Immune activation and CD8+ T-cell differentiation towards senescence in HIV-1 infection

Abstract

Progress in the fight against the HIV/AIDS epidemic is hindered by our failure to elucidate the precise reasons for the onset of immunodeficiency in HIV-1 infection. Increasing evidence suggests that elevated immune activation is associated with poor outcome in HIV-1 pathogenesis. However, the basis of this association remains unclear. Through ex vivo analysis of virus-specific CD8(+) T-cells and the use of an in vitro model of naïve CD8(+) T-cell priming, we show that the activation level and the differentiation state of T-cells are closely related. Acute HIV-1 infection induces massive activation of CD8(+) T-cells, affecting many cell populations, not only those specific for HIV-1, which results in further differentiation of these cells. HIV disease progression correlates with increased proportions of highly differentiated CD8(+) T-cells, which exhibit characteristics of replicative senescence and probably indicate a decline in T-cell competence of the infected person. The differentiation of CD8(+) and CD4(+) T-cells towards a state of replicative senescence is a natural process. It can be driven by excessive levels of immune stimulation. This may be part of the mechanism through which HIV-1-mediated immune activation exhausts the capacity of the immune system.

Figure 1. CD8⁺ T-Cell Activation during Acute HIV-1 Infection

(A) Percentages of activated CD38⁺ cells (gated on whole CD8⁺ T-cells, HIV tetramer-positive CD8⁺ T-cells, or whole CD4⁺ T-cells) in donors during acute HIV-1 infection and later postacute on ART (n = 12); healthy donors (n = 11) and untreated donors with nonprogressing chronic infection (n = 12) are also shown. (B and C) CD38 and Ki67 expression on CD8⁺ T-cell subsets defined by CD45RA/CD62L (B) or CD28/CD27 (C) expression, shown in one single donor from acute to postacute (on ART) HIV-1 infection. Percentages of positive cells are shown. Means (± SEM) of CD38⁺ and Ki67⁺ CD8⁺ T-cells for ten patients are also shown; statistics concern CD38 expression. (D) Staining for the activation marker CD38 on CMV-, EBV-, or influenza A virus-specific CD8⁺ T-cells during acute and postacute (on ART) HIV-1 infection in a single donor. Percentages of CD38⁺ tetramer-positive CD8⁺ T-cells are shown. Data on all donors (see Table 1) are also shown. (E) Activation (CD38 and Ki67 staining) of CMV-specific CD8⁺ T-cells or whole CD8⁺ T-cell population during acute and postacute (on ART) HIV-1 infection in a single donor. Percentages of cells present in quadrants are shown. Statistics: * p < 0.002, ** p < 0.01, NS = nonsignificant, with the nonparametric Mann–Whitney test.

Figure 2. In Vitro Priming of Antigen-Specific CD8⁺ T-Cells

(A) Representative stainings for melan-A-specific CD8⁺ T-cells following priming of naïve cells from healthy donor PBMCs by autologous mature DCs loaded with various concentrations of antigen. Cells are gated on lymphocytes 47 d after priming. Percentages of melan-A tetramer-positive CD8⁺ T-cells are shown. (B) Percentages of melan-A-specific CD8⁺ T-cells over time following priming at day 0 with mature DCs loaded with various concentrations of antigen, with no restimulation or with restimulation using mature DCs at day 25. The legend indicates the concentration of melan-A–peptide used in microgram per milliliter; populations generated with 0 or 10⁻³ μg/ml of antigen are plotted on the right-hand side Y axis. (C) Percentages of melan-A tetramer-positive CD8⁺ T-cells expressing granzyme A, Ki67, CD62L, or CD57 according to antigen concentration used, at day 30 following priming. Ki67 and CD57 expressions are plotted on the right-hand side Y axis. (D) CD28 and CD27 expression on melan-A tetramer-positive CD8⁺ T-cells in PBMC (day 0), and over time following priming with 1 μg/ml of antigen. Percentages of cells present in quadrants are shown. The model of CD8⁺ T-cell differentiation based on CD28 and CD27 expression is illustrated (top left panel). (E) Distribution of the melan-A-specific CD8⁺ T-cells into the distinct differentiated subsets according to antigen concentration used, at day 47 following priming. Similar observations were made whether the cells were subjected to a second round of stimulation or not. Data are representative of three independent experiments.

Figure 3. Activation and Differentiation of Antigen-Specific CD8⁺ T-Cells during HIV-1 Infection

(A) Representative staining for the differentiation marker CD27 on three HIV-specific (HLA-B8 nef, HLA-A2 p17, and HLA-B8 p24) populations in a single HIV-1-infected donor. Numbers show percentages of tetramer-positive CD8⁺ T-cells (outside the quadrants) and percentages of CD27⁻ tetramer-positive cells (inside the quadrants). (B) Correlation between size (percentage of tetramer-positive CD8⁺ T-cells) and differentiation (percentages of CD27⁻ tetramer-positive cells) of CD8⁺ T-cells specific for HIV antigens (including HLA-A2 p17, pol, HLA-B7 nef, gp41, HLA-B8 nef, p24, and HLA-B57 p24) (open circles), CMV antigens (including HLA-A2, B7, and B35 pp65) (filled circles), EBV (HLA-A2 BMLF1, HLA-B8 BZLF1, EBNA3A) (filled squares), and influenza (HLA-A2 matrix) (open squares) antigens or all antigens together. These populations were studied in individuals with chronic infection for HIV, CMV, or EBV (independently from clinical status). P values were obtained using the nonparametric Spearman rank correlation test. (C) CD28 and CD27 expression on whole, HIV nef-, or p24-specific CD8⁺ T-cells during acute and postacute (on ART) HIV-1 infection in a single donor. (D) CD28 and CD27 expression on CMV-, EBV-, or influenza-specific CD8⁺ T-cells during acute and postacute (on ART) HIV-1 infection in a single donor. Percentages of cells present in quadrants are shown.

Figure 4. CD8⁺ T-Cell Differentiation and HIV-1 Disease Progression

(A) Distribution of the CD8⁺ T-cell population in differentiated subsets (CD28⁺/CD27⁺ early, CD28⁻/CD27⁺ intermediate, and CD28⁻/CD27⁻ late) through the course of HIV-1 infection. Abbreviations: H, healthy (n = 15); A, acute HIV infection (n = 11); C, chronic HIV infection nonprogressor (no ART; n = 14); P, chronic HIV infection with signs of disease progression (no ART; n = 10). Statistics: * p < 0.0001 with the ANOVA test and p < 0.005 between each group. (B) Percentages of CD27⁻ CD8⁺ T-cells that are specific for HLA-B8 HIV (nef) or HLA-A2 CMV in HIV-1-infected individuals at different stages of infection. Statistics: ** p < 0.005 with the nonparametric Mann–Whitney test. (C) Inverse correlation between CD4⁺ T-cell counts and percentage of highly differentiated CD27⁻ cells in the whole CD8⁺ T-cell population of HIV-1-infected donors during chronic infection (untreated nonprogressors and progressors). The p value was obtained using the nonparametric Spearman rank correlation test.

Figure 5. CD8⁺ T-Cell Differentiation and Senescence

(A) Expression of the replicative senescence-associated marker CD57 on antigen-experienced CD8⁺ T-cell subsets. The percentage and mean fluorescence intensity for the CD57⁺ cells are shown for one single donor. Data on several donors (HIV-1-infected or healthy) are also shown (n = 24). (B) Expression of CD57 on CD8⁺ T-cells (whole population or antigen-specific) from acute to postacute (on ART) HIV-1 infection. (C) CD69 expression and CFSE proliferation profile for CD8⁺ T-cell subsets gated on the basis of CD57 and CD27 expression following stimulation with anti-CD3 antibodies. PBMCs were analysed for CD69 expression after 18 h and CFSE labeling after 6 d. Percentages of proliferating cells (with background subtracted) are indicated. Representative results from three experiments (one HIV-infected and two healthy donors) are shown. (D) Telomere length measurement by flow FISH on naïve and antigen-experienced CD8⁺ T-cell subsets FACS-sorted on the basis of CD57, CD27, CCR7, and CD45RA expression. The average length of telomeres was obtained by substracting the mean fluorescence of the background control (no probe; open histogram) from the mean fluorescence obtained from cells hybridised with the FITC-labeled telomere probe (gray histogram). Representative results from two experiments (on healthy donors) are shown. (E) CD57 and perforin expression in the CD8⁺ T-cell population dissected into naïve (CD27^+high, perforin-negative), antigen-experienced CD27⁺ (perforin^low), and antigen-experienced CD27⁻ perforin^low or perforin^high subsets. The percentage and mean fluorescence intensity for the CD57⁺ cells are indicated. (F) Representative staining for perforin and CD57 in CD8⁺ T-cells from a HIV-1-infected or a healthy donor. Percentages of cells present in the top quadrants are shown. (G) Representative staining for perforin and CD57 in CD4⁺ T-cells from an HIV-1-infected or a healthy donor. Percentages of cells present in the top quadrants are shown.