The interaction of a T cell receptor, a class II histocompatibility molecule and antigen is essential for immune recognition. Diversity of both the T cell receptor and the class II molecule is involved in generating the specificity of the response. While the mechanisms which generate potential diversity are clearly defined, the forces which shape the T cell receptors available for interaction with antigen are not yet completely understood in the human. Although many aspects of diversity impinge on this interaction, this study will focus on the importance of germline diversity (repertoire) of T cell receptor genes in a specific cellular immune response and will examine the following: (1) The effect of the repertoire on the functional specificity of alloreactive T lymphocyte clones as measured by the patterns of recognition of an HLA-typed panel; (2) The effect of the repertoire on the primary structure of T cell receptors expressed by the above alloreactive clones as measured by cDNA sequence analysis; and (3) The effect of nonMHC, nonT cell receptor components (genetic/environmental) on the functional specificity and structure of T cell receptors utilized by the above alloreactive clones. The T cell response to an allogeneic human class II molecule will be used to sample the available T cell, repertoire as a model of an antigen recognition. To control the influence of thymic selection by MHC on the expressed T cell receptors, HLA identical siblings will be used as responders in the study. By RFLP analysis with T cell receptor V segment probes, the responders carry different combinations of parental T cell receptor beta chain genes. The stimulator (an HLA recombinant sibling of the responders) shares one HLA haplotype with the responders and differs only at the class II region of the second HLA haplotype. Although the stimulator and responders differ at DR, DQ and DP loci, the priming and selection protocols used will preferentially generate clones which recognize the DR molecule. In order to further focus the DR-specific T cell response on a small number of stimulatory determinants, the priming combination has been selected so that responders and stimulator express DR variants which only differ by three amino acids. With this protocol, the T cell response can be focused on a limited and precisely defined molecular difference and, thereby, restrict the heterogeneity of that response. On a functional level, the specificity and heterogeneity of alloreactive T cell clones generated with this priming combination will be characterized using an HLA-typed panel and monoclonal antibody blocking studies. On a molecular level, heterogeneity of the T cell response and specific T cell receptor gene usage will be studied by cDNA sequencing of variable regions of T cell receptor alpha and beta genes. Segments, if any, which are differentially utilized will be examined using hybridization studies with cloned genes and sequence specific probes and DNA sequence analysis to detect deletion or alteration of genetic segments in one of the responders.
