In the thymus, positive and negative selection shape the T cell repertoire. It has previously been shown that positive selection, like negative selection, is the result of the interaction of the TCR with self-peptides bound to MHC. However, little is known about the number or nature of the self-peptide ligands that mediate positive selection in vivo. We devised a novel assay with enhanced sensitivity for low affinity TCR ligands to identify self-peptides that may be biologically relevant. At least eight Kb-bound self-peptides were detected by this assay using thymocytes bearing the OT-I TCR (specific for OVAp/Kb). The sequence of one of these peptides was determined using the recently developed technique of membrane preconcentration-capillary electrophoresis-tandem mass spectrometry. This peptide, CPα1, has limited sequence similarity to OVAp, yet was found to induce positive selection of OT-I thymocytes in fetal thymic organ culture.
Bibliographical noteFunding Information:
Correspondence should be addressed to K. A. H. (e-mail, hog email@example.com). The authors wish to thank Eric Becken and Scott Pearcy for technical assistance, Ellen Robey for providing the CD8αβ transgenic mice, Anton Berns for providing the TAP o mice, and Mike Bevan and John Cooper for advice and helpful discussion. We would also like to thank Sasha Rudensky and Paul de Roos for initial help in developing the immunoprecipitation and HPLC protocols. This work was supported by the Arthritis Foundation (K. A. H.), the American Cancer Society (K. A. H.), the Leukemia Society (S. C. J.), the Mayo Foundation, Beckman Instruments, and Finnegan MAT. M. C. H. was supported by a grant from the National Institutes of Health (GM38542) to J.A. Cooper, Department of Cell Biology and Physiology, Washington University School of Medicine.