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 d eterminants 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 paramet ers in the recognition of these cells by CTL. We show that induction o f K(b) in CMT.64 cells with IFN-gamma results in molecules capable of presenting VSV epitopes to the appropriate CTL. However, cells untreat ed with IFN-gamma and infected with VSV are not recognized by VSV-spec ific CTL. This study reveals that beta2m3 is synthesized in limiting a mounts in uninduced CMT.64 and becomes highly expressed after IFN-gamm a induction. Thus, the limiting amount of beta2m expressed in uninduce d cells may partially explain the inability of the cells to present vi ral components for CTL recognition. This concept is reinforced by the experiments identifying two functional effects upon the addition of im munogenic peptides to uninduced CMT.64 cells: at high peptide concentr ations in excess of 5 nM, CMT.64 cells are recognized efficiently afte r 5 min of incubation; at the limiting peptide concentration of 500 pM , uninduced CMT.64 cells are only recognized providing beta2m is added before or simultaneously with the antigenic peptide. BFA, an inhibito r of protein transport, and emetine, an inhibitor of protein synthesis , were used to show that at high peptide concentrations, 25 muM, recog nition takes place after the peptide has stabilized the limited amount of newly arriving MHC/beta2m complexes, devoid of peptides, at the ce ll surface of uninduced CMT.64 cells. These experiments thereby exclud e the possibility that peptides are taken up into CMT.64 cells for ass embly, transport and surface expression of functional MHC/beta2m/pepti de complexes. In summary, our data expands previous research showing t he importance of exogenous beta2m in sensitizing cells for CTL recogni tion with peptides added exogenously. These functional experiments als o imply that the concentration of endogenous beta2m may regulate the a mount of MHC class I expressed at the cell surface and receptive to ex ogenous peptides. Finally, the phenotype of CMT.64 cells we describe p rovides evidence of the complexity of the Ag-presenting capacity of th is cell line not previously identified in other studies on these cells , thus revising our understanding of the Ag-processing deficiency in t hese cells.