T-helper cell-mediated proliferation and cytokine responses against recombinant Merkel cell polyomavirus-like particles

Abstract

The newly discovered Merkel Cell Polyomavirus (MCPyV) resides in approximately 80% of Merkel cell carcinomas (MCC). Causal role of MCPyV for this rare and aggressive skin cancer is suggested by monoclonal integration and truncation of large T (LT) viral antigen in MCC cells. The mutated MCPyV has recently been found in highly purified leukemic cells from patients with chronic lymphocytic leukemia (CLL), suggesting a pathogenic role also in CLL. About 50-80% of adults display MCPyV-specific antibodies. The humoral immunity does not protect against the development of MCC, as neutralizing MCPyV antibodies occur in higher levels among MCC patients than healthy controls. Impaired T-cell immunity has been linked with aggressive MCC behavior. Therefore, cellular immunity appears to be important in MCPyV infection surveillance. In order to elucidate the role of MCPyV-specific Th-cell immunity, peripheral blood mononuclear cells (PBMC) of healthy adults were stimulated with MCPyV VP1 virus-like particles (VLPs), using human bocavirus (HBoV) VLPs and Candida albicans antigen as positive controls. Proliferation, IFN-γ, IL-13 and IL-10 responses were examined in 15 MCPyV-seropositive and 15 seronegative volunteers. With the MCPyV antigen, significantly stronger Th-cell responses were found in MCPyV-seropositive than MCPyV-seronegative subjects, whereas with the control antigens, the responses were statistically similar. The most readily detectable cytokine was IFN-γ. The MCPyV antigen tended to induce stronger IFN-γ responses than HBoV VLP antigen. Taken together, MCPyV-specific Th-cells elicit vigorous IFN-γ responses. IFN-γ being a cytokine with major antiviral and tumor suppressing functions, Th-cells are suggested to be important mediators of MCPyV-specific immune surveillance.

Figure 1. MCPyV VP1–specific T cell responses of MCPyV seropositive individuals after T cell subset depletion.

PBMC of four MCPyV seropositive (P1 to P4) and one seronegative subject with strong MCPyV-specific CMI (N1) were depleted of either CD4+ or CD8+ T cells and stimulated with MCPyV VP1-VLPs (2.5 µg/ml). Proliferation (panel A) and cytokine (IFN-γ, IL-13 and IL-10) responses (panel B, C, D) were studied by thymidine incorporation and ELISA, respectively.

Figure 2. Effect of HLA class II-specific monoclonal antibodies (MAbs) on MCPyV-VP1 specific proliferation and cytokine responses.

PBMC from four MCPyV seropositive subjects (P1 to P3 and P5) and from a seronegative subject (N1) were incubated either with a HLA class II-specific blocking MAb or with an isotype-matched control MAb. The effect of these MAbs on MCPyV-specific (2.5 µg/ml) proliferation (panel A) and cytokine (panels B, C and D) responses are shown. Subjects P1 to P3 and N1 are same than in Figure 1.

Figure 3. Characterization of MCPyV VP1 antigens.

Silver staining of capsid protein (panel A) in 10% SDS PAGE. Lane 1: molecular weight markers, lane 2: MCPyV VP1 capsid antigen. Dot blotting (panel B) for MCPyV antigen, studied with MCPyV-IgG positive (I) and negative (II) sera. Electron microscopy of sterile MCPyV particles (panel C) purified by caesium chloride density gradient ultracentrifugation, with 200 nm scale bar shown.