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Comparative Study
. 2005 May 31;102(22):7928-33.
doi: 10.1073/pnas.0502255102. Epub 2005 May 18.

Activated Antigen-Presenting Cells Select and Present Chemically Modified Peptides Recognized by Unique CD4 T Cells

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Free PMC article
Comparative Study

Activated Antigen-Presenting Cells Select and Present Chemically Modified Peptides Recognized by Unique CD4 T Cells

Jeremy Herzog et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

CD4 T cells recognized posttranslationally modified peptides of the protein hen egg-white lysozyme (HEL), consisting of nitration of tyrosines and modifications of tryptophans in the T cell contact residues of the peptides. T cells were directed against modifications of a chemically dominant HEL peptide as well as a minor HEL peptide, bound to the class II histocompatibility molecule I-A(k). The modified peptides were generated in vivo after immunization with native HEL molecules or were generated ex vivo by peroxynitrite treatment of HEL. Moreover, antigen-presenting cells (APC), either macrophages or dendritic cells activated in culture or in vivo, generated the modified HEL epitopes that stimulated the T cells. In transgenic mice expressing HEL, the T cells to the modified epitopes escaped negative selection and were found, albeit fewer in number than in normal mice. Infection with Listeria monocytogenes of the transgenic HEL mice generated APC containing the modifications. T cells to modified epitopes induced by activation of APC may be a component of antimicrobial immunity and autoimmune reactions.

Figures

Fig. 1.
Fig. 1.
The AJ.S7 T cells react with nTyr-53 residue of the 52–60 family, and the 2–5 T cells also react with Trp-62. The C3.F6 APC line was used to present the antigens. HEL+PN refers to HEL treated with peroxynitrite. (A) The response of the AJ.S7 T cell hybridoma. In other experiments, peptide 48–61 treated with peroxynitrite stimulated at a half-maximal response of 0.01 μM (and a top response of 21,500 cpm), but the treatment of the peptide in which the Tyr-53 was changed to alanine did not stimulate. AJ.S7 did not respond when tested with an unrelated peptide but had a nTyr (a transferrin peptide 103–117: KGTDY*QLNQLEGKKG0). (B) The response of the 2–5 hybridoma that recognizes nTyr-53 and changes in the Trp-62. This hybridoma recognizes HEL, the 48–62 or 48–63 peptides treated with peroxynitrite, but not the 48–61 peptide (which lacks the Trp-62) or the 48–62 peptide, in which the Trp-62 has been changed to alanine. (C) The response of the 2–5 hybridoma to 48–62. Indicated are the peptides containing the residues at positions 52 and 62, all tested at 1 μM. Tx, treatment; PN, peroxynitrite.
Fig. 2.
Fig. 2.
Analysis of two other T cells reactive with nTyr-53 of the 52–60 peptide family. The APC was the C3.F6 line. (A) Clone H recognizes peroxynitrite-treated 48–62. Clone H recognizes equally 48–61 or 48–62; i.e., the Trp-62 is irrelevant. (B) Recognition of 48–61 in which the tyrosine has been modified, as indicated. The recognition of nTyr is specific. (C and D) The response of NO75 and clone H, respectively, to a number of peptides, explained in the text.
Fig. 3.
Fig. 3.
Hybridoma 1H5 responds to Trp-28 of the 20–35 peptide. A representative experiment of three in which 1H5 was tested against 20–35 modified peptides (YRGYSLGNWVCAA; see Results). Underlined are the amino acids subject to modification. Peptides were tested at 1 μM. Tx, treatment; PN, peroxynitrite. The APC was C3.F6. In a different experiment, the T cells responded to a peroxynitrite-treated peptide in which the Tyr-23 was changed to phenylalanine (46,500 cpm vs. 51,250 in controls).
Fig. 4.
Fig. 4.
APC present the modified peptides. For each of these experiments, the T cell hybridoma used was clone H cultured for 24 h with the indicated amounts of HEL. (A) Peritoneal macrophages harvested 3 days after Con A injection of mice. (B) The same cells after 3 days of culture treated as explained in the key. (C) Peritoneal macrophages from normal mice cultured, as indicated. (D) Exudates from mHEL transgenic mice after injection of Con A or infection with L. monocytogenes (LM), both i.p. (E and F) Flow cytometry plots of peritoneal exudate cells obtained 3 days after Con A. (E) An irrelevant monoclonal antibody, clone A4-8 reactive with listeriolysin O. (F) Anti-nTyr clone ANY590.
Fig. 5.
Fig. 5.
Spleen APC and cultured DC present the modified peptides. Clone H was tested after 24 h of incubation. (A) The response of CD11b+ cells harvested from spleens of normal mice. (B and C) The responses of DC cultured from bone marrow with granulocyte/macrophage colony-stimulated factor and treated as indicated in the keys.
Fig. 6.
Fig. 6.
Peritoneal macrophages present the modified peptides when cultured with either peptide 48–61 or HEL. In all experiments, the hybridoma is clone H added for 24 h to the culture of the APC. (A) Peritoneal macrophages obtained after Con A injection of the mice and fixed before addition of the peptide. 48–61* is peptide 48–61 with nTyr-53. (B) The same cells, but tested without fixation. (C) The response of B cell lymphoma C3.F6 to the indicated peptides.
Fig. 7.
Fig. 7.
Limiting dilution analysis of popliteal nodes after immunization with 10 nmol of HEL. Two sets of mice mHEL transgenic or normal were immunized with peroxynitrite-treated HEL (PN-HEL) or HEL. See Table 1.

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