20 h of incubation of PBMCs in the presence of the indicated HIV-1-derived peptides. **, P < 0.005 compared with unstimulated PBMCs. (E) Each bar represents the mean ± SEM IFN-γ⁺ NK cell percentages for each cohort after stimulation with the indicated peptides. (F) Positive correlation between the concentration of IL-2 in the supernatant and the percentages of IFN-γ⁺ NK cells after 20 h of incubation of PBMCs in the presence of Gag peptides (Spearman's rank correlation test). Data are reported after background subtraction. Horizontal lines indicate the medians. Statistical differences with P < 0.05 are indicated and were determined using the nonparametric Wilcoxon signed-rank test.

FIG 2

Reconstitution of IL-2 production by therapeutic vaccination leads to enhanced NK cell function. Proportions of CD4⁺ T cells that produce IL-2 (A) and of NK cells that produce IFN-γ (B) quantified by intracellular cytokine staining in response to Gp120- and Nef-derived HIV-1 peptides before (W0) and after (W6/W14) vaccination. The plots on the left represent the vaccine group, while the plots on the right represent the control group. Values are represented before and after vaccination for each subject. Statistical differences with P < 0.05 are indicated and were determined using the nonparametric Wilcoxon signed-rank test. Data are reported after background subtraction. (C) Percentages of IFN-γ⁺ NK cells in PBMCs and CD4⁺ T-cell-depleted PBMCs from 4 vaccinated subjects postvaccination, stimulated with either HIV-1 Gp120 peptides (left) or CMV pp65 peptides (right). Statistical differences with P < 0.05 are indicated and were determined using the nonparametric Wilcoxon signed-rank test. Data are reported after background subtraction. (D) Flow cytometry panels show representative examples of IFN-γ production by NK cells after intracellular cytokine staining of PBMCs that were left unstimulated or were stimulated with Gp120 in the presence of either isotype antibody control or IL-2 and IL-12 blocking antibodies, as indicated, in a vaccinated individual at 6 weeks postimmunization. (E) Relative percentages of IFN-γ⁺ NK cells after stimulation of PBMCs from 8 study subjects postvaccination with Gp120-derived peptides in the presence of isotype control antibody, IL-2 neutralizing antibody, or IL-2 and IL-12 neutralizing antibodies, as indicated. Values are expressed relative to the average percentage of IFN-γ⁺ NK cells in the absence of blocking antibodies after background subtraction, which was given the arbitrary value of 100%. Displayed values are the means ± SEM. Statistical differences with P < 0.05 are indicated and were determined using the nonparametric Wilcoxon signed-rank test.