Pre-T Cell Receptors (Pre-TCRs) Leverage Vβ Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands

Abstract

Edited by Peter CresswellThe pre-T cell receptor (pre-TCR) is a pT␣-␤ heterodimer functioning in early ␣␤ T cell development. Although once thought to be ligand-autonomous, recent studies show that preTCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the ␣␤TCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the C␤ FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCR␣␤ and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic V␤ patch apposition. This patch functions in the pre-TCR as a surrogate V␣ domain, fostering ligand promiscuity to favor development of ␤ chains with self-reactivity but is occluded by ␣ subunit replacement of pT␣ upon ␣␤TCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor ␣␤TCR repertoire tuning via the pre-TCR.The mammalian adaptive immune system protects its host against infectious diseases as well as tumors in a highly specific manner. At the core of ␣␤ T lymphocyte recognition is self-versus non-self-discrimination, a functionality endowed by clonal cell-surface T cell receptors (TCRs) 4 (1-3). In the mammalian thymus, the millions of distinct TCRs expressed create a repertoire that is refined to eliminate unwanted autoreactive specificities prior to export into the peripheral lymphoid compartment (Ref. 4 and references therein).The earliest thymocytes, termed double negative (DN1-4), lack both CD4 and CD8 and expression of ␣␤TCR complexes (hereafter termed ␣␤TCRs) (5). Within the DN3 stage, a pre-TCR complex is generated comprised of a variable TCR␤ chain disulfide-linked to the invariant pT␣ subunit. In turn, the pT␣-␤ heterodimer is noncovalently complexed with the same CD3 dimers as found in the ␣␤TCR, namely CD3⑀␥, CD3⑀␦, and CD3 (1, 2). This pre-TCR complex triggers cellular survival and expansion and, importantly, induces expression of CD4 and CD8 co-receptors so that the thymocytes transit to the DP (CD4 ϩ CD8 ϩ ) thymocyte stage where rearrangement of the TCR␣ gene occurs. Only at the DP stage is the ␣␤TCR expressed. The pre-TCR signaling process, termed ␤ selection, also controls allelic exclusion of the TCR␤ locus in a given cell (6). Pre-TCR signaling components include tyrosine kinases Lck, Fyn, with Notch-1, Notch-1 ligand DL4, interleukin 7, and CXCR4 supporting pre-TCR function (5, 10). Although ␣␤TCR DP thymocyte selection processes involve pMHC-dependent posit...

Mechanosensing drives acuity of αβ T-cell recognition

“…6B. Concordance of force direction resulting from actin motion and optical trap as applied to the TCR could facilitate release of the force-dependent structural transition of the TCR αβ heterodimer-pMHC complex (33,52). In contrast, the structural extension would persist in the case of a pMHC-arrayed bead, slowly relaxing back to trap center.…”

“…On the other hand, higher force regimes also fail to trigger. Although we believe that these TCRpMHC molecular interactions are capable of supporting conformational change, they may either rapidly rupture or not allow repetitive hopping back and forth between conformational states seen in our single-molecule experiments (52). Actomyosin-based transport acts as a force regulator, maintaining the TCR-pMHC system within this window optimal for conformational transitioning.…”

“…In contrast, without direct connection to the cytoskeleton to sustain load and drive force generation, the TCR-pMHC bond simply unbinds or displaces within the fluid-like membrane, never achieving critical load thresholds required for mechanosensorbased activation, including bond strengthening, conformational change, and other push and pull motions (33,52). A fluid-like microenvironment does not have the densely organized actin machinery of a motile cell, and thus, second wave signaling after TCR-pMHC triggering is muted.…”

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

“…Single-molecule force spectroscopy has been employed previously to distinguish the nature of proteinligand bonds (37) and hence infer multi-modality or conformational transitions involved in protein-ligand binding interactions (50). Single-molecule force spectroscopy in conjunction with steered MD simulations has been employed extensively to study superior mechanical stability (51), characterize the force-unfolding behavior (52), and resolve multiple binding modes of cohesin-dockerin complexes (53).…”

“…However, the application of AFM-based force spectroscopy to study CBM-cellulose binding has revealed challenges in distinguishing specific vs non-specific interactions (54). Here, we have developed a novel single-molecule optical tweezer-based bond rupture assay with piconewton (pN) force resolution and millisecond (ms) time resolution (50), to understand the heterogeneity of CBM binding behavior and provide a firm molecular basis for using a certain adsorption model to interpret classical 'pull-down' assay data. CBM1 showed multimodal force-lifetime behavior towards both cellulose I and cellulose III, as evidenced by the inability to fit this dataset to either single or double exponential decay functions.…”