CD4+ CD25+ regulatory T cells impair HIV-1-specific CD4 T cell responses by upregulating interleukin-10 production in monocytes

Abstract

T cell dysfunction in the presence of ongoing antigen exposure is a cardinal feature of chronic viral infections with persistent high viremia, including HIV-1. Although interleukin-10 (IL-10) has been implicated as an important mediator of this T cell dysfunction, the regulation of IL-10 production in chronic HIV-1 infection remains poorly understood. We demonstrated that IL-10 is elevated in the plasma of individuals with chronic HIV-1 infection and that blockade of IL-10 signaling results in a restoration of HIV-1-specific CD4 T cell proliferation, gamma interferon (IFN-γ) secretion, and, to a lesser extent, IL-2 production. Whereas IL-10 blockade leads to restoration of IFN-γ secretion by HIV-1-specific CD4 T cells in all categories of subjects investigated, significant enhancement of IL-2 production and improved proliferation of CD4 T helper cells are restricted to viremic individuals. In peripheral blood mononuclear cells (PBMCs), this IL-10 is produced primarily by CD14(+) monocytes, but its production is tightly controlled by regulatory T cells (Tregs), which produce little IL-10 directly. When Tregs are depleted from PBMCs of viremic individuals, the effect of the IL-10 signaling blockade is abolished and IL-10 production by monocytes decreases, while the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), increases. The regulation of IL-10 by Tregs appears to be mediated primarily by contact or paracrine-dependent mechanisms which involve IL-27. This work describes a novel mechanism by which regulatory T cells control IL-10 production and contribute to dysfunctional HIV-1-specific CD4 T cell help in chronic HIV-1 infection and provides a unique mechanistic insight into the role of regulatory T cells in immune exhaustion.

Fig 1

Elevated IL-10 plasma levels in chronic HIV-1 infection and the effects of IL-10Rα blockade. (A) Plasma IL-10 levels were measured in HIV-1-seronegative individuals (HIV-1−; n = 10), in individuals with chronic HIV-1 infection and spontaneous control of viremia (Cont; n = 13), in patients receiving treatment with antiretroviral therapy (ART; n = 10), and in patients with chronic, untreated infection (CU; n = 21). Intergroup comparisons were performed by Kruskal-Wallis test (*, P < 0.05). Solid lines indicate medians; n.s., nonsignificant (P > 0.05). (B) CD8-depleted PBMCs were stimulated with recombinant HIV-1 p24 protein in the presence of an isotype control antibody or an IL-10Rα-blocking antibody. Proliferation was then measured in a 7-day CFSE assay, and net proliferation was calculated by subtracting the background in the absence of antigen. The fold increase in proliferation was calculated by taking the ratio of the net percentage of CD4⁺ CFSE^lo cells treated with blocking antibody versus the percentage of those treated with an isotype control antibody (***, P < 0.001). The dashed line indicates no effect (fold increase = 1). (C and D) CD8-depleted PBMCs were stimulated with HIV-1 p24 in the presence of an isotype control or IL-10Rα-blocking antibody, and IFN-γ (C) and IL-2 (D) were measured in supernatants 3 days after stimulation. The increase in IFN-γ production was greater than that of IL-2 following IL-10Rα blockade for all subjects (E), including HIV-1 controllers (F), subjects on ART (G), and those with chronic, untreated infection (H).

Fig 2

CD25⁺ cell depletion abrogates the effects of IL-10Rα blockade. PBMCs from 12 chronically infected, untreated individuals were depleted of CD8 cells and then further depleted with anti-CD25 antibody-coated beads or were mock depleted. (A) A representative FACS plot of the depletion efficiency of CD25⁺ FoxP3⁺ cells is shown. (B) CD25-depleted or mock-depleted cells were stimulated with recombinant HIV-1 p24 protein in the presence of IL-10Rα-blocking antibody or an isotype control. CD4 T cell proliferation was then measured using a CFSE assay. An increase in CD4 T cell proliferation was seen with IL-10Rα blockade (C, left panel), and CD25 depletion (C, middle panel), but IL-10Rα blockade had minimal impact when the assays were performed with CD25-depleted PBMCs (C, right panel). (D) The fold increase in CD4 T cell proliferation in response to HIV-1 p24 stimulation with IL-10Rα blockade was decreased following depletion of CD25⁺ cells (median,1.8× increase without depletion and 1.1× with depletion).

Fig 3

IL-10 production by cellular subsets. (A) CD4⁺ T cells, CD8⁺ T cells, CD25⁺ FoxP3⁺ Tregs, CD14⁺ monocytes, CD19⁺ B cells, CD11c⁺ mDCs, and CD123⁺ pDCs from chronically HIV-1-infected, untreated subjects were analyzed for spontneous production of IL-10 by ICS. (B) Analysis of IL-10 secretion in unstimulated PBMC subsets in 15 untreated, chronically HIV-1-infected individuals. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 4

Treg depletion decreases IL-10 production by monocytes. Magnetic beads coated with antibodies against CD14, CD19, or CD25 were used to deplete specific cellular subsets from PBMCs from 15 chronically HIV-1-infected, untreated subjects. (A) Representative FACS plots are shown. (B) IL-10 in tissue culture supernatants was measured after 3 days of culture. IL-10 levels were normalized to those of mock-depleted PBMCs for each condition. *, P < 0.05; ***, P < 0.001. (C and D) Depletion of CD25⁺ cells resulted in a decrease in IL-10 production by CD14⁺ monocytes and a simultaneous increase in TNF-α production as measured by ICS. Representative FACS plots are shown (C) as well as group statistics for a cohort of 8 chronically infected, untreated individuals (D).

Fig 5

Induction of IL-10 in monocytes involves contact-dependent Treg activity and is also modulated by IL-27. CD14⁺ monocytes and CD25^hi CD127^lo Tregs from 8 untreated chronically HIV-1-infected subjects were purified by FACS sorting. Cell subsets were then cultured alone or in cocultures, and IL-10 was measured in supernatants after 3 days of incubation. (A) IL-10 production by Tregs and monocytes cultured alone. (B, C, and D) Monocytes were either cultured alone or cocultured with Tregs in direct contact (Mono+Treg) or separated by a Transwell membrane (Mono/Treg). (E) PBMCs from 8 untreated chronically infected individuals were cultured in the presence or absence of recombinant IL-27 receptor protein (IL-27R), and IL-10 was measured in the supernatants. (F) The same experiments were performed with the same persons after CD25 depletion. (G) Production of IL-27 in sorted CD14 monocytes and CD4 T cells was assessed by quantitative PCR. (H) IL-27 production was assessed in supernatants of purified monocytes cultured alone, purified Tregs cultured alone, or Tregs cultured with monocytes.