This project is focused on functional studies of T cell receptor recognition of autoantigens and how this leads to autoimmune disease. Our current approaches include: 1) the characterization of T cell receptors derived from autoreactive T cell clones, functionally and structurally; 2) the development of transgenic mouse strains that constitutively express a limited TCR repertoire consisting of autoreactive T cell receptors; 3) the functional characterization of these autoreactive TCR transgenic mouse strains; 4) the characterization of the antigenic peptides recognized by the TCR transgenic mouse strains both functionally and biochemically; and 5) the in vivo characterization of processing of autoantigens as this process relates to T lymphocyte selection and development of autoimmune disease. We have cloned and expressed two different TCR from T cell clones that show specificity for two peptides from the gastric H/K ATPase. On transfer to immunodeficient animals, one of these clones causes a Th1 type disease, and the other a Th2-like disease. The Th2 disease is characterized by T cells producing IL4, IL5, and IL13 and leukocyte infiltrates in the gastric mucosa. Transgenic animals expressing the TCR from each of these clones have been produced and have been analyzed. The transgenic derived from the Th1 clone, TXA23, develops a fulminant autoimmune gastritis within 10 days of birth. The transgenic derived from the Th2 clone, TXA51, has a less fulminant disease. This second model offers to provide insight into how inflammatory (Th1) cytokines influence autoimmune disease in a manner distinct from Th2 cytokines. Cells taken from the Th2 diseased animals can be maintained in vitro as Th2 cells, or if stimulated with progressive doses of antigenic peptide presented by dendritic cells, can differentiate into Th1 cells. Several hypotheses concerning the differential cytokine and disease profiles of the two transgenic strains have been investigated: 1) is the intrinsic affinity of the TXA23 TCR greater for its MHC/peptide complex than that of the TXA51 TCR; and 2) is the efficiency of the processing and presentation of the antigenic peptide seen by TXA23 better than that of the peptide seen by TXA51. Careful functional dose-response experiments and IAd/peptide tetramer staining experiments are consistent with the view that the TXA51 TCR affinity for peptide/MHC is greater than that of TXA23 TCR. Cell transfer experiments indicate that TXA23 CD4 T cells transferred to normal mice proliferate extensively in the gastric lymph node while TXA51 cells fail to proliferate. Thus, the simplest explanation is that the affinity differences do not explain the functional phenomena, and that antigen processing and presentation differences do. This view is confirmed by quantitative measures of the stability of the antigenic peptide/MHC complex and will be further examined by direct measurement of the binding of engineered TXA23 and TXA51 TCR for the MHC/peptide complexes. Presently, we have successfully produced both TCR in sufficient quantity and purity to initiate these binding experiments. ? ? Advances include:1) the development of a useful whole animal model for organ specific autoimmunity in our animals that are transgenic for the ATPase reactive TCR--these animals develop a fulminant autoimmune disease completely spontaneously without priming with antigen; 2) the demonstration that a Th2 disease, considerably milder in phenotype has developed in an animal that has a different TCR receptor; 3) the demonstration that the Th1 or Th2 phenotype of the disease-causing cells can be changed by changing the antigen density of antigen presenting cells. We have recently made considerable progress in: 1) fine structure mapping of the epitopic residues of the H/K ATPase as seen by the MHC-II IA-d restricting element; 2) developing MHC-II tetramers of IA-d that can be loaded with the epitopic peptides to use as crucial reagents in stimulating and visualizing the autoreactive T cells in these autoimmune animals; 3) the determination of the X-ray structure of the TXA23 TCR (the structure has been solved by molecular replacement and refinement is almost complete); 4) the successful engineering of a recombinant form of the TXA51 TCR, and the purification of this TCR (binding studies are underway, and crystallization trials are in progress);5) successful engineering, purification, crystallization, and X-ray structure determination of the MHC/peptide complex of IAd bound to the PLL (H/K ATPase alpha chain residues 889-900). This last achievement provides a definitive structural context resolving ambiguities that were indicated by the functional studies done with various sets of related synthetic peptides.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Intramural Research (Z01)
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Morozov, Giora I; Zhao, Huaying; Mage, Michael G et al. (2016) Interaction of TAPBPR, a tapasin homolog, with MHC-I molecules promotes peptide editing. Proc Natl Acad Sci U S A 113:E1006-15
Margulies, David H (2009) Home schooling of NK cells. Immunity 30:313-5
Hu, Jin-Shan; Plaksin, Daniel; Margulies, David H (2005) Backbone and side chain resonance assignmentsof a TRAV14-3 mouse T cell receptor domain. J Biomol NMR 31:271-2
Margulies, David H (2005) Monoclonal antibodies: producing magic bullets by somatic cell hybridization. J Immunol 174:2451-2
Candon, Sophie; McHugh, Rebecca S; Foucras, Gilles et al. (2004) Spontaneous organ-specific Th2-mediated autoimmunity in TCR transgenic mice. J Immunol 172:2917-24
Kuribayashi, Hideki; Wakabayashi, Ayako; Shimizu, Masumi et al. (2004) Resistance to viral infection by intraepithelial lymphocytes in HIV-1 P18-I10-specific T-cell receptor transgenic mice. Biochem Biophys Res Commun 316:356-63
Dam, Julie; Guan, Rongjin; Natarajan, Kannan et al. (2003) Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b). Nat Immunol 4:1213-22
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Margulies, David H (2003) CD28, costimulator or agonist receptor? J Exp Med 197:949-53
Margulies, David H (2003) Molecular interactions: stiff or floppy (or somewhere in between?). Immunity 19:772-4

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