T cell receptor (TCR) gamma delta is expressed on a population of TCR alpha beta-T lymphocytes. Like TCR alpha and beta, the TCR gamma and delta genes are assembled by somatic arrangement of germ-line gene segments. We have previously characterized the structure and diversity of TCR gamma and delta proteins and genes. Here we wish to generate tetanus toxoid (TT)- specific human gamma-delta T cell clones by in vitro stimulation of gamma-delta T cell-enriched peripheral blood lymphocytes. The fine specificity of T cell clones will be examined by determining the TT peptides that recognize. these T cell clones will be analyzed for self-restriction by their ability to respond to TT presented on autologous and allogeneic antigen presenting cells (APC). If self- restriction exists, a role for MHC class I and II molecules will be examined by determining the TT peptides that they recognize. These T cell clones will be analyzed for self-restriction by their ability to respond to Tt presented on autologous and allogeneic antigen presenting cells (APC). If self-restriction exists, a role for MHC class I and II molecules will be examined using a panel of APCs of known HLA haplotypes, and blocking experiments with anti-hla monoclonal antibodies. Should the restricting elements not be the known class I or class II molecules, we will characterize them further to define their structure. We then wish to examine the TCR gamma and delta V and J segment usage and the V-J junctional sequences of these TT-specific gamma-delta T cell clones by Southern and Northern blot analyses, and the polymerase chain reaction. These analyses should allow us to correlate gene segment usage with TT or self-restricting element recognition. We will then isolate and sequence TCR gamma and delta cDNAs from selected T cell clones, and transfect them by electroporation into a TCR- recipient cell line to reconstitute antigen recognition. Further, we will alter the TCR chains by exchanging their gene segments and by in vitro mutagenesis to examine the contribution of various gene segments or certain sequence motifs to recognition. The ultimate goal recognition by the gamma-delta T cell receptor.
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