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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032494-09
Application #
6510693
Study Section
Experimental Immunology Study Section (EI)
Program Officer
Macchiarini, Francesca
Project Start
1994-09-30
Project End
2004-06-30
Budget Start
2002-07-01
Budget End
2003-06-30
Support Year
9
Fiscal Year
2002
Total Cost
$284,596
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Pathology
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
Vergara-Silva, Andrea; Schaefer, Katherine L; Berg, Leslie J (2002) Compartmentalized Eph receptor and ephrin expression in the thymus. Mech Dev 119 Suppl 1:S225-9
Vergara-Silva, Andrea; Schaefer, Katherine L; Berg, Leslie J (2002) Compartmentalized Eph receptor and ephrin expression in the thymus. Gene Expr Patterns 2:261-5
Berg, L J; Kang, J (2001) Molecular determinants of TCR expression and selection. Curr Opin Immunol 13:232-41
Bunnell, S C; Diehn, M; Yaffe, M B et al. (2000) Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade. J Biol Chem 275:2219-30
Yelon, D; Schaefer, K L; Berg, L J (1999) Alterations in CD4-binding regions of the MHC class II molecule I-Ek do not impede CD4+ T cell development. J Immunol 162:1348-58
Yelon, D; Berg, L J (1997) Structurally similar TCRs differ in their efficiency of positive selection. J Immunol 158:5219-28
Thomis, D C; Lee, W; Berg, L J (1997) T cells from Jak3-deficient mice have intact TCR signaling, but increased apoptosis. J Immunol 159:4708-19
Yelon, D; Spain, L M; Lim, K et al. (1996) Alterations in CD4 dependence accompany T cell development and differentiation. Int Immunol 8:1077-90
Lieberman, S A; Spain, L M; Wang, L et al. (1995) Enhanced T cell maturation and altered lineage commitment in T cell receptor/CD4-transgenic mice. Cell Immunol 162:56-67