Molecular Genetic Analysis Of Lymphocyte Function

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

As indicated above, our studies have resulted in a detailed understanding of the three-dimensional structure and structural changes that accompany MHC-I interaction with its chaperone, TAPBPR, and with the release of the chaperone from the MHC-I molecule on interaction with peptide. Our recently published experiments extend this analysis to the interaction of the MHC-I interaction with tapasin, the crucial chaperone of the peptide loading complex (PLC). To this end, we have for the first time characterized a molecular complex of a human MHC-I molecule, HLA-B*44:05 with the human chaperone, tapasin. Additional studies include the in vivo characterization of a pan-anti-MHC-I monoclonal antibody in stimulating anti-viral and anti-tumor immune responses. Structural studies of the antibody/MHC complex have defined the binding site of the monoclonal antibody, and provide insight into the mechanism of its biological function in activation of NK cells. In Part 2 of the studies of MHC-I/peptide interactions, we have explored the role that the anti-retroviral drug, abacavir, plays in binding to MHC-I and distorting the self-peptide repertoire bound by susceptible MHC-I alleles. In particular, we examined the biological effects of abacavir binding to HLA-B*57:01 in a model animal system that we have developed. Thus, abacavir alone, when bound by HLA-B*57:01 in a transgenic animal, can elicit a neoantigen T cell response, dependent on regulation of CD4 T cells. This animal model system provides an explanation for the severe hypersensitivity reactions that are observed in a high proportion of HLA-B*57:01 individuals who receive the drug. The initial studies were performed with HLA-B*57:01 transgenic animals on a normal mouse MHC background. Recent collaborative studies of a similar drug/MHC-I interaction involving the antibiotic, flucloxacillin, demonstrate a related but distinct mechanism for this particular hypersensitivity reaction. The third part of this project is focused on structural and functional studies of T cell receptor recognition of antigens, how this leads to T cell signaling, and how this leads to autoimmune disease. Here, to provide a baseline for understanding antigen-specific structural changes in the T cell receptor (TCR), we have determined the X-ray structure of a virus specific, MHC-I-restricted TCR, as well as its complex with its MHC-I/viral antigen ligand. Remarkably, although the MHC/peptide complex has a relatively rigid structure, the TCR shows great movement of its CDR3 alpha and beta loops, indicative of a fly-casting mechanism for ligand engagement. NMR experiments have characterized allosteric effects of the TCR on peptide/MHC interaction. Current efforts are geared to apply our recently acquired expertise in cryo-EM structure determination to the MHC/TCR complex.