Scientific Abstract
Proposal No. IBD-0094
Principal Investigator: Victor M. Morales, Ph.D.
Current Applicant Organization: University of Rochester (New York, U.S.A.)
Original Applicant Organization: Boston University Medical Center (Massachusetts, U.S.A.)
Project Title: Peptides recognized by T cell clones from inflammatory bowel disease patients
Period of Award: February 1, 2004 – September 30, 2006
Inflammatory bowel disease (IBD) is considered to be the result of an exaggerated response to intestinal antigens of either endogenous or microbial origin. In Crohn’s Disease (CD), CD4+ T cells play an important role in this immune response. The antigens that activate the Th1 cells and trigger a cascade of inflammation leading to destruction of the mucosal tissues are currently unknown. Identification of the source of antigenic peptides that activate the Th1 CD4+ T cells in CD patients will help our understanding of the mechanisms of disease initiation and progression, as well as the development of therapeutic treatments.
In this proposal, we describe research designed to identify and isolate T cell clones relevant to CD from intestinal surgical samples of CD subjects selected by their preponderance in affected versus healthy tissue areas. The dominant representation of clones in the whole population of T cells in the samples will be assessed using a quantitative real-time PCR technique. Following identification of the antigenic contact region, the T cell receptor (TCR) α and β chains from dominant T cell clones, the coding sequences for these proteins will be PCR amplified, subcloned as fusion constructs to mouse constant TCR α and β regions, and expressed in a mouse hybridoma cell line.
In this way, we will reconstruct human TCRs in a cell line with long-term growth capability. Each chimeric TCR clone will be tested against autologous Epstein Barr virus (EBV) transformed B cells that have been given a pool of peptides from a positional scanning synthetic combinatorial peptide library (PS-SCL). This type of peptide library is formed by pools of synthetic peptides in which each pool contains peptides with random amino acid sequences except for one single fixed position. The IL-2 production by T cells, which have exposed to antigen presenting cells loaded with one of the peptide library pools, will be scored and a matrix of stimulation data will be constructed. The analysis of the stimulation matrix will yield an optimal stimulatory peptide sequence. A search of all the available databases will be performed to identify proteins that have portions homologous to the optimal peptide. The coding sequence for these proteins will be obtained, synthesized as recombinant proteins and tested for stimulation of the chimeric TCR clones. By these experiments, potential antigenic determinants for dominant CD tissue CD4+ T cell clones will be determined.