CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status

Abstract

Human immunodeficiency virus (HIV) disease is associated with loss of CD4(+) T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4(+) T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25(+)CD4(+) regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25(+)CD4(+) T cells significantly suppressed cellular proliferation and cytokine production by CD4(+) and CD8(+) T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-beta independent. Individuals with strong HIV-specific CD25(+) T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4(+): CD8(+) T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25(+)CD4(+) T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.

Figure 1.

CD25⁺CD4⁺ T cells in PBMCs from HIV-infected individuals express phenotypic markers consistent with CD25⁺ T reg cells: quantification of total CD25⁺CD4⁺ T reg cells in PBMCs of HIV-infected subjects by surface expression of CD25^+hi. (A) FoxP3 protein expression, detected by Western blot, is comparable in fresh CD25⁺CD4⁺ T cells isolated from HIV-infected versus HIV-uninfected individuals Fresh CD25⁻CD4⁺ T cells, CD8⁺ T cells, and monocytes do not express FoxP3. (B, top) Representative example of FACS^® profile and gates used to quantify CD25^+hiCD4⁺CD45RO⁺ T cells. Freshly isolated PBMCs were gated on CD45RO⁺CD3⁺ and analyzed for CD4 and CD25 expression. CD25^+hi gates were set so that ≤0.05% of all PBMC subsets, other than CD4⁺ T cells, fell to the right of the CD25^+hi gate (dashed line). (bottom) Comparison of the frequency of CD25^+hi cells within the CD4⁺CD45RO⁺ T cell subset of PBMCs from HIV-infected (n = 38) versus uninfected (n = 20) subjects. (C) The frequency of and the (D) absolute number of CD25^+hi versus total CD4⁺ memory T cells in PBMCs isolated from 38 HIV-infected individuals representing a broad percentage range of CD4⁺ memory T cells.

Figure 2.

Functional analyses of the CD25⁺ memory CD4⁺ T cell subset from HIV-infected individuals. (A) Comparison of the HIV p24 proliferation SI of total versus CD25⁺ cell subset-depleted CD4⁺ memory T cells isolated from HIV-infected individuals with strong (top, HIV p24 LPA Suppressors) or no/weak (bottom, HIV p24 LPA Non-suppressors) HIV-specific CD25⁺ T reg cell suppressor activity. The geometric mean HIV p24 SI of total and CD25⁺ cellular subset-depleted populations from LPA suppressors versus LPA nonsuppressors was 5.6 and 20.4 versus 5.3 and 3.6, respectively. (B) HIV p24 SI in more extensive LPA assays using PBMCs and CD4⁺ memory T cell subsets from LPA suppressors (n = 19). Data are represented as mean HIV p24 SI ± SD. (C) Comparison of the suppressive activity of CD25⁺CD4⁺ memory T cells isolated from (top) HIV-uninfected individuals (n = 6) versus all HIV⁺ subjects (n = 9) and (bottom) from HIV⁺ subjects comparing HIV p24 LPA suppressors, nonsuppressors, and nonresponders (n = 3 for each category) under polyclonal (pool of allogeneic PBMCs) stimulation conditions. Only CD25⁺ memory CD4⁺ T cells from HIV p24 LPA nonresponders exhibited significantly (P ≤ 0.04) weaker polyclonal T reg cell activity as compared with HIV-uninfected subjects or other groups of HIV⁺ subjects. Data are represented as mean ± SD percent inhibition of control (CD25⁻ subset) SI. (D) Comparison of cytokine/chemokine production by anti-CD3–stimulated CD25⁺ and CD25⁻ subsets of CD4⁺ memory cells isolated from HIV-uninfected individuals (n = 6) and HIV p24 LPA suppressors, nonsuppressors, and nonresponders (n = 4 for each category).

Figure 3.

CD25⁺CD4⁺ memory T cell–mediated suppression of HIV p24-stimulated proliferation is cell contact dependent and IL-10 and TGF-β independent. (A) CD25⁻ ± CD25⁺ memory CD4⁺ T cells from four p24 LPA suppressors were stimulated with HIV p24 (plus 10% CD2⁻ PBMCs) either together (no transwell) or separated by a 0.4 μM transwell insert to prevent cell–cell contact. Data are presented as mean ± SD percent inhibition of HIV p24-stimulated proliferation achieved in the presence, as compared with the absence, of the CD25⁺ subset. (B) A representative example of an HIV p24 LPA assay of PBMC subsets isolated from an HIV p24 LPA Suppressor conducted in the presence or absence of neutralizing anti–human IL-10 or TGF-β antibodies; data are presented as mean cpm ± SD of triplicate wells.

Figure 4.

Suppression of HIV p24-stimulated proliferation by CD25⁺CD4⁺ memory T cells is associated with inhibition of IL-2 production. (A) IL-2 levels present in supernatants of an HIV p24 LPA using CD4⁺ memory subsets isolated from an HIV p24 LPA suppressor. Data are representative of 19 independent experiments; the limit of detection for this assay was 15 pg/ml. (B) IL-2 ICC assay of total versus CD25⁺ cell-depleted memory CD4⁺ T cells (plus 10% CD2⁻ PBMCs as APCs) isolated from an HIV p24 LPA suppressor stimulated with p24 protein for 13 h. Data are representative of eight independent experiments.

Figure 5.

Depletion of CD25⁺ cells enhances the frequency of CD8⁺ T cells producing IFN-γ in response to HIV Gag peptides. (A) CD25⁺ PBMCs are primarily CD4⁺ memory T cells. PBMCs were stained for CD25 and CD3; CD3⁺ and CD3⁻CD25⁺ populations (top) were analyzed (bottom) for the expression of other markers identifying the major PBMC cellular subsets. Approximately 90% of CD25⁺ PBMCs are CD3⁺ and the majority of these are CD4⁺CD45RO⁺ cells (bottom right); the remainder of the CD25⁺ PBMCs are primarily CD19⁺ B cells (bottom left). Data are representative of 25 independent experiments. (B) The frequency of CD8⁺ T cells producing IFN-γ after 6 h of exposure to HIV Gag peptides in unfractionated versus CD25⁺ cell-depleted PBMCs isolated from an HIV p24 LPA suppressor. Data are representative of nine independent experiments.

Figure 6.

CD25⁺, but not CD25⁻, CD4⁺ T cells suppress the proliferation of perforin-expressing effector CD8⁺ T cells cultured in the presence of autologous HIV super-infected target cells. (A) HIV super-infected target cells were added to CFSE-labeled CD8⁺ T cells alone, CFSE-labeled CD8⁺ T cells plus CD25⁻CD4⁺ T cells, or CFSE-labeled CD8⁺ T cells plus CD25⁺CD4⁺ T cells. Cultures were stained periodically for CD69 and CD3, and CFSE intensity was assessed over the course of 3–10 d after coculture (left). At the time by which at least four division cycles could be visualized (6–10 d), CD8 ⁺ cells from each culture condition were analyzed for perforin expression and for cellular division by CFSE (right). Values representing the percent CFSE^lo (dividing cells) perforin⁺ cells within the total CFSE⁺ perforin⁺ cellular population are indicated. Data are representative of six independent experiments conducted using cells from HIV p24 LPA suppressors. (B) Mean ± SD percent dividing cells (CFSE^lo) within the perforin⁺ CFSE⁺ (CD8⁺) T cell population in experiments with cells isolated from HIV p24 LPA suppressors with normal HIV disease progression (left, n = 6) or from long-term nonprogressors (LTNPs; right, n = 2). In experiments with cells from HIV p24 LPA suppressors, perforin⁺ CD8⁺ T cells cultured with CD25⁺CD4⁺ T cells proliferated to a significantly lesser degree in response to HIV-infected target cells than did those with target cells alone or those cultured with CD25⁻CD4⁺ T cells (*, P < 0.03).