The function of natural killer (NK) and myeloid cells depends on the balance of a number of activating and inhibitory receptors. Natural cytotoxicity receptors (NCR) are the major receptors responsible for NK cell-mediated lysis of tumor and viral-infected cells. In the past few years, we have characterized the structure and function of several inhibitory as well as activating NK cell receptors. These include the crystal structures of CD94, KIR2DL2 and its complex with HLA-Cw3, NKG2D and its complex with ULBP3, NKp46 and TREM-1. Despite the progress made in understanding the function of NK receptors in the past few years, the ligands recognized by NCR remain unknown. Our effort of understanding the function of NCR has led a structural solution for NKp30. The structure revealed striking resemblence between NKp30 and CTLA-4 family receptors and enabled us to propose a potential ligand binding site.The ability of NK cells to distinguish self versus non-self cells is particularly critical in tissue transplantation and tumor rejection.UL16 binding proteins (ULBPs) are a family of cell surface proteins present in transformed and stressed cells and ligands for NKG2D. Soluble NKG2D ligands have been found in sera from cancer patients with their protein concentrations correlated with poor cancer prognosis. Here we show that human tumor cells lost their surface ULBP2, but not ULBP1 and ULBP3 expressions during NK-mediated cytolysis. The shedding of ULBP2 was induced by target cell apoptosis and a result of metalloproteinase cleavage. Inhibition of ULBP2 shedding by a metalloproteinase inhibitor BB-94 resulted in down-regulations of NKG2D expression and cytokine production on NK cells. Thus, the NKG2D ligand shedding enables NK cells to optimize their function by selectively engaging live target cells. This may be a mechanism for NK cells to distinguish live versus killed cells. In collaboration with Dr. Alfred Singer's group at NCI, we investigated the determinants for T cell receptor specificity against MHC ligands. The central feature of T cell biology is that T cells selected from the thymus utilize their antigen receptors (TCR) to specifically recognize antigenic peptides presented by the major histocompatibility complex (MHC), a characteristic referred to as MHC-restriction. However, the mechanisms leading to MHC-restriction of T cells are not fully understood. Previous structural studies of TCR and peptide/MHC complexes suggested that MHC-restriction is intrinsic to TCR structures as a result of germline-encoded CDR1 and CDR2 loops that have evolved to specifically promote contacts with MHC. In contrast, emerging evidence has shown that MHC-restriction is imposed by thymic selection in that coreceptor-independent TCR signaling in the thymus permits selection of TCRs that recognize MHC-independent ligands. Further deep sequencing analysis revealed that the overall germline V gene usage was similar in the peripheral TCR repertoire of both wild type B6 and Quad_KO mice. Nevertheless, individual TCR clones were selected at remarkably different frequencies in the presence or absence of MHC, further demonstrating the ligand specificity of TCRs was imposed by the thymic selection. The comparisons of TCR sequences and their frequencies between MHC-restricted and independent repertoires enabled us to delineate any amino acid sequence preferences both in germline-encoded CDR1 and 2, and in non-germline derived CDR3 regions specific for MHC-recognition. In an attempt to characterize the usage of both germline and non-germline regions of TCR in response to MHC, we carried out RNA-seq based deep sequencing on TCR chains from both MHC-independent and MHC-restricted animals. To address the impact of thymic selection to TCR repertoires, we also sequenced TCR repertoire from pre-selection double negative (DN) B6 thymocytes. These comparative repertoire analyses of polyclonal TCRs from different mouse strains revealed that MHC-restricted but not independent repertoires share greater number of public TCR sequences. They selectively enrich CDR3 sequences containing smaller, hydrogen-bonding amino acids but disfavor larger, hydrophobic amino acids and exclude cysteine residues in their MHC-binding site. In contrast, MHC-independent TCRs exhibiting antibody-like CDR3 amino acid compositions. The selective preference of residue composition in CDR3 but not in germline-encoded CDR1 and 2 demonstrates that MHC specificity is not intrinsic to germline-encoded TCR sequences but results from ligand-specific selections. In addition to comparative repertoire analyses, we also report the first crystal structures of MHC-independent TCRs (A11 and B12A),both recognized mPVR as their activation and selection ligand. B12A and A11 closely resemble the conventional MHC-restricted TCRs. The structural comparison of these MHC-independent TCR with those of MHC-restricted ones illustrated the lack of structural changes in CDR1 and 2, demonstrating the structures of germline V genes could not pre-determine TCR ligand specificity. Moreover, B12A and A11 TCRs recognized different epitopes on mPVR, reminiscent of antibody-antigen recognition. In summary, our results suggest that within the pre-selected repertoire TCRs are capable of recognizing a huge diversity of ligand structures and highlight the role of thymic selection in determining the TCR specificities. The comparison of TCR amino acid sequences of various pre-selection repertoires with those of mature repertoires from multiple MHC-specific strains as well as MHC-independent (MHCi) animals showed that MHC-specific thymic selection affected only non-germline encoded CDR3, restricting both their length and usage of specific amino acids, but did not affect TCR V-gene usage nor V-J pairing. Violation of these constraints may result in T cells to fail positive selection from unfavorable interactions with MHC or undergo clonal deletion due to self-reactivity.

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