The function of the immune system in health is critically dependent upon the appropriate recognition of foreign antigen by the antigen receptor of the T lymphocytes of the host. The T cell receptor (alpha-beta-TCR) must therefore discriminate between those antigens which are """"""""self"""""""" and those antigens which are foreign or """"""""non-self"""""""". This self/non-self discrimination is generated within the thymus where T cells undergo positive selection to generate a repertoire which recognizes foreign antigen only in the context of self-MHC and negative selection to from a repertoire those T cells with self-reactive specificities. We propose to test the hypothesis that positive selection and negative selection act on discreet populations in thymic development and therefore may be defined functionally and phenotypically. We will further test the hypothesis that the physiologic outcome of positive vs. negative selection is determined by the signal transduction properties of the alpha-beta-TCR and the co- receptor molecules CD4 and CD8 in these discreet populations. We will first (Aim 1) identify and isolate the developing T cell subpopulations which are subject to positive vs. negative selection. Negative selection will be analyzed in normal mice by the functional assays of [Ca++]i mobilization and deletion in thymic organ culture in response to ligation of the alpha-beta-TCR. Positive selection will be analyzed in those cells resistant to deletion by analysis of the Vbeta repertoire in """"""""overselecting"""""""" genetic backgrounds. Thymic subpopulations will also be isolated and analyzed from alpha-beta-TCR transgenic mice bred onto selecting or non-selecting backgrounds. We propose to use these isolated subpopulations from normal and transgenic mice to identify unique surface markers, to define the ontogeny of positive and negative selection, and to development a system for positive selection in thymic organ culture. With isolated and characterized subpopulations of immature thymocytes in hand from Aim 1, which are susceptible to either positive or negative selection, we will then in Aim 2 determine the early signal transduction events of [Ca++]i mobilization, zeta chain expression and tyrosine phosphorylation, and activation of the tyrosine kinase p56lck, in order to begin to understand those signalling events which determine the selective event in thymocyte maturation, as we have in our previous studies defined [Ca++]i mobilization and coupling of the TCR complex as critical to the deletion event. These experiments will employ mouse T cells, T and B cell hybridomas, and alpha-beta-TCR transgenic mice in conjunction with a number of experimental methods including biochemical analysis of [Ca++]i mobilization, receptor expression, protein phosphorylation, protein kinase activation, and gene expression. The proposed studies should define the specific thymic subpopulations in which positive and negative selection occur, as well as the early signal transduction events which result in a mature thymocyte population competent to discriminate between """"""""self"""""""" and """"""""non-self"""""""". This will represent a major advance in our understanding of T cell development which will be crucial to the development of therapeutic intervention to treat and prevent the breakdown in these processes which occurs in autoimmune disease.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Immunobiology Study Section (IMB)
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National Jewish Health
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