Class II in the Absence of Invariant Chain on Uveal Melanomas - A Pathway to Present Endogenous Tumor Antigens

Abstract

Abstract: : Purpose: A major cause for tumor cell-based vaccine failure is the inability to stimulate Class II restricted helper T cells against endogenous tumor antigens. This is due, in part, to the presence of invariant chain that associates with Class II α and ß subunits in the endoplasmic reticulum. Invariant chain performs two functions: (i) stabilizes α ß heterodimers, and (ii) blocks endogenously synthesized peptides from binding Class II. Studies using invariant chain negative murine tumor cells transfected with Class II demonstrate expression of functional Class II heterodimers that: (i) present endogenous peptides, (ii) activate CD4⁺ T cells, and (iii) induce protective immunity. Similar studies using human tumors have not been performed, since many human tumors express invariant chain when stimulated with exogenous cytokines. Interestingly, some uveal melanomas fail to express either Class II, or invariant chain, even after treatment with exogenous IFN-γ. We hypothesize that genetically engineered Class II positive / invariant chain negative uveal melanomas will express stable cell-surface Class II α ß heterodimers that present endogenous tumor antigens. Methods: To introduce Class II DRα and DRß chain genes, we constructed a bicistronic retroviral vector containing both DRα 0101 and DRß 0101 genes on one expression cassette, which increases the chance of subunit dimerization. Results: Western blot analysis revealed tumor cells were negative for α / ß chains and invariant chain, while transfected tumor cells expressed high levels of α / ß chains, but no invariant chain. Western blots probed with antibodies specific for α / ß heterodimers revealed significant heterodimers only in transfected tumor cells. Biochemical and flow cytometric analysis confirmed these heterodimers were expressed on the cell surface. Cell surface proteins on transfected tumor cells were biotinylated and probed by Western analysis for Class II αß chains, revealing stable heterodimers. Flow cytometry also revealed cell surface Class II expression. Conclusions: We conclude that stable Class II heterodimers are expressed on transduced uveal melanoma cells and predict these Class II heterodimers will present endogenous tumor antigens due to the absence of invariant chain. Our data implies retroviral transduced uveal melanoma cells can be used to successfully activate T helper cells that may contribute to effective anti-tumor immunity. NEI RO1-EY9294.

Keywords: antigen presentation/processing • melanoma • gene transfer/gene therapy