William C. Raschke, Ph.D., Professor

Immunology Program

MOLECULAR INTERACTIONS IN IMMUNE RESPONSES - THE ROLE OF CD45

CD45 has the interesting properties of (a) expression restricted to nucleated hematopoietic cells, (b) a highly abundant cell surface glycoprotein occupying an estimated 10% of the lymphocyte cell surface, (c) multiple isoforms with differ in the extracellular domain, (d) strictly regulated expression of the different isoforms in the various hematopoietic lineages, (e) a large cytoplasmic domain with protein tyrosine phosphatase (PTPase) activity, (f) a role in T and B cell development, and (g) essential involvement in signal transduction as a positive regulator of antigen receptor signaling in T and B cells. As a result, CD45 has received considerable attention as evidence accumulates for its role in signal transduction during immune responses, in lymphocyte development and in various disease states. Many studies have demonstrated the CD45 molecule has a critical role in the function of cells expressing it.

To analyze the contributions of the different isoforms, the PTPase activity and other components of the CD45 molecule to molecular interactions and functional properties of the cells, we developed a minigene expression system. A reliable expression vector for CD45 had been long sought by many labs for the study of this critical molecule in hematopoietic cells. A key feature of the minigene is the inclusion of the genomic sequence between exons 3 and 9 in an otherwise cDNA construct. This genomic region contains the exons and introns in the region of alternative splicing. We have demonstrated this minigene functions like the endogenous gene in that it provides correctly regulated isoform expression in the various leukocyte lineages and reconstitutes the functional capabilities of leukocytes lacking a functional endogenous CD45 gene. These findings derive from in vitro studies using cell lines and in vivo from the use of this minigene to produce transgenic mice. The minigene restores leukocyte functional capacity and lymphocyte development in an immunodeficient CD45 knockout mouse. Not only are the functions of individual leukocyte subpopulations restored, but also the mice survive infection by pathogens, such as influenza and murine gammaherpesvirus, which are lethal to the CD45 knockout mice.

We have used these mice to generate mutations in key regions of the molecule for the study of their roles in CD45 function. Transgenic mice have been made with three of these mutations. The deletion of the cytoplasmic domain in the minigene, which removes the PTPase region and intracellular interaction components of the molecule, produces knockout mice carrying this transgene with the same immunodeficient properties as the parent knockout strain. Furthermore, we showed a mutation that alters a single amino acid, the active site cysteine of one of the two PTPase domains to a serine, not only disrupts immune cell function but also results in the immune system developmental defects seen in the CD45 deficient mice. However, the mutation of a postulated regulatory site of tyrosine phosphorylation to phenylalanine has the same immune restorative properties as the wild type minigene in transgenic knockout mice.

Humans lacking functional CD45 have severe combined immunodeficiency and die at an early age. We are currently investigating means of using a similarly constructed human minigene for a therapeutic application to the human disease. The minigene provides a means to evaluate the contribution of O-linked glycosylation in the dimerization of CD45, which controls the level of PTPase activity. O-linked glycosylation, which occurs mainly in the region encoded by the alternatively spliced exons, has been strongly implicated in this role. The three transgenic strains we have produced carrying the wild-type minigene express different levels of CD45. One of the predictions from the dimerization model of CD45 function is that the different levels of CD45 expression will have functional consequences. Our transgenic strains allow the investigation of this prediction.