The Roles of a Positively Selecting Self-peptide in the Thymus and in the Periphery

Positive selection of CD4+ T cells requires the interaction of the T cell receptor (TCR) of double positive (DP) thymocytes with self-peptide bound MHC class II molecules that are presented on thymic cortical epithelia cells. Only the interaction with appropriate strength of signals can promote DP thymocytes to complete positive selection. However, the precise biophysical nature of this interaction with regard to degree of specificity of positive selection remains unresolved. We recently identified a naturally occurring positively selecting ligand, gp250, which can induce positive selection of AND TCR (MCC/I-Ek specific). We screened for MHC class II positively selecting ligands, using thymocytes from four I-Ek restricted TCRtg mice and a large panel of self-peptides. One peptide, gp250, induced positive selection of AND CD4+ T cells, had no homology with the agonist ligand, and was recognized with a high degree of specificity. On peripheral AND CD4+ T cells, gp250 acted as a co-agonist to initiate T cell activation and enhance survival. Thus, positively selecting ligands play both a critical role in thymocyte development and in the activation and maintenance of peripheral T cells. Furthermore, in DP thymocytes, the affinity of TCR-self-peptide/MHC interaction determines T cell fate. It remains unknown how such weak interactions translate into biochemical signals and gene expression required for positive selection, nor how peptide specificity is maintained. To address the question with our novel gp250-AND system, we first determined the binding affinity of gp250 peptides for AND TCRs. By surface plasmon resonance and tetramer decay analysis, gp250/I-Ek showed expectedly yet still surprisingly weak binding affinity to AND TCR with an estimated affinity weaker than 500 µM. Importantly, using Fura-2 based single cell calcium imaging, we analyzed calcium profiles of DP AND T cells in respond to plate-bound I-Ek/Ig dimers loaded with gp250 peptide or agonist MCC peptide. A strong and sustained calcium influx was induced after gp250/I-Ek engagement, whereas MCC/I-Ek triggered a strong but transient calcium influx. The weak interaction of gp250/I-Ek and AND TCR was also sufficient to induce downstream signaling events involved in positive selection, including the phosphorylation of Zap-70 and Erk. After 7-hour stimulation with plate-bound I-Ek/Ig dimers, transcriptional analysis shows 28 genes were specifically and exclusively upregulated under gp250/I-Ek stimulation, and not by MCC/I-Ek. We identify a voltage-gated Na+ channel (VGSC), composed of a pore-forming SCN5A subunit and a regulatory SCN4B subunit, essential in the positive selection of CD4+ T cells. We hypothesized positive selection signals through TCR activate VGSCs, depolarizing the membrane and activating L-type voltage-gated Ca2+ channel (CaV) to induce sustained Ca2+ flux. Pharmacological inhibition of the VGSC pore impaired in vitro positive selection and diminished DP thymocytes to induce Ca2+ influx in respond to positive selection signals. Treatment with CaV inhibitors blocked CD4+ T cell differentiation, and attenuated the sustained Ca2+ flux induced by positively selecting ligand stimulation. Peripheral AND CD4+ T cells transfected with the VGSC, which they normally do not express, gained the ability to respond to positively selecting ligands, directly demonstrating that expression of the VGSC was responsible for increased sensitivity to weak TCR ligands. In lentiviral bone marrow reconstitution, shRNA knockdown of scn5a specifically inhibited thymic selection of CD4+ T cells. This novel finding of an active VGSC in thymocytes provides a mechanism by which a weak positive selecting signal can induce the sustained Ca2+ signals required for CD4+ T cell development. To determine if gp250 self-peptide may alter the antigen specificities of post-selection T cell repertoire, we generated a transgenic mouse line that expresses gp250/I-Ek as the only MHC class II molecule. In these mice, we observed significant numbers of T cells that developed in the thymus and appeared in the periphery where they expressed a full complement of TCR Vb genes. The post-selection mature CD4+ T cells showed some skewing in TCRb usages, including Vb2+, Vb4+, Vb8.1+ and Vb8.2+. Interestingly, the gp250-mediated positive selection substantially elevated the frequency of MCC-tetramer bound CD4+ T cells. In gp250 single chain mice, the Va11+ Vb3+ and Va11+ Vb14+ populations were increased. The increased Va11+ Vb3+ and Va11+ Vb14+ populations may contribute directly to the enhanced MCC-specific CD4+ frequency in gp250 single chain mice. Our data suggested that gp250 ligand has the capability to select many different TCRs, and may serve as a good selecting ligand for Va11+ Vb3+ and Va11+ Vb14+ populations to augment peripheral T cell specificities toward the MCC antigen