The first part of this project has been directed to understand the breadth of the CD8+ T cell immune response to immunization with a single gene product, the HIV envelope glycoprotein. Within the context of the immunodominant V3 loop peptide, we observed that minor variations of the peptide in sequence and length permitted detection of distinct populations of reactive T cells. Tetramer analysis and T cell receptor V region sequencing showed that distinct clones were stimulated by recombinant adenovirus 5 (rAd5) alone compared to DNA/rAd5 or recombinant Bacille Calmette-Guerin (rBCG)/rAd5. We explored this phenomenon in greater detail by determining the X-ray crystallographic structure of several of the MHC/peptide complexes recognized by the reactive T cells, and observed that minor differences in the sequence or length of bound peptide nevertheless give rise to major conformational differences in the MHC/peptide complex, explaining the observed variation in the evoked T cell responses. In particular, major differences in solvent exposure and conformation of amino acid side chains of the phenylalanine and tyrosine residues of the peptide IGPGRAFYA as compared to IGPGRAFYTI, when bound to the MHC restricting element, H-2Dd, were observed. The families of TCR that differentially react with these distinct peptide/MHC complexes are different in Valpha and Vbeta sequence as well. These results suggest that differences in the efficacy of alternative prime boost vaccines result from the induction of distinct T cell responses that arise from the differential vector-induced presentation of antigen. Thus, although the use of MHC/peptide tetramers is a valuable and sensitive way to assess T cell responses, a set of variant peptides must be evaluated to be certain that major aspects of particular responses are not overlooked. ? ? ? The second part of 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-like disease is characterized by T cells producing IL4, IL5, and IL13 and shows 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 agressive 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, for the first part of the project on MHC/TCR interactions: 1) the demonstration that different DNA and recombinant BCG vaccines, although they encode the same glycoprotein antigen, elicit distinct populations of CD8 T cells as evidences by their fine specificity; 2) that the different T cell responses can be rationalized by differences in the molecular structure of MHC/peptide complexes that differ minimally in peptide sequence and/or length; and 3) that some of these T cell populations are distinct in terms of their fine specificity. ? ? With respect to the second part of this project, advances include: 1) the development of a unique whole animal model for organ specific, spontaneous autoimmunity; 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, by cell transfer studies, that the phenotypic difference in the autoimmune gastritis in the Th1-like as compared with the Th2-like disease is likely due to differences in the presentation of the different peptide antigens, rather than differences in the stability of the peptide/MHC complexes or differences in the affinity of the TCR for the MHC/peptide complex.
Showing the most recent 10 out of 34 publications
