Stanley G. Nathenson, M.D. - US grants

The H-2 MHC class I system plays a crucial role in the cell mediated immune response against cell-associated foreign antigens. The long-term research objectives of this proposal are to continue studies to attempt to understand the basis and purpose of polymorphism and diversity in this system, and to determine the structural features of the MHC class I molecules important for their immunological function in the presentation of foreign antigens, i.e. MHC restriction. Studies on the question of polymorphism and diversity will be approached by continuing analyses of the mechanism of genetic recombination or gene conversion which has been shown to generate the in vivo MHC class I Kb mutants, a system which serves as a model for the mechanisms operating at the population level to generate diversity in the H-2 alleles. The details of class I gene conversion will be studied by molecular analysis of Kb molecules of additional in vivo bm variants, as well as through the study of an in vitro system in which DNA constructs will be utilized to measure interaction of class I H-2 DNA sequences. To analyze the structural features of class I molecules which are responsible for their immunological role, we propose to continue our studies of a series of somatic cell variants expressing altered Kb gene products. Such studies will be directed towards defining the target sites on the Kb molecule which are important for recognition by allogeneic T cells and by cytolytic T cells which recognize foreign antigens plus self MHC molecules. Where appropriate, specific Kbm mutant genes will be constructed by site directed mutagenesis for study in transfected cells. We will prepare large amounts of a few wild type and mutant Kb class I molecules for X-ray diffraction analysis to determine the three- dimensional structure.

Six investigators will continue to study the cell and molecular biology of lymphoid cells. Cultured cell lines and somatic mutants derived from them will be used wherever possible. Immunologically important molecules from parental and mutant cells will be characterized structurally and functionally. Amongst the questions being addressed are: the origins and targets of NK cells, the biochemical basis of macrophage functions including anti-bacterial activity, the mechanism of unresponsiveness in leprosy, the nature and role of cross reactive idiotypes of anti-DNA antibodies in lupus, the molecular basis of MHC polymorphism and the structural basis of MHC function in cell-cell interactions and antigen presentation, and the molecular basis of the somatic generation of antibody diversity and gene rearrangement associated with B cell differentiation.

DESCRIPTION: (Adapted from the investigator's abstract) This proposal is a competitive renewal of the previous grant, AI 07289, a long term project supported by a ten year Merit Award. The aims of this competitive renewal have been expanded from the original application to take advantage of progress in these investigations as well as in the field. These studies focus on the question of how foreign peptide epitopes are selected, and complexed by the MHC molecule for antigen presentation, and how the T cell receptor recognizes the peptide epitope in the context of MHC. The general aims are to use the techniques of immunobiology, biochemistry, molecular genetics and three dimensional structure analysis, (l) to understand the mechanism by which peptides of appropriate length and sequence are selected for and bound by the MHC presenting molecules and (2) to identify at the structural level, the features of the T cell receptor involved in recognizing these peptides in the MHC target molecules. Specifically, it is proposed to continue the studies begun in the previous grant period on the structural basis for the function of class I molecules in peptide presentation. These will include a series of related projects utilizing either three-dimensional structure analysis or in vitro, biochemical and physical-chemical procedures to probe the structural impact of MHC polymorphism, the mechanism for editing and binding peptides, and the role and interactions of chaperones in the proper folding of MHC class I complexes. These studies are relevant to peptide vaccine development. T-cell receptor function/structure analyses will be complemented by studies to determine the 3D structure of the T-cell receptor in order to understand the structural basis for the events of T-cell recognition of peptide presented by MHC, studies that are relevant to T-cell development, immunity to infectious diseases and autoimmunity.

DESCRIPTION (provided by the applicant): Together with signaling through the T cell antigen receptor, costimulation is essential for a robust and properly regulated immune response. The engagement of CD28 and ICOS with the B7 isoforms and B7H, respectively, on antigen presenting cells, provide the stimulatory signals that direct T cell proliferation and cytokine production. Engagement of CTLA-4 and PD-1 with the B7 and PD-L isoforms, respectively, on antigen presenting cells, provides inhibitory signals required for attenuation of the T cell response and the induction of peripheral tolerance. As a consequence of their fundamental roles in modulating T cell responsiveness, these costimulatory molecules and their associated signaling pathways are active targets for the development of therapeutics to treat a wide range of human pathologies, including cancer, autoimmunity, and graft rejection. Our recent structural studies have identified a highly ordered, alternating network of CTLA-4 and B7-2 homodimers that for the first time provides a model for the organization of these molecules and their associated signaling partners within the T cell/antigen presenting cell immunological synapse. These observations have generated a number of directly testable hypotheses regarding the molecular mechanisms of CTLA-4 costimulation. Our overall goals are to understand the features of the CTLA-4 and B7 homodimers that contribute to signaling, the importance of the periodic organization of the CTLA-4/B7 assembly for function, and the generality of these features in the signaling mechanisms of the other costimulatory receptor-ligand pairs. We have adopted a multidisciplinary approach that combines three-dimensional structural, biochemical, and cell biological information with appropriate mammalian models so as to determine in vivo structure-function correlations for the CD28/CTLA-4/ICOS family of costimulatory molecules. Accordingly, our Specific Aims are: 1) Three dimensional structure determination of the costimulatory receptor-ligand pairs; 2) Characterization of the biologically relevant oligomeric states of the costimulatory molecules; 3) Identification of the functional requirements associated with dimeric organization that are relevant to costimulation; 4) Examination of the organization of costimulatory molecules at the immunological synapse.

DESCRIPTION (provided by applicant): Autoimmune diabetes in both humans and nonobese diabetic (NOD) mice results from T cell-mediated autoimmune destruction of insulin-producing pancreatic beta cells. Both class I major histocompatibility complex (MHC)-restricted and class II MHC-restricted T cells are involved. However, T cell receptors (TCRs) restricted to recognition of autoantigenic peptides presented by the trimeric class I molecule, consisting of MHC heavy chain, beta2-microglobulin (beta2m), and peptide, are absolutely required for the initiation of disease in the NOD mouse. Autoimmune diabetes is a polygenic disease, with particular MHC haplotypes providing the primary genetic component of susceptibility in both humans and NOD mice. Recently, (beta2m) was identified as a diabetes susceptibility gene in NOD mice, the first such gene to be identified that maps outside of the MHC region, lying within the Idd13 locus on Chromosome 2. Two beta2m alleles are widespread throughout the common laboratory mouse strains. When the a2ma allele found in NOD mice is replaced by the a2mb allele, development of diabetes is prevented. The amino acid sequence difference between these two allelic proteins resides in a single exchange of Asp ("a" isoform) for Ala ("b" isoform) at position 85. A number of previous serological and T cell recognition studies, using a variety of MHC allelic products and a2m proteins from different species, have suggested significant conformational flexibility of the class I molecule dependent on the particular beta2m present in the complex. Based on these findings, it can be hypothesized that class I MHC molecules containing beta2ma might exhibit an altered conformation as compared to those containing beta2mb. Such changes could exert effects on both selection of autoreactive T cells and presentation of autoantigenic peptides. The overall goal of this proposal is to test this hypothesis by systematically examining the structural, biochemical, and biological properties of disease-relevant MHC-peptide complexes containing the two beta2m isoforms. Four Specific Aims are proposed: (1) Structural, thermodynamic, and dynamic characterization of the isolated allelic beta2m proteins, using X-ray diffraction analysis, chemical and thermal denaturation, and amide proton exchange; (2) Similar characterization of the diabetes-related MHC/peptide complexes, including measurements of beta2m and peptide exchange rates; (3) Structural, biochemical, and dynamic characterization of the diabetes-related TCR/MHC-peptide complexes containing the two isoforms Of beta2m, using X ray diffraction analysis, amide proton exchange, and surface plasmon resonance; and (4) Cellular analysis of T Cell recognition and TCR dwell time using MHC-peptide complexes containing the two different beta2m isoforms. Completion of the proposed Aims should allow identification of the molecular and atomic determinants that result in beta2m-dependent susceptibility or protection against disease.

DESCRIPTION (provided by applicant): Autoimmune diabetes in both humans and nonobese diabetic (NOD) mice results from T cell-mediated autoimmune destruction of insulin-producing pancreatic beta cells. Both class I major histocompatibility complex (MHC)-restricted and class II MHC-restricted T cells are involved. However, T cell receptors (TCRs) restricted to recognition of autoantigenic peptides presented by the trimeric class I molecule, consisting of MHC heavy chain, beta2-microglobulin (beta2m), and peptide, are absolutely required for the initiation of disease in the NOD mouse. Autoimmune diabetes is a polygenic disease, with particular MHC haplotypes providing the primary genetic component of susceptibility in both humans and NOD mice. Recently, (beta2m) was identified as a diabetes susceptibility gene in NOD mice, the first such gene to be identified that maps outside of the MHC region, lying within the Idd13 locus on Chromosome 2. Two beta2m alleles are widespread throughout the common laboratory mouse strains. When the a2ma allele found in NOD mice is replaced by the a2mb allele, development of diabetes is prevented. The amino acid sequence difference between these two allelic proteins resides in a single exchange of Asp ("a" isoform) for Ala ("b" isoform) at position 85. A number of previous serological and T cell recognition studies, using a variety of MHC allelic products and a2m proteins from different species, have suggested significant conformational flexibility of the class I molecule dependent on the particular beta2m present in the complex. Based on these findings, it can be hypothesized that class I MHC molecules containing beta2ma might exhibit an altered conformation as compared to those containing beta2mb. Such changes could exert effects on both selection of autoreactive T cells and presentation of autoantigenic peptides. The overall goal of this proposal is to test this hypothesis by systematically examining the structural, biochemical, and biological properties of disease-relevant MHC-peptide complexes containing the two beta2m isoforms. Four Specific Aims are proposed: (1) Structural, thermodynamic, and dynamic characterization of the isolated allelic beta2m proteins, using X-ray diffraction analysis, chemical and thermal denaturation, and amide proton exchange; (2) Similar characterization of the diabetes-related MHC/peptide complexes, including measurements of beta2m and peptide exchange rates; (3) Structural, biochemical, and dynamic characterization of the diabetes-related TCR/MHC-peptide complexes containing the two isoforms Of beta2m, using X ray diffraction analysis, amide proton exchange, and surface plasmon resonance; and (4) Cellular analysis of T Cell recognition and TCR dwell time using MHC-peptide complexes containing the two different beta2m isoforms. Completion of the proposed Aims should allow identification of the molecular and atomic determinants that result in beta2m-dependent susceptibility or protection against disease.