Identification of an I-Ed-restricted T-cell epitope of Escherichia coli outer membrane protein F.
A predominant T-cell epitope of Escherichia coli outer membrane protein F (OmpF) that encompasses amino acids 295 to 314 was identified in H-2(d) mice. BALB/c-derived T-cell hybridomas generated against this region were CD3(+), CD4(+), CD8(-), and T-cell receptor alphabeta(+) and secreted TH-1-associated cytokines (interleukin-2 [IL-2] and gamma interferon), but not a TH-2-associated cytokine (IL-4), when restimulated with peptide 295-314. Class II(+) mouse lymphoma (A20) cells, but not class II(-) mouse mastocytoma (P815) cells, supported IL-2 secretion of hybridomas when substituted for syngeneic splenocytes as antigen-presenting cells (APCs). Antibodies specific for I-E(d) blocked IL-2 secretion by hybridomas, but I-A(d)-specific antiserum did not. When transfected L cells expressing I-A(d) (AalphaAbeta(d)), I-E(d) (EalphaEbeta(d)), or the hybrid molecule I-EalphaAbeta(d) were used as APCs, hybridomas recognized peptide only when presented by the I-E(d)-transfected cells. When peptide 295-314 truncated at either the C or the N terminus of the sequence was used, the minimal epitope was determined. Critical residues were determined by using alanine-substituted peptide analogues. T-cell hybridomas were only stimulated by peptides that encompassed amino acids 295 to 303 (9-mer), and the core sequence required a minimum of three additional amino acids at either the amino or the carboxy terminus to induce IL-2 secretion. Critical residues were determined to be phenylalanine at position 295, threonine at position 300, and tyrosines at positions 301 and 302. This study is the first to identify a minimal T-cell epitope and major histocompatibility complex restriction element of the OmpF protein and confirms previous observations that there is considerable degeneracy in the length of peptides that can bind I-E(d) and variability in the amino acid composition of the C and N termini of these peptides.
Williams
Kristina M
KM
Center for Food Safety and Applied Nutrition, Immunobiology Branch, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA. kwillia2@cfsan.fda.gov
Bigley
Elmer C
EC
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000939:Epitopes / Q000276:immunology*; D004926:Escherichia coli / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006825:Hybridomas / Q000276:immunology; D008285:Major Histocompatibility Complex / Q000276:immunology; D051379:Mice; D018272:Porins / Q000276:immunology* / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*
D000596:Amino Acids; D000939:Epitopes; D000949:Histocompatibility Antigens Class II; C024138:I-E-antigen; C109237:OmpF protein; D018272:Porins