Analysis of kinetics parameters on T cell recognition to viral infection

T cell plays an important role in clearance of viral infections and development of memory population for a rapid immune response in the case of secondary infections. T cells utilize T cell receptor (TCR) to recognize viral antigens in the form of peptide major histocompatibility complex (pMHC) as its ligand. When viral mutations occur, TCR recognition is impaired for its ligand and in result, lowers immune cell lytic response. Conventionally, TCR binding kinetics to its ligands is linked to TCR binding propensity stemming from 3D assays, providing numerical values in relation to its strength. However, characterizing the interaction of viral variant peptides to TCR is poorly understood and providing a unique perspective in understanding the interaction provides potential solutions to vaccine development, especially for chronic viral infections. In this study, we characterize Cytomegalovirus (CMV)-specific T cell 2D effective binding affinity to both WT and viral variants. Additionally, we characterized to elucidate the importance of specific amino acids found on the peptide influences recognition, thereby potentially bridging gap to understand the mechanics of how specific recognition motifs influence functionality. As T cell gets activated following peptide recognition, multiple signaling pathways take place in order to invoke an effective functional output. To invoke such a response, T cells first translate from naïve to activated state and then returning to homeostasis over the course of an immune response. Although the entire process takes several days, how recognition dynamics is influenced in the context of viral clearance and how that leads into developing memory population has not been fully elucidated. To understand these phenomenon, we primarily evaluated a single transgenic T cell population over the course of an acute lymphocytic choriomeningitis virus (LCMV) viral infection to characterize and understand the dynamics of the T cell function and development that is influenced by organ compartmentalization. Our results highlight an important aspect on how TCR propensity is influenced by the microenvironments during viral clearance and in the occurrence of viral mutation, how characterizing TCR recognition provides new insights on impaired binding kinetic and functional profile.Ph.D

Effects of Anchor Structure and Glycosylation of Fcγ Receptor III on Ligand Binding Affinity

Isoforms of the Fcγ receptor III (FcγRIII or CD16) are cell surface receptors for the Fc portion of IgG and important regulators of humoral immune responses. Different ligand binding kinetics of FcγRIII isoforms are obtained in three dimensions by surface plasmon resonance and in two dimensions by a micropipette adhesion frequency assay. We show that the anchor structure of CD16 isoforms isolated from the cell membrane affects their binding affinities in a ligand-specific manner. Changing the receptor anchor structure from full to partial to none decreases the ligand binding affinity for human IgG1 (hIgG1) but increases it for murine IgG2a (mIgG2a). Removing N-glycosylation from the CD16 protein core by tunicamycin also increases the ligand binding affinity. Molecular dynamics simulations indicate that deglycosylation at Asn-163 of CD16 removes the steric hindrance for the CD16-hIgG1 Fc binding and thus increases the binding affinity. These results highlight an unexpected sensitivity of ligand binding to the receptor anchor structure and glycosylation and suggest their respective roles in controlling allosterically the conformation of the ligand binding pocket of CD16

Hotspot autoimmune T cell receptor binding underlies pathogen and insulin peptide cross-reactivity

The cross-reactivity of T cells with pathogen- and self-derived peptides has been implicated as a pathway involved in the development of autoimmunity. However, the mechanisms that allow the clonal T cell antigen receptor (TCR) to functionally engage multiple peptide–major histocompatibility complexes (pMHC) are unclear. Here, we studied multiligand discrimination by a human, preproinsulin reactive, MHC class-I–restricted CD8+ T cell clone (1E6) that can recognize over 1 million different peptides. We generated high-resolution structures of the 1E6 TCR bound to 7 altered peptide ligands, including a pathogen-derived peptide that was an order of magnitude more potent than the natural self-peptide. Evaluation of these structures demonstrated that binding was stabilized through a conserved lock-and-key–like minimal binding footprint that enables 1E6 TCR to tolerate vast numbers of substitutions outside of this so-called hotspot. Highly potent antigens of the 1E6 TCR engaged with a strong antipathogen-like binding affinity; this engagement was governed though an energetic switch from an enthalpically to entropically driven interaction compared with the natural autoimmune ligand. Together, these data highlight how T cell cross-reactivity with pathogen-derived antigens might break self-tolerance to induce autoimmune disease

A TCR mechanotransduction signaling loop induces negative selection in the thymus

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR–pMHC–CD8 trimolecular ‘catch bonds’, whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR–pMHC and pMHC–CD8 bimolecular ‘slip bonds’. Catch bonds were not intrinsic to either the TCR–pMHC or the pMHC–CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC–CD8 interaction to the intracellular interaction of CD8 with TCR–CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans–cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus

A TCR mechanotransduction signaling loop induces negative selection in the thymus

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR–pMHC–CD8 trimolecular ‘catch bonds’, whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR–pMHC and pMHC–CD8 bimolecular ‘slip bonds’. Catch bonds were not intrinsic to either the TCR–pMHC or the pMHC–CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC–CD8 interaction to the intracellular interaction of CD8 with TCR–CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans–cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus