Identification of a Naturally Occurring Ligand for Thymic Positive Selection

Abstract

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 K(b)-bound self-peptides were detected by this assay using thymocytes bearing the OT-I TCR (specific for OVAp/K(b)). The sequence of one of these peptides was determined using the recently developed technique of membrane preconcentration-capillary electrophoresis-tandem mass spectrometry. This peptide, CP alpha1, has limited sequence similarity to OVAp, yet was found to induce positive selection of OT-I thymocytes in fetal thymic organ culture.

Positive Selection Is Limited by Available Peptide-Dependent MHC Conformations

“…Thymocytes (5 ϫ 10 5 ) from OT-I, CD8tg, ␤ 2 M 0 mice were isolated from 4-to 6-week-old mice and cocultured with the peritoneal exudate cells for 16 -20 h at 37°C. Cells were stained for expression of CD4 (PE-RM4-5) and the non-Tg-encoded CD8.2 (biotin-2.43), followed by streptavidin-FITC, and analyzed as described previously (11).…”

“…The dulling assay measures activation-induced coreceptor down-regulation on immature DP thymocytes. This assay has been successfully utilized to find positive selection ligands in the past (11). In a DP dulling assay, peritoneal macrophages are used as APC for thymocytes from preselection TCR Tg mice.…”

“…In a DP dulling assay, peritoneal macrophages are used as APC for thymocytes from preselection TCR Tg mice. In this case the OT-I, ␤ 2 -microglobulin-deficient thymocytes also express a CD8 transgene which further enhances their ability to detect weak ligands (11). In this assay, TCR stimulation leads to the decreased surface expression of CD4 and CD8 which is measured after 24 h of culture (11,36).…”

“…In this case the OT-I, ␤ 2 -microglobulin-deficient thymocytes also express a CD8 transgene which further enhances their ability to detect weak ligands (11). In this assay, TCR stimulation leads to the decreased surface expression of CD4 and CD8 which is measured after 24 h of culture (11,36). Although this assay detects both positive (weak) and negative (strong) selection ligands, those peptides that stimulate thymocytes but not CTL are good candidates for positive selection.…”

“…Perhaps K bm8 simply does not bind any of the self peptides that OT-I utilizes for selection. We have estimated that the number of such peptides is small and have determined the sequence of one of these (11). This peptide, CP␣1, is naturally processed and presented from the ubiquitously expressed F-actin capping protein.…”