IFN-gamma-induced recognition of the antigen-processing variant CMT.64 by cytolytic T cells can be replaced by sequential addition of beta 2 microglobulin and antigenic peptides.

The present study describes the functional nature of the MHC class I determinants expressed in CMT.64 cells and was undertaken to define and further analyze the deficiency in the cell line CMT.64 in the hope of elucidating the relative functional importance of constituent parameters in the recognition of these cells by CTL. We show that induction of Kb in CMT.64 cells with IFN-gamma results in molecules capable of presenting VSV epitopes to the appropriate CTL. However, cells untreated with IFN-gamma and infected with VSV are not recognized by VSV-specific CTL. This study reveals that beta 2 m3 is synthesized in limiting amounts in uninduced CMT.64 and becomes highly expressed after IFN-gamma induction. Thus, the limiting amount of beta 2 m expressed in uninduced cells may partially explain the inability of the cells to present viral components of CTL recognition. This concept is reinforced by the experiments identifying two functional effects upon the addition of immunogenic peptides to uninduced CMT.64 cells: at high peptide concentrations in excess of 5 nM, CMT.64 cells are recognized efficiently after 5 min of incubation; at the limiting peptide concentration of 500 pM, uninduced CMT.64 cells are only recognized providing beta 2m is added before or simultaneously with the antigenic peptide. BFA, an inhibitor of protein transport, and emetine, an inhibitor of protein synthesis, were used to show that at high peptide concentrations, 25 microM, recognition takes place after the peptide has stabilized the limited amount of newly arriving MHC/beta 2m complexes, devoid of peptides, at the cell surface of uninduced CMT.64 cells. These experiments thereby exclude the possibility that peptides are taken up into CMT.64 cells for assembly, transport and surface expression of functional MHC/beta 2m/peptide complexes. In summary, our data expands previous research showing the importance of exogenous beta 2m in sensitizing cells for CTL recognition with peptides added exogenously. These functional experiments also imply that the concentration of endogenous beta 2m may regulate the amount of MHC class I expressed at the cell surface and receptive to exogenous peptides. Finally, the phenotype of CMT.64 cells we describe provides evidence of the complexity of the Ag-presenting capacity of this cell line not previously identified in other studies on these cells, thus revising our understanding of the Ag-processing deficiency in these cells.