Identification of an I-Ed-Restricted T-Cell Epitope of Escherichia coli Outer Membrane Protein F

AUTOR(ES)

Williams, Kristina M.

FONTE

American Society for Microbiology

RESUMO

A predominant T-cell epitope of Escherichia coli outer membrane protein F (OmpF) that encompasses amino acids 295 to 314 was identified in H-2d mice. BALB/c-derived T-cell hybridomas generated against this region were CD3+, CD4+, CD8−, and T-cell receptor αβ+ 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-Ed blocked IL-2 secretion by hybridomas, but I-Ad-specific antiserum did not. When transfected L cells expressing I-Ad (AαAβd), I-Ed (EαEβd), or the hybrid molecule I-EαAβd were used as APCs, hybridomas recognized peptide only when presented by the I-Ed-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-Ed and variability in the amino acid composition of the C and N termini of these peptides.

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