Analysis of CD45 Alternative Exon Expression in Murine and Human CD4+ T Cell Subpopulations: a Thesis
Graduate School of Biomedical Sciences, Program in Immunology and Virology
Antigens, CD45; Exons; Leukocytes; RNA, Messenger; T-Lymphocytes; Academic Dissertations
Leukocytes express a family of high molecular weight glycoproteins called leukocyte common antigens (CD45) which have tyrosine phosphatase activity and are involved in phosphotyrosine signal transduction. Antibodies to different CD45 isoforms distinguish functionally different CD4+ T cell subsets in humans, rats, and mice. Selected protein isoforms are expressed through a process of exon splicing which is cell-type and differentiation-state specific. Splicing of the three variable exons, A, B, and C, which encode amino acids located near the extracellular amino terminus of the protein, potentially results in generation of eight different mRNA transcripts. The purpose of this study was to determine the relative levels of all eight different CD45 transcripts present in a panel of murine CD4+ T cell lines and normal murine and human CD4+ T cell subsets separated with antibodies to CD45 variable exons. I show, as expected, that the broad features of CD45 surface isoform expression in these cells can be accounted for by the relative amounts of the eight differentially spliced transcripts. Unexpectedly, all the differences in CD45 isoform expression among the CD4+ T cell subpopulations that I measured could be accounted for by differences in the overall level of variable exon expression. I did not see differences among T cell populations in the relative expression of particular variable exons. Exon B was always found in greater abundance than exons C or A. Of the dual exon species, only AB and BC were found in CD4+ T cells. The AC species was undetectable. Human CD4+ T cells, especially those in the naive subset, express higher levels of CD45 variable exons than murine CD4+ T cells.
In unrelated studies, I have generated a rat-mouse hybridoma which secretes a rat IgG antibody reactive with mouse CD45. I show that the monoclonal antibody, 25D10, defines a novel epitope consistent with a post-translational modification of CD45, similar but distinct from the epitope recognized by monoclonal antibody RA3.6B2 (anti-B220). This conclusion is based on evidence that it precipitates similar molecular weight bands from cells as does a framework monoclonal antibody to CD45, yet has a distinct cell surface expression as determined by flow cytometric analysis. It stains activated Th cell lines at a higher intensity than resting Th cells, stains 60-70% of splenocytes, and 25-30% of lymph node cells. It stains all class II positive cells but not freshly isolated CD4+, CD8+ T cells or CD45 transfected fibroblasts.
Rogers, PR. Analysis of CD45 Alternative Exon Expression in Murine and Human CD4+ T Cell Subpopulations: a Thesis. (1993). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 282. http://escholarship.umassmed.edu/gsbs_diss/282
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