Modulation of monocyte/macrophage function by human CD4+CD25+ regulatory T cells

Abstract

The suppressive effects of CD4+CD25+ regulatory T cells (Tregs) on T cells have been well documented. Here we investigated whether human CD4+CD25+ Tregs can inhibit the proinflammatory properties of monocytes/macrophages. Monocytes and T cells were isolated from peripheral blood of healthy volunteers by magnetic cell separation and cocultured for 40 h. Monocytes were analyzed directly for cytokine production and phenotypic changes or repurified and used in T-cell stimulation and lipopolysaccharide challenge assays. Coculture with CD4+CD25+ Tregs induced minimal cytokine production in monocytes, whereas coculture with CD4+CD25- T cells resulted in large amounts of proinflammatory (tumor necrosis factor-alpha, interferon-gamma, interleukin-6) and regulatory (interleukin-10) cytokines. Importantly, when these CD4+CD25+ Treg-treated monocytes were repurified after coculture and challenged with lipopolysaccharide, they were severely inhibited in their capacity to produce tumor necrosis factor-alpha and interleukin-6 compared with control-treated monocytes. In addition, monocytes that were precultured with CD4+CD25+ Tregs displayed limited upregulation of human leukocyte antigen class II, CD40 and CD80, and downregulation of CD86 compared with control-treated monocytes. This altered phenotype had functional consequences, as shown by the reduction in T cell-stimulatory capacity of Treg-treated monocytes. Together, these data demonstrate that CD4+CD25+ Tregs can exert direct suppressive effects on monocytes/macrophages, thereby affecting subsequent innate and adaptive immune responses.

Figure 1. CD4+CD25+ Tregs are anergic and do not induce pro-inflammatory cytokine production.

CD14+ monocytes were cultured with anti-CD3 mAb in the absence (hatched bars) or presence of either CD4+CD25− T cells (black bars) or CD4+CD25+ Tregs (open bars). A. T cell proliferation was measured by ³H-thymidine incorporation (mean ± SEM, n=16 independent experiments). B-D. IFN-γ (n=12), TNF-α (n=16), and IL-10 (n=13) production was measured in culture supernatants collected 40 hours after the start of the culture. * p<0.05, ** p<0.005 and *** p<0.0005 compared to monocytes cultured with CD4+CD25− T cells.

Figure 2. CD4+CD25+ Tregs suppress T cell proliferation and pro-inflammatory cytokine production by monocytes/macrophages and CD4+CD25− T cells.

Monocytes were cultured with anti-CD3 mAb and autologous CD4+CD25− T cells with or without autologous CD4+CD25+ T cells at a 1:1 ratio (open symbols). Alternatively, responses were compared between monocytes cultured with CD4+CD25− T cells and monocytes cultured with unseparated CD4+ T cells i.e. the naturally occurring mix of CD4+CD25− and CD4+CD25+ T cells (solid symbols). T cell proliferation and cytokine production were measured as described in the legend to Figure 1. Each symbol reflects a different experiment using a different donor; the symbols correspond between the four analyses (IL-10 was not detected in two out of nine experiments).

Figure 3. CD4+CD25+ Tregs inhibit activation of monocytes/macrophages.

CD14+ monocytes were cultured in the presence of anti-CD3 mAb without T cells (hatched bars), with CD4+CD25− T cells (black bars) or CD4+CD25+ T cells (open bars). After 40 hours the cells were collected and labeled for FACS analysis. Monocytes were gated based on their forward/side scatter and low expression of CD4. A-D. Expression of CD40, HLA II, CD80 and CD86 was analyzed using CellQuest software and depicted as geometric mean fluorescence intensity (MFI). Data represent the mean ± SEM of 10 independent experiments each with cells of a different donor.

Figure 4. CD14+ monocytes were cultured in the presence of anti-CD3 mAb without T cells (hatched bars), with CD4+CD25− T cells (black bars) or CD4+CD25+ Tregs (open bars).

After 40 hours, CD14+ monocytes were re-purified (see Materials and Methods for details). The antigen-presenting capacity was investigated by adding re-purified monocytes to autologous responder CD4+CD25− T cells in the presence of PPD (A) or by using the monocytes as allogeneic stimulators to CD4+CD25− T cells obtained from a different donor (B). T cell proliferation was measured after 5 and 7 days respectively. T cell proliferation in the absence of stimulation was less than 1,000 cpm. One out of three independent experiments is shown.

Figure 5. CD4+CD25+ Tregs modulate the monocyte/macrophage cytokine profile in response to LPS.

CD14+ monocytes were cultured without T cells (hatched bars), with CD4+CD25− T cells (black bars) or CD4+CD25+ Tregs (open bars) in the presence of anti-CD3 mAb. After 40 hours monocytes were re-purified and LPS was added to the cultures. Culture supernatants were collected after 48 hours for detection of TNF-α (A, n=6), IL-6 (B, n=5) and IL-10 (C, n=6). Data are depicted as mean cytokine production ± SEM (pg/ml).