(Adapted from the applicant's abstract and Specific Aims.) The generation of the T cell receptor repertoire is a critical factor in determining immune responses. A system for studying T cell development and the selection mechanisms shaping the T cell repertoire using 2B4 T cell receptor (TCR) transgenic mice have been developed. In these mice, over 95 percent of the developing T cells express a single TCR specificity (cytochrome c plus I-Ek). The previous genetic experiments using this system have demonstrated that T cell maturation requires an interaction between the TCR and I-Ek molecules present on thymic stromal cells. Furthermore, this interaction determines the differentiation of immature thymocytes into mature CD4+ helper T cells, rather than CD8+ cytotoxic T cells. Using site directed mutagenesis and an in vitro functional assay, two independent sites for CD4 interaction on the beta chain of the MHC class II-Ek molecule have been recently identified. A double mutant carrying both of these alterations will be introduced into the germline of mice. Transgenic mice carrying the mutant E-beta gene will be crossed to the 2B4 TCR transgenic mice to assess the role of MHC class II/CD4 interaction during positive and negative selection. In addition, the role of this interaction in the commitment of differentiating T cells to the helper versus cytotoxic lineage will be evaluated. T cell development in vitro has been studied in thymic organ cultures derived from the 2B4 TCR transgenic mice. It was found that both the interactions resulting in the elimination of self-reactive T cells (negative selection) and the differentiation of immature thymocytes (positive selection) can be faithfully reproduced in this in vitro system. Using this approach, the goal is to understand which intercellular interactions can induce differentiation, which can induce cell death, and whether specific thymic stromal cell types are necessary for each of these interactions. Thymic organ cultures derived from I- E-2B4 TCR transgenic mice will be supplemented with exogenously-derived cells, or cells plus peptides, in an attempt to generate the missing signal required to induce T cell differentiation or cell death. Specifically, Specific Aim 1 proposes to analyze the role of MHC class II/CD4 interactions during the thymic selection process. Specific Aim 2 proposes to study the thymocyte/antigen presenting cell interactions responsible for inducing positive and negative selection. Specific Aim 3 proposes to study the coordinate regulation of helper versus cytotoxic T cell function with CD4 versus CD8 lineage commitment. This information could enhance our understanding of T cell recognition and the establishment of self-tolerance.

R01AI032494

1994-09-30

2004-06-30