Human thymocytes and T cell lines, bearing an immature phenotype, express CR2-like molecules whose characteristics suggest differences from the CR2 found on B cells. In thymocytes, these receptors allow infection by EBV and synergistically with IL-2 induce thymocyte proliferation. Furthermore, infected thymocytes induce tumors when injected into SCID mice. The leukemic T cell line Jurkat expresses CR2-like transcripts which, upon preliminary sequencing of the coding cDNA's, suggest that they represent differentially spliced products of CR2. Jurkat cells display differential binding of anti-CR2 antibodies and EBV, the latter being able to infect the cells (as determined by expression of EBNA-1 ). Another leukemic cell line, HSB-2, has no reactivity with anti-CR2 antibodies, but yet binds EBV very strongly and becomes infected. HSB-2 expresses a 5.2 kb message which cross-reacts with CR2 cDNA. We propose to further characterize the thymocyte sub-population that expresses the CR2-like receptors by using two-color immunofluorescence analysis, anti-T cell antibodies, EBV, and C3. The functional effects of EBV on thymocytes will be determined by assessing their relative frequency of infection and compare it to that of B cells, testing for transcription of EBV-antigens (e.g. EBNA-1, and -2, LMP), looking for initiation of events that characterize T cell activation (e.g. intracellular Ca++, phosphoinositide biosynthesis, proliferation), and by measuring induction of IL-2-receptor expression. The same parameters will be assessed in order to determine the biologic effects of C3 on thymocytes. These experiments will give us insights into the biological significance of these CR2-like receptors. We'll exploit the ability of EBV to infect thymocytes in order to generate in vitro cell lines. Establishment of in vitro cell lines will be also attempted by taking advantage of the preliminary data that EBV-cultured thymocytes produce tumors in SCID mice. In addition, HTLV-1 induced thymocyte lines will be used. Continuous cell lines will provide useful material for the biochemical analysis of the CR2-like molecules. In this context, immunoprecipitations with anti-CR2 antibodies, tryptic peptide mapping and microsenquencing will be used to compare the CR2-like receptors on T cells to the well-characterized CR2. If time permits, any unique tryptic peptide sequences may be utilized in order to generate anti-peptide antibodies. Generation of monoclonal antibodies to the CR2- like receptors may also be considered. Analysis of isolated cDNA clones from a Jurkat library, coding for CR2- like products, will be pursued in terms of sequencing, comparison to B cell CR2, and transient transfection in order to study biologic activity. Finally, RNA transcripts from the HSB-2 cell line and thymocytes will be analyzed by hybridization with B cell CR2-cDNA and by PCR under various conditions of stringency.
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