HIV-specific regulatory T cells are associated with higher CD4 cell counts in primary infection

Abstract

Objective: Expansion of regulatory T (Treg) cells has been described in chronically HIV-infected individuals. We investigated whether HIV-suppressive Treg could be detected during primary HIV infection (PHI).

Methods: Seventeen patients diagnosed early after PHI (median: 13 days; 1-55) were studied. Median CD4 cell count was 480 cells/microl (33-1306) and plasma HIV RNA levels ranged between 3.3 and 5.7 log10 copies/ml. Suppressive capacity of blood purified CD4CD25 was evaluated in a coculture assay. Fox-p3, IL-2 and IL-10 were quantified by reverse transcriptase (RT)-PCR and intracellular staining of ex vivo and activated CD4+CD25 T cells.

Results: The frequency of CD4CD127CD25 T cells among CD4 T cells was lower in patients with PHI compared with chronic patients (n = 19). They exhibited a phenotype of memory T cells and expressed constitutively FoxP3. Similar to chronic patients, Treg from patients with PHI inhibited the proliferation of purified tuberculin (PPD) and HIV p24 activated CD4CD25 T cells. CD4CD25 T cells from patients with PHI responded specifically to p24 stimulation by expressing IL-10. In untreated patients with PHI, the frequency as well as HIV-specific activity of Treg decreased during a 24-month follow-up. A positive correlation between percentages of Treg and both CD4 cell counts and the magnitude of p24-specific suppressive activity at diagnosis of PHI was found.

Conclusion: Our data showed that HIV drives Treg, as PHI and these cells persist throughout the course of the infection. A correlation between the frequency of Treg and CD4 T-cell counts suggest that these cells may impact on the immune activation set point at PHI diagnosis.

Figure 1. Characteristics of CD4⁺CD25^high T cells in PHI patients

A) Phenotype of CD4 T lymphocytes in a representative PHI patient. B) Expression of Foxp3 and CD127 markers on gated CD4⁺CD25⁻, CD4⁺CD25^low and CD4⁺CD25^high gated T Cells. C) The median percentages of CD4⁺CD25^high in PHI patients at diagnosis (n=17) and chronic c-ART treated patients (n=19). The frequency of CD4⁺CD25^high T cells was evaluated on freshly blood samples after combining a gating on lymphocytes and on CD4⁺CD3⁺ T cells. D) Phenotype of CD4⁺CD25⁻ and CD4⁺CD25^high T cell subsets in PHI patients (n=13). Median percentages of CD4⁺CD25− and CD4⁺CD25^high gated T cells expressing the different markers are indicated. Comparison between CD4⁺CD25⁻ and CD4⁺CD25^high subsets are indicated (* P< 0.05; ** P< 0.01; *** P<0.001; Mann Whitney rank sum test).

Figure 2. CD4⁺CD25⁺ T cells from PHI and chronic HIV-infected patients are hypo-responsive to antigen specific stimulation and suppress CD4+CD25− proliferation

A) 5x10⁴ purified CD4⁺CD25⁻ (white bars) and CD4⁺CD25⁺ T cells (black bars) were stimulated for 5 days with 5μg/ml of plate bound either with anti-CD3 mAb and soluble anti-CD28 mAb (5μg/ml), 5μg/ml of purified PPD, p24 or CMV antigen. (³H) thymidine was added during the last 16 hours of culture. Results are expressed as median of stimulation index from 9 PHI and 8 chronic HIV infected patients. B) Peripheral CD4⁺CD25⁺ T cells from PHI (n=8) and chronic HIV-infected patients (n=6) suppress similarly CD4⁺CD25⁻ T cell proliferation in response to PPD and HIV p24 protein. Purified CD4⁺CD25⁻ (5.10⁴) cells were incubated at different ratio with CD4⁺CD25⁺ T cells and autologous monocytes for 5 days with either 5μg/ml PPD or p24, before adding 0.5 μCi of thymidine for the last 16 hours of culture. Results are expressed as median values of the stimulation index. (* P< 0.05; ** P< 0.01, Mann Whitney rank sum test).

Figure 3. HIV-specific production of IL-10 and IL-2 by CD4⁺CD25^high T cells

A) Frequency of CD4⁺CD25⁻ and CD4⁺CD25^high producing IL-2 (left) or IL-10 (right) following either polyclonal or p24 stimulation. Cells (5.10⁴/well) were incubated for 24 hours alone or in presence of either 5μg/ml plate bound anti-CD3 mAb and soluble anti-CD28 or of p24 antigen (5 μg/ml) and 5.10³ monocytes/well, before assessment of intracellular production of cytokines by flow cytometry. Changes from baseline in the mean frequencies of cells producing cytokines following stimulation are indicated. Results from 6 patients studied are presented. B) Quantitative RT-PCR analyses of Foxp3 (left panel), IL-2 (middle) and IL-10 (right) expression of CD4⁺CD25⁻ and CD4⁺CD25^high after purification and non-stimulated (NS), or stimulated either with anti-CD3 and anti-CD28 antibodies or p24 antigen. Mean from 5 patients are presented. Ct values were normalized to mRNA for SF3A1 expression. (* P< 0.05; ** P< 0.01, Mann Whitney rank sum test).

Figure 4. Evolution of the frequency and suppressive activity of Treg in PHI patients

A) Frequency of CD4⁺CD25^high T cells at diagnosis and month 24 in 6 PHI patients who remained untreated during the follow up. Evaluation of the suppressive activity of purified CD4⁺CD25^high T cells on PPD (B) and p24 (C) stimulated CD4⁺CD25⁻ T cells (values at a 1/4 ratio of CD4⁺CD25^high/CD4⁺CD25⁻ T cells are shown). Mean percentages of inhibition of experiments performed in 6 patients are shown. (* P< 0.05, Mann Whitney rank sum test).

Figure 5. Correlation between the frequency and activity of CD4⁺CD25^high T cells and absolute CD4 cell counts in PHI patients

The correlations at diagnosis between total number of CD4⁺CD25^high T cells and CD4 cell counts or plasma HIV RNA values (A), expression of activation marker HLA-DR on CD4 T cells (B) inhibition of CD4⁺CD25⁻ proliferation in the presence of p24 and tuberculin (C) were investigated using a Spearman rank correlation coefficient test.