T lymphocytes are essential for the proper regulation of an immune response and recognize antigen via the T-cell receptor complex, a group of six distinct transmembrane polypeptides. Engagement of the T-cell receptor complex results in the activation of protein tyrosine kinases which results in the phosphorylation of a number of different substrates ultimately leading to a cascade of events culminating in changes in cellular metabolism, cytokine production and proliferation. Two members of the Src-family, p56lck and p59fyn, have been implicated in T-cell receptor signal transduction and both are positively regulated by the transmembrane protein tyrosine phosphatase, CD45. CD45- deficient T-cell clones fail to efficiently activate p56lck and p59fyn and, correspondingly, fail to respond to antigen challenge. Recently, the development of CD45-deficient mice has demonstrated that CD45 expression is critical for the development of the immune system. CD45-deficient mice are arrested in thymocyte development at a stage where cells express both CD4 and CD8. Consequently, these mice have few peripheral T-cells. The CD45-deficient cells and mice provide an important experimental model to further evaluate the regulation of CD45 function. This application addresses the role of CD45 extracellular domain in regulating CD45 function.
Specific Aim 1 examines whether CD45 extracellular domain is required for thymocyte development. Transgenic mice will be generated that thymically express CD45 with a deleted extracellular domain. These transgenic mice will be crossed with the CD45-deficient mice and the ability to rescue thymocyte development determined.
Specific Aims 2 and 3 examines whether there are differences between lymphocytes that express different CD45 isoforms. There are multiple isoforms of CD45 that differ in the length of an amino-terminal region which contains multiple O-linked carbohydrate sites. CD45 isoform expression is highly regulated in lymphocyte development and activation, and the patterns of expression are conserved in vertebrate evolution, implying the CD45 isoform expression is important to function.
Specific Aim 2 proposes to express the high and the low molecular weight CD45 isoform in CD45-deficient T-cell clones. The ability of these cells to respond to different antigen-presenting cells will be determined.
Specific Aim 3 proposed to develop CD45 isoform- specific mice by developing transgenic mice that express either the high or low molecular weight isoform, using the CD45 promoter, and crossing with the CD45-deficient mice. CD45 isoform specific mice will be examined for thymocyte development, circulation and compartmentalization of lymphocytes and the ability of lymphocytes to respond to antigen receptor stimulation. This application proposes experiments to understand the role of CD45 extracellular domain in lymphocyte development and activation and will provide information on how transmembrane protein tyrosine phosphatases regulate cells.
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