Biological effect of varying peptide binding affinity to BoLA-DRB3.2*2703.

摘要

The major histocompatibility complex (MHC) class I and class II molecules bind selectively to different antigenic peptides and present peptides to T-cell receptors. In mice and humans, the nature of peptide binding affinity (low and high) to MHC II molecules influences T helper 1/T helper 2 responses by inducing distinctive cytokine expression. To examine the effects of antigen binding affinity to bovine MHC, the BoLA-DRB3.2*2703 allele, previously associated with mastitis in Canadian Holsteins, was extracted and purified from transfected BoLA-DRB3.2*2703 L929. Subsequently, a selection of self (bovine MHC-DQ and bovine fibrinogen fragments) and non-self overlapping peptides from the core region of ovalbumin (OVA), VP2 and VP4 peptides from foot and mouth disease virus (FMDV) were used in competitive binding assays. Briefly, a biotinylated self-peptide with high affinity, a bovine MHC-DQ fragment, was incubated with purified BoLA-DRB3.2*2703 molecules in the presence of various concentrations of competing peptides. Binding was detected using Western blotting and enhanced chemiluminescence. Binding affinity determined based on IC50 (concentration required to inhibit 50% of binding), ranged from 26.92 to >320 µM. T-cells from a homozygous cow (BoLA-DRB3.2*2703), were stimulated in vitro and cell proliferation induced by the above peptides using transfected L929 cells as antigen presenting cells. The results indicate association between binding affinity and the induction of T-cell proliferation.