Fine mapping of T-cell determinants of bovine beta-lactoglobulin

Abstract

T-cell recognition sites, i.e. T-cell determinants, of bovine beta-lactoglobulin, a major allergen in milk, were analyzed in detail. For this purpose, we prepared primary cultures of lymph node cells from three strains of mice, C57BL/6 (H-2b), C3H/HeN (H-2k), and BALB/c (H-2d), and examined the proliferative response of these cells to a complete set of overlapping 15-mer peptides which covered the entire sequence of beta-lactoglobulin by shifting in single amino acid steps. We were able to determine the putative core sequence of each T-cell determinant and estimate its relative importance. In the case of C57BL/6 mice, dominant, subdominant, and minor determinants were identified as residues 122-130, 16-26, and 108-122, respectively, as represented by their core sequences. Each determinant peptide induced the production of interferon-gamma, the amount of which showed a correlation with the intensity of the proliferative response induced by each determinant. In the case of C3H/HeN mice, a dominant determinant comprised of residues 140-148 was identified together with three subdominant and two minor determinants. Dominant T-cell determinants recognized in BALB/c mice were identified as residues 67-75, 71-79, and 80-88, and six other regions were identified as subdominant determinants. Comparisons between our results and the determinants predicted from relevant MHC-binding motifs reported to date revealed the inadequacy of the motifs in predicting even the dominant determinants. The information obtained by complete mapping of T-cell determinants as done in this study is expected to be helpful in establishment and evaluation of new prediction methods and also may contribute to the development of a new approach to control immune responses by manipulation of the T-cell determinants of allergens.