Biochemical characterization of a high molecular weight thymoma antigen presented by class II MHC on third party APCs

My project has focused on the identification of class II-restricted tumor antigen genes from the EL4 thymoma, which is devoid of class I1 MHC. l%r this purpose [ have been using an Ab class II MtlC-restricted T cell clone, bEZ89.3, generated against EL4 thymoma. This T-cell clone has been fused with a fusion partner, resulting a lacZ-inducible hybrid cell. I have demonstrated that the antigen recognized by bEZ89.3 is secreted into the cell culture medium by EL4 thymoma. This antigen can be presented by the third party professional APC, such as B6 spleen cells or B6 peritoneal macrophages, in a T-cell functional assay. Secreted proteins from EL4 thymoma are being analyzed in biochemical methods to identify the antigen. By using specific molecular weight cutoff filters, I have also shown that this tumor antigen is larger than 50KD in size. Metabolic labeling of the EL4 thymoma, protein gel analysis, enzymatic digests, and HPLC are employed to further characterize and identi' this tumor antigen/gene. The identification of this class II-restricted antigen/gene(s) that is different from the large number of normal genes will not only reveal the effector role of class II:restricted T cells in tumor recognition and clearance but also serve as a model to study tumor antigens secreted by class II MHC-negative tumors and presented by normal third-party class II-expressing antigen presenting cells.