Potent immunosuppressive activities of cytomegalovirus-encoded interleukin-10

Abstract

Cytomegalovirus (CMV) has highly evolved mechanisms for avoiding detection by the host immune system. Recently, in the genomes of human and primate CMV, a novel gene comprising segments of noncontiguous open reading frames was identified and found to have limited predicted homology to endogenous cellular interleukin-10 (IL-10). Here we investigate the biological activities of the CMV IL-10-like gene product and show it to possess potent immunosuppressive properties. Both purified bacterium-derived recombinant CMV IL-10 and CMV IL-10 expressed in supernatants of human cells were found to inhibit proliferation of mitogen-stimulated peripheral blood mononuclear cells (PBMCs), with specific activity comparable to that of recombinant human IL-10. In addition, CMV IL-10 expressed from human cells inhibited cytokine synthesis, as treatment of stimulated PBMCs and monocytes with CMV IL-10 led to a marked decrease in production of proinflammatory cytokines. Finally, CMV IL-10 was observed to decrease cell surface expression of both major histocompatibility complex (MHC) class I and class II molecules, while conversely increasing expression of the nonclassical MHC allele HLA-G. These results demonstrate for the first time that CMV has a biologically active IL-10 homolog that may contribute to immune evasion during virus infection.

FIG. 1.

Supernatant CMV IL-10 inhibits PBMC proliferation. (A) Western blot analysis of supernatants from transfected HEK 293 cells with an anti-poly-His antibody. Cells were transiently transfected with pcDNA3.1-m/H-RhCMV IL-10, pcDNA3.1-m/H-HCMV IL-10, or an empty vector control (mock). MW, molecular weight marker (sizes at right, in thousands). (B) PBMCs from healthy human donors were stimulated with PHA for 72 h in the presence of RhCMV IL-10 or mock supernatants. Proliferation was measured by [³H]thymidine uptake during the final 18 h of culture. RhCMV IL-10 was immunoprecipitated from conditioned medium with an anti-poly-His antibody (CMV IL-10 + Ab). Error bars, standard deviations. (C) PBMCs from nine human donors were tested for effects on proliferation in the presence of HCMV IL-10-conditioned medium or recombinant human IL-10 (1 μg/ml). Results are expressed as percent increase in proliferation relative to control cultures from the same donor. The horizontal bar indicates mean change in proliferation for all donors.

FIG. 2.

CMV IL-10 inhibits cytokine production by activated human PBMCs. PHA-stimulated PBMCs were incubated in the presence of mock-, RhCMV IL-10-, or HCMV IL-10-conditioned medium or recombinant human IL-10 (1 μg/ml). Supernatants were harvested after 48 h and assayed for IFN-γ production by sandwich ELISA.

FIG. 3.

HCMV IL-10 inhibits expression of proinflammatory cytokines by human monocytes. Monocytes were stimulated with LPS and incubated in the presence of mock- or HCMV IL-10-conditioned medium or recombinant human IL-10 (rhIL-10) (1 μg/ml). Supernatants were harvested after 48 h and assayed for IL-1α, IL-6, GM-CSF, and TNF-α production by sandwich ELISA. Error bars, standard deviations.

FIG. 4.

MHC expression is altered by RhCMV IL-10. Flow cytometric analysis of LPS-stimulated monocytes cultured in the presence of mock- or RhCMV IL-10-conditioned medium. After 48 h the cells were stained for cell surface expression of CD54 (ICAM), MHC class I (HLA-A, -B, and -C), MHC class II (HLA-DR), and the nonclassical class I MHC molecule HLA-G. Filled histograms represent staining with the indicated antibody; open histograms indicate isotype controls. The mean fluorescence intensity is indicated in the upper right corner of each plot.

FIG. 5.

Recombinant HCMV IL-10 inhibits PBMC proliferation. PBMCs from healthy human donors were stimulated with PHA for 72 h in the presence of serial dilutions of recombinant HCMV IL-10 (B and D [black bars]) or recombinant human IL-10 (A and C [black bars]). Proliferation was measured by [³H]thymidine uptake during the final 18 h of culture. Activity of recombinant proteins was abolished by the presence of monoclonal anti-human IL-10 receptor antibody at 15 μg/ml (C and D [gray bars]). Error bars, standard deviations.