Pre-TCR Mechanotransduction: From Biology to Structure

ABSTRACT – PROJECT 2 A multi-step process in the thymus programs αβ T-lineage cells to distinguish self from non-self. Two sets of receptors are involved in this thymic education, namely pre-T cell receptors (preTCRs) at the early CD4−CD8−double-negative (DN) thymocyte stage followed by αβ T cell receptors (TCRs) beginning at the CD4+CD8+double-positive (DP) thymocyte stage. Signaling through the preTCR, a CD3-associated heterodimer comprising an invariant pTα chain and a clone-specific β chain, is a critical early checkpoint in thymocyte development within the T cell lineage. PreTCRs arrayed on DN thymocytes ligate peptides bound to major histocompatibility complex molecules (pMHC) on thymic stroma as do αβ TCRs that appear on DP thymocytes but use a related yet distinct molecular docking strategy. Employing synchronized fetal thymic progenitor cultures differing in the presence or absence of pMHC on support stroma and single-cell transcriptomes, we observed that although MHC-negative stroma fosters αβ T cell differentiation, the absence of preTCR-pMHC interactions leads to deviant thymocyte transcriptional programming associated with dedifferentiation and antecedent characteristics of T cell lymphoblastic and myeloid malignancies. Compensatory upregulation of diverse MHC class Ib proteins in B2m/H2-Ab1-MHC-knockout mice partially safeguards thymocyte progression in vivo but disseminated DP thymic tumors develop with aging. Thus, beside promoting β chain repertoire broadening for subsequent αβ T cell receptor utilization, preTCR-pMHC interactions limit cellular plasticity to facilitate normal thymocyte differentiation. In Aim 1 we shall correlate preTCR-pMHC bond biophysical properties described in Project 1 required for successful navigation through the β selection checkpoint with signaling, repertoire selection and thymocyte progression using our toolbox of RNA-Seq, clonotype analyses and functional studies. In conjunction with X-ray crystallographic studies of preTCRβ-pMHC, NMR methods involving select labeling, chemical shift and structural dynamics (Core B-C) and molecular dynamics under force (MDf) (Project 3) insights on the ligand binding interface, load pathways linked to bond formation and developmental progression shall be provided. Aim 2 will utilize the computational power of Project 3 to predict various altered peptide ligands (APL) in silico to be assessed experimentally for efficacy in directing repertoire development from preTCR to TCR to foster immunoprotective influenza A virus (IAV) specificities. In Aim 3 we shall establishing a facile in vitro model system to support selection and repertoire development from HSC through to CD4SP/CD8SP thymocytes, since progression through to the SP stage is inadequate in the current in vitro epithelial system offering a complete developmental trajectory analysis with translational significance. The completion of these aims will provide a greater in-depth understanding of the role of the preTCR in T cell development, offer insights into how the preTCR pretests β chains for incorporation into the αβ TCR repertoire and biological and atomistic detailing of developmental control of thymocyte repertoire generation.