Abstract
Intracellular processing of the breakpoint products of the bcr-abl fusion gene may generate novel peptides that, if capable of binding to HLA class I molecules, would be potential targets for a cytotoxic T lymphocyte (CTL) response. In humans, peptides derived from the e1a2 p190 breakpoint have generated peptide-specific MHC class II proliferative responses. Short peptides have shown binding to MHC class I molecules, although processing and presentation of endogenous p190 protein has not been shown. In a Kd Balb/c mouse model, we tested the hypothesis that single, key amino acids near the breakpoint could be the reason of lack of immunogenicity of the p190 breakpoint peptides. We synthesized a native peptide from the 190 breakpoint (AFHGDAEAL) as well as a synthetic mutated peptide (AYHGDAEAL), which showed excellent predicted binding on the BIMAS algorithm (1152 and 2880 respectively), although in vivo experiments did not show any specific CTL response. In order to assess if the lack of immunogenicity in vivo was due to the absence of binding to the MHC class I molecule rather than to poor TCR interactions, we designed a series of peptides where neutral amino acid, alanine, substitutions were introduced at different potential binding sites in the synthetic peptide: at position three (AYAGDAEAL), position four (AYHADAEAL), position five (AYHGAAEAL) and position seven (AYHGDAAAL). The binding of these altered peptides to H2 class I was assessed using a MHC stabilization assay on T2-Kd cells (TAP deficient cells). In spite of the good computer prediction for binding the MHC stabilization assay did not show evidence of binding of the native and synthetic peptides (<1.9 over the background). In contrast, alanine substitution in position five resulted in the best binding (3.5x over the background). Alanine substitutions in positions three, four and seven also showed improved binding to Kd molecules (2.6x, 3.1x and 3x over the background respectively). These results were confirmed by immunization in vivo with these altered peptides. Only the peptide with the alanine substitution in position five generated a immune response in a CD8 gamma-IFN Elispot assay. Since the altered peptides with the alanine substitutions in positions three, four and seven have shown binding to the Kd molecules, but not a CTL response, change of the aspartic acid in position five near to the fusion breakpoint allows appropriate presentation and recognition of the sequence by the TCR.
Transplantation Tolerance to a Single Noninherited MHC Class I Maternal Alloantigen Studied in a TCR-Transgenic Mouse Model
