Construction of a Novel System to Analyze Recognition of MHC Class II-Restricted Epitopes by CD4+ T Cell Hybridoma Clones in vitro

The Simian Virus 40 large tumor antigen (SV40 Tag) is an excellent model system for studies in cancer immunology because it is strongly immunogenic, but capable of causing tumors when expressed as a transgene in vivo when protected by immune tolerance. The CD8+ T cell response to Tag in the C57BL/6 murine system is directed against four well-characterized epitopes. However, the CD4+ T cell response in this system remains uncharacterized. Recent work using Tag-specific CD4+ hybridoma clones and immune splenocytes to screen a 175 member, overlapping 15-mer peptide library corresponding to the 708 amino acid Tag protein sequence have identified three CD4+ epitopes. In order to complement this peptide-based screening system, we are constructing a system in which candidate epitopes can be amplified by PCR and inserted into a modified pGLO plasmid, forming an amino terminal extension of the green fluorescent protein (GFP). Such plasmids will be transformed into E. coli. Induction of the fusion gene with arabinose should fill the bacteria with glowing fusion protein. Incubation of arabinose-induced (glowing) bacteria with cultured murine dendritic cells (BMDCs) results in uptake which is readily observed as the phagocytes also begin to glow; glowing BMDCs containing E. coli DH5a expressing unmodified GFP do not stimulate hybridoma clones. Antigen processing should release the epitope peptide from the amino terminus of the fusion protein for presentation to added NFAT-lacZ+ CD4+ T cell hybridoma clones. This system should provide an excellent complement to the current methods which rely on incubation of MHC class IIpositive APCs with synthetic peptides because it more closely resembles in vivo presentation. Efforts to generate and test the GFP fusion protein system will be described. The system will initially be tested by inserting the LT529-543 epitope and detecting it with appropriate hybridoma clones