Biochemistry and Pharmacology - Research Publications
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ItemPresentation of newly synthesized glycoproteins to CD4+ T lymphocytes. An analysis using influenza hemagglutinin transport mutants.(Rockefeller University Press, 1993-04-01)Human lymphoblastoid cells transiently expressing the hemagglutinin (HA) glycoprotein of influenza virus are rapidly and efficiently recognized by CD4+ HA-specific T lymphocytes. This endogenous presentation pathway is sensitive to chloroquine and is therefore likely related to the classical class II major histocompatibility complex (MHC) exogenous pathway of antigen presentation. In this study we have examined a series of transport-defective HA mutants. We correlate the intracellular fate of the native antigen with its presentation characteristics. We have found that the native antigen must enter the secretory pathway since a cytosolic form is not presented. However, surface expression and normal trafficking through the Golgi apparatus are not required for efficient presentation. Instead, escape of native antigen from the endoplasmic reticulum appears to be both necessary and sufficient for gaining access to a compartment where antigen is processed and binds class II MHC molecules.
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ItemCHANGES IN THE REPERTOIRE OF PEPTIDES BOUND TO HLA-B27 SUBTYPES AND TO SITE-SPECIFIC MUTANTS INSIDE AND OUTSIDE POCKET-B(ROCKEFELLER UNIV PRESS, 1993-03-01)HLA-B27 subtypes share many structural features, including their pocket B, which interacts with a conserved Arg residue at the second position of B*2705-bound peptides. Subtypes differ among each other at other locations in the peptide binding site. In this study, metabolic labeling and radiochemical pool sequencing were used to address the following issues: (a) presence of the Arg 2 (R2) motif among peptides bound to the various HLA-B27 subtypes; (b) influence of mutations inside and outside pocket B on this motif; and (c) the degree of similarity among the peptide pools bound to the various B27 subtypes. Sequencing of Arg-labeled peptide pools extracted from B*2701 to B*2706, and from two site-directed mutants of B*2705 with changes outside pocket B, indicated that all of these molecules bind peptides with Arg at position 2. Peptides from several mutants with changes altering the structure of pocket B, and from one mutant at the pocket B rim, also retained the R2 motif. However, this was absent in the peptide pool extracted from the M45 mutant, in which the negative charge of pocket B, conferred to HLA-B27 by Glu45, was canceled. These results indicate that alterations outside pocket B, and even disruption of the network of hydrogen bonds that stabilizes Arg binding in pocket B, do not impair binding of peptides bearing the R2 motif, but a nonconservative substitution at position 45 does. As a substantial fraction of anti-B*2705 cytotoxic T lymphocyte (CTL) clones crossreact with the M45 mutant (Villadangos, J., B. Galocha, D. López, V. Calvo, and J. A. López de Castro. 1992. J. Immunol. 149:505) this result suggest that determinant mimicry by nonidentical peptides may frequently account for unexpected CTL crossreactions. Metabolic labeling with various other amino acids and radiochemical sequencing revealed similarities, but also substantial differences, among the peptide pools from the various HLA-B27 subtypes. This strongly suggests that many peptides bind to multiple subtypes, but significant subsets of peptides bound to a given HLA-B27 subtype do not bind to other subtypes or do so with greatly altered efficiency. These results indicate the importance of polymorphism outside pocket B in modulating peptide binding to HLA-B27.
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ItemAN AUTOIMMUNE-DISEASE WITH MULTIPLE MOLECULAR TARGETS ABROGATED BY THE TRANSGENIC EXPRESSION OF A SINGLE AUTOANTIGEN IN THE THYMUS(ROCKEFELLER UNIV PRESS, 1993-08-01)Many autoimmune diseases are characterized by autoantibody reactivities to multiple cellular antigens. Autoantigens are commonly defined as targets of the autoimmune B cell response, but the role, if any, of these autoantigens in T cell-mediated autoimmune diseases is generally unknown. Murine experimental autoimmune gastritis is a CD4+ T cell-mediated organ-specific autoimmune disease induced by neonatal thymectomy of BALB/c mice. The murine disease is similar to human autoimmune gastritis and pernicious anemia, and is characterized by parietal and chief cell loss, submucosal mononuclear cell infiltrates, and autoantibodies to the alpha and beta subunits of the gastric H/K ATPase. However, the specificity of T cells that cause the disease is not known. To examine the role of the H/K ATPase in this T cell-mediated disease, transgenic mice were generated that express the beta subunit of the H/K ATPase under the control of the major histocompatibility complex class II I-Ek alpha promoter. We show that transgenic expression of the gastric H/K ATPase beta subunit specifically prevents the onset of autoimmune gastritis after neonatal thymectomy. In addition, thymocyte transfer experiments suggest that tolerance of pathogenic autoreactive T cells is induced within the thymus of the transgenic mice. We conclude that the beta subunit of the gastric H/K ATPase is a major T cell target in autoimmune gastritis and that thymic expression of a single autoantigen can abrogate an autoimmune response to multiple autoantigens.