Tumor recognition by cytotoxic T cells. : Definition of new tumor antigens and the effect of interleukin-10 on antigen presentation

Abstract: The tumor antigens against which T cell responses have been demonstrated most frequently are not tumor specific but are also expressed on normal tissues. This observation has been interpreted as evidence for breaking of immunological tolerance to normal cellular proteins. In the first part of this thesis we define HLA-A2 restricted CTL epitopes from the Melanocortin 1 Receptor (MC1R) which is expressed on cells of melanocytic origin, including melanoma cells. Peptides derived from this protein were selected on the basis of HLA-A2 binding motifs and tested for their HLA-A2 binding capacity. Three high- or intermediate binding nonamers were found to induce peptide-specific CTL from PBMC of healthy HLA-A2+ donors after in vi~ro stimulation with peptide-pulsed antigen presenting cells (APC). The CTL elicited against MCIR derived peptides could recognize HLA A2+/MC1R expressing melanomas. Using a similar protocol, four new epitopes derived from the proto-oncogene Her2/neu were identified. The CTL raised from ascites fluid of patient with ovarian carcinomas against these HLA-A2+ restricted peptides could recognize naturally processed peptides from HLA-A2+ tumors and from cell lines co-transfected with the antigenic protein gene and HLA-A2. Furthermore, we screened for the expression of MC1R in normal tissues and found this protein to be express to a low degree in adrenal gland and activated monocytes. The screening of anti-melanoma CTL from TIL or PBMC of melanoma patients revealed the presence of anti-MC1R CTL precursors in 50% of the patients, indicating that MC1R is an immunodominant melanoma antigen. Taken together, our findings have implications in relation both to autoimmunity as well as immunotherapy of malignant melanomas and carcinomas. The majority of human tumors are defective in their MHC class I antigen presenting capacity. In the second part of this thesis we have studied the role of Interleukin-10 (IL-10). We showed that IL-10 inhibits antigen presentation to specific CD8+ cytotoxic T Iymphocytes in melanomas. Furthermore, we demonstrated that pre treatment of the mouse Iymphoma RMA with IL-10 resulted in a dose dependent inhibition of Iysis by CTL. However, IL-10 treatment of RMA led to an increased sensitivity to Iysis by NK cells. RMA cells showed an IL-10 dependent downregulation of H-2 expression which could be normalized by addition of H-2 binding peptides, indicating that IL-10 exerts a post transcriptional effect on H-2 expression. Oligopeptides are delivered to the secretory pathway by the TAP protein complex. Relative to normal cells, TAP-deficient cells express substantially lower levels of intracellular antigens to CTL. We demonstrated that IL-10 expression in the RMA Iymphoma and other murine tumors inhibits the TAP-dependent translocation of peptides to the endoplasmic reticulum (ER), resulting in a low expression of cell surface MHC class I molecules. This finding is explained by a down regulation of expression of TAPI and TAP2, observed in IL-10 transfected murine tumor cells. In the J558L plasmacytoma cell line constitutively expressing high levels of IL-10, an increased TAP-dependent translocation of peptides and expression of cell surface MHC class I could be induced by IL-10 anti-sense expression. The effect of IL-10 on a biological relevant system was demonstrated using the NK sensitive prototype tumor YAC-1. Our studies showed that the NK sensitivity of this cell line correlates with the capability of the cells to produce IL-10. IL-10 is the first example of a cytokine which can decrease the expression and function of the TAP1/2 molecular complex, and in more general terms the first example of a cytokine with an inhibitory effect on MHC class I mediated antigen presentation.