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Infection and Immunity, July 2004, p. 3907-3913, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.3907-3913.2004

Identification of an I-E^d-Restricted T-Cell Epitope of Escherichia coli Outer Membrane Protein F

Kristina M. Williams^* and Elmer C. Bigley III

Immunobiology Branch, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, Maryland 20708

Received 8 January 2004/ Returned for modification 10 February 2004/ Accepted 22 March 2004

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 ß⁺ 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 (AAß^d), I-E^d (EEß^d), or the hybrid molecule I-EAß^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.

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* Corresponding author. Mailing address: Food and Drug Administration, Center for Food Safety and Applied Nutrition, Immunobiology Branch, 8301 Muirkirk Rd., Laurel, MD 20708. Phone: (301) 827-8458. Fax: (301) 594-0517. E-mail: kwillia2{at}cfsan.fda.gov.

Editor: A. D. O'Brien

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Infection and Immunity, July 2004, p. 3907-3913, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.3907-3913.2004