HIV controllers maintain a population of highly efficient Th1 effector cells in contrast to patients treated in the long term

Abstract

HIV controllers are rare individuals who spontaneously control HIV replication in the absence of antiretroviral therapy. To identify parameters of the CD4 response that may contribute to viral control rather than merely reflect a persistently low viremia, we compared the T helper profiles in two groups of patients with more than 10 years of viral suppression: HIV controllers from the Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) CO18 cohort (n = 26) and efficiently treated patients (n = 16). Cells specific for immunodominant Gag and cytomegalovirus (CMV) peptides were evaluated for the production of 10 cytokines and cytotoxicity markers and were also directly quantified ex vivo by major histocompatibility complex (MHC) class II tetramer staining. HIV controller CD4(+) T cells were characterized by a higher frequency of gamma interferon (IFN-γ) production, perforin(+)/CD107a(+) expression, and polyfunctionality in response to Gag peptides. While interleukin 4 (IL-4), IL-17, and IL-21 production did not differ between groups, the cells of treated patients produced more IL-10 in response to Gag and CMV peptides, pointing to persistent negative immunoregulation after long-term antiretroviral therapy. Gag293 tetramer-positive cells were detected at a high frequency (0.12%) and correlated positively with IFN-γ-producing CD4(+) T cells in the controller group (R = 0.73; P = 0.003). Tetramer-positive cells were fewer in the highly active antiretroviral therapy (HAART) group (0.04%) and did not correlate with IFN-γ production, supporting the notion of a persistent immune dysfunction in HIV-specific CD4(+) T cells of treated patients. In conclusion, HIV controllers maintained a population of highly efficient Th1 effectors directed against Gag in spite of a persistently low antigenemia, while patients treated in the long term showed a loss of CD4 effector functions.

Fig 1

Intracellular cytokine production in patients' CD4⁺ T cells stimulated with Gag peptides. Intracellular cytokine staining (ICS) was done on PBMC stimulated overnight with the immunodominant peptides Gag161, Gag263, and Gag293. Cytokine production was evaluated in the live CD3⁺ CD4⁺ population. (A) Examples of ICS in CD4⁺ T cells from patients stimulated with Gag293, Gag263, or Gag161. The first 7 plots show the production of TNF-α, MIP-1β, IL-2, IL-4, IL-17, IL-21, and IL-10 combined with that of IFN-γ. The last plot on the right, second row, shows the combined expression of total intracellular perforin and of the degranulation marker CD107a, which was used to determine the cytotoxic potential of CD4⁺ T cells. The patient identification number and the peptide used for stimulation are indicated above each plot. (B) Quantitation of the percentage of cytokine-producing CD4⁺ T cells detected in HIV controllers (HIC) and treated (HAART) patients. The summed percentages of cytokine-positive cells in response to Gag161, Gag263, and Gag293 are listed. Horizontal bars indicate the median values. Significant P values (<0.05) obtained with the nonparametric Mann-Whitney U test are shown.

Fig 2

Analysis of intracellular cytokine production to individual Gag and CMV peptides. (A and C) The median percentage of cytokine-producing cells induced by each of the three immunodominant Gag peptides is represented in stacked columns (Gag293, gray; Gag263, black; Gag161, white). (B and D) Cytokine production induced by optimal CMV peptides chosen in the function of patients' HLA-DR alleles are represented by the hatched bars. Median cytokine responses that did not reach the detection threshold are represented by the midpoint between the threshold value of 0.01% and zero (0.005%). Significant differences obtained in the Mann-Whitney U test are indicated by the asterisks.

Fig 3

Polyfunctionality of Gag293-specific CD4⁺ T cells. (A and B) Polyfunctionality analysis for the CD107a/IFN-γ/IL-2/MIP-1β/TNF-α panel. The colors in the pie charts represent the proportions of monofunctional cells (dark blue), bifunctional cells (light blue), trifunctional cells (green), quadrifunctional cells (yellow), and pentafunctional cells (orange) within the population of Gag293-specific CD4⁺ T cells, as determined by ICS. The bar chart shows the frequency of Gag293-specific cells in each functional category, with red bars corresponding to HIV controllers (HIC) and dark blue bars corresponding to treated patients (HAART). *, significant difference in bifunctional cells between the HIC and HAART groups (P < 0.05). (C and D) Polyfunctionality analysis for the IFN-γ/IL-10/IL-17/IL-21 panel. The colors in the pie charts represent the proportions of monofunctional cells (dark blue), bifunctional cells (light blue), trifunctional cells (green), and quadrifunctional cells (yellow) within the population of Gag293-specific CD4⁺ T cells. The bar chart shows the frequency of Gag293-specific cells in each functional category. These analyses were carried out with the SPICE 5.21 program.

Fig 4

Detection of Gag293-specific CD4⁺ T cells by MHC class II tetramer staining. (A) Gating strategy to detect Gag293-specific cells in CD4⁺ T cells. Tetramer staining was analyzed in viable CD3⁺ CD8⁻ CD14⁻ CD19⁻ CD4⁺ lymphocytes, starting from a minimum of 6 × 10⁶ PBMC. (B) Examples of detection of Gag293-specific cells with 4 different tetramers based on the DRB1*0101, DRB1*0701, DRB1*1101, and DRB5*0101 molecules. For each tetramer, the percentage of tetramer-positive (Tet⁺) cells in CD4⁺ T cells is indicated for one healthy donor (HD), one treated patient (HAART), and one HIV controller (HIC). A negative control consisting of CD4⁺ T cells labeled with a CLIP-loaded tetramer is shown for each of the HIV controllers tested (HIC-ctrl, right column).

Fig 5

Frequency and phenotype of Gag293-specific tetramer-positive CD4⁺ T cells in HIV controllers (HIC) and treated patients (HAART). (A) The percentages of tetramer-positive (Tet⁺) CD4⁺ T cells specific for Gag293 were compared in the HIC and HAART groups. (B) The percentages of Tet⁺ cells specific for optimal CMV pp65 peptides were compared in the HIC and HAART groups. (C and D) The percentages of central memory (CM) (CD45RA⁻ CCR7⁺) and effector memory (EM) (CD45RA⁻ CCR7⁻) CD4⁺ T cells were analyzed in the Gag293 Tet⁺ population (C) and the CMV Tet⁺ population (D). (E and F) The percentages of CD27⁺ cells were analyzed in the CM and EM Gag293 Tet⁺ populations (E) and in the CM and EM CMV Tet⁺ populations (F). (G and H) The percentages of CD38⁺ cells were analyzed in the CM and EM Gag293 Tet⁺ populations (G) and in the CM and EM CMV Tet⁺ populations (H). The horizontal bars indicate the median values. Significant P values (<0.05) obtained with the nonparametric Mann-Whitney U test are shown.

Fig 6

Correlations between the percentages of Gag293-specific cells detected by tetramer staining and by intracellular cytokine staining (ICS). (A) Correlation between the percentage of Gag293 tetramer-positive CD4⁺ T cells (%Gag293 Tet⁺) (x axis) and the percentage of IFN-γ secreting CD4⁺ T cells detected by ICS after Gag293 stimulation (%IFN-γ⁺) (y axis) in the HIV controller (HIC) group. (B) Correlation between the percentages of Gag293 Tet⁺ cells and Gag293-specific IFN-γ secreting CD4⁺ T cells in the HAART group. (C) Correlation between the percentages of Gag293 Tet⁺ cells and Gag293-specific IL-17-γ-secreting CD4⁺ T cells in the HIC group. (D) Correlation between the percentages of Gag293 Tet⁺ cells and Gag293-specific IL-17-γ-secreting CD4⁺ T cells in the HAART group. The nonparametric Spearman correlation coefficient R and the P value are shown for each correlation.