Detection of shared MHC-restricted human melanoma antigens after vaccinia virus-mediated transduction of genes coding for HLA

Abstract

To detect shared human melanoma Ag that are recognized by HLA-A2 restricted, melanoma-specific CTL derived from tumor infiltrating lymphocytes, we have developed a convenient method to insert and express foreign HLA genes capable of presenting Ag on target cell lines. Seventeen melanoma cell lines and 11 nonmelanoma cell lines were infected with recombinant vaccinia virus containing the HLA-A2.1 gene. Infection by the vaccinia virus resulted in expression of functional HLA-A2 molecules on the cell surface of virtually 100% of infected cells within a 3.5-h period. The results showed that 11 of 17 (65%) naturally HLA-A2- melanoma cell lines were specifically lysed by the HLA-A2-restricted, melanoma-specific TIL after infection with the vaccinia-HLA-A2.1 virus. None of the nine human nonmelanoma cell lines tested (three colon cancer, four breast cancer, or two immortalized non-tumor cell lines) or two murine melanoma cell lines were lysed by the HLA-A2-restricted TIL after vaccinia-HLA-A2.1 infection. Coinfection of the vaccinia virus containing the beta 2-microglobulin gene with the vaccinia-HLA-A2.1 virus increased the surface expression of HLA-A2 and subsequent lysis by melanoma-specific tumor infiltrating lymphocytes. With this new method we could extend previous findings demonstrating that shared melanoma Ag recognized by HLA-A2-restricted tumor infiltrating lymphocytes exist among melanoma cells from different patients regardless of HLA type. These Ag represent excellent candidates for the development of vaccines to induce T cell responses for the immunotherapy of patients with melanoma.

FIGURE 1

Time kinetics of surface expression of HLA-A2 molecule after infection with vac-A2. Flow cytometric analysis of two melanoma cell lines, 397 and 928 mel, at various times after infection with vac-A2 shows expression of HLA-A2 on the cell surface (with anti-HLA-A2 mAb, BB7.2, solid line) as early as 3.5 h after the time of infection. Anti-Thy-1.2 mAb was used as a control (dashed line).

FIGURE 2

Coinfection of melanoma cell lines with vac-A2 and vac-β2 m increased surface expression of HLA-A2. Two melanoma cell lines, 888 and 928 mel, were incubated either without vac, with vac-A2 or with vac-A2 and vac-β2 m for 3.5 h and stained with anti-HLA-A2 mAb (BB7.2, solid line) or anti-Thy-1.2 mAb (dashed line). Surface expression of HLA-A2 were analyzed by flow cytometry. Peak and mean channel number of cells stained with anti-HLA-A2 mAb are follows: 501 mel (HLA-A2⁺ control, not shown) 64, 138; 888 mel, 4, 6; 888 mel + vac-A2, 8, 11; 888 mel + vac-A2 + vac-β2 m, 37, 33; 928 mel, 4, 6; 928 mel+vac-A2, 17, 18; 928 mel + vac-A2 + vac-β2 m, 26, 28. Coinfection of vac-β2 m with vac-A2 increased HLA-A2 surface expression.