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Original Article

^a Laboratory of Immunobiology and Department of Cancer Immunology & AIDS, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, MA 02115
^b Microchemistry and Proteomics Analysis Facility, Harvard University, Cambridge, MA 02138
Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02115.617-632-3351617-632-3412

ellis_reinherz{at}dfci.harvard.edu

Peptide fragments of self-proteins bound to major histocompatibility complex molecules within the thymus are important for positively selecting T cell receptor (TCR)-bearing CD4⁺CD8⁺ double positive (DP) thymocytes for further maturation. The relationship between naturally processed thymic self-peptides and TCR-specific cognate peptides is unknown. Here we employ HPLC purification of peptides released from H-2K^b molecules of the C57BL/6 thymus in conjunction with mass spectrometry (MS) and functional profiling to identify a naturally processed K^b-bound peptide positively selecting the N15 TCR specific for the vesicular stomatitis virus octapeptide (VSV8) bound to K^b. The selecting peptide was identified in 1 of 80 HPLC fractions and shown by tandem MS (MS/MS) sequencing to correspond to residues 68–75 of the MLRQ subunit of the widely expressed mitochondrial NADH ubiquinone oxidoreductase (NUbO_68–75). Of note, the peptide differs at six of its eight residues from the cognate peptide VSV8 and functions as a weak agonist for mature CD8 single positive (SP) N15 T cells, with activity 10,000-fold less than VSV8. In N15 transgenic (tg) recombinase activating gene 2^–/– transporter associated with antigen processing 1^–/– fetal thymic organ culture, NUbO_68–75 induces phenotypic and functional differentiation of N15 TCR bearing CD8 SP thymocytes. Failure of NUbO_68–75 to support differentiation of a second K^b-restricted TCR indicates that its inductive effects are not general.

Key Words: positive selection • thymocyte development • CTL • naturally processed peptides • TAP-1^–/–

© 2001 The Rockefeller University Press

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