94 research outputs found
Regulation of T-cell antigen recognition by melanoma tumor microenvironment and TCR-CD3 ectodomain interaction
No full textDespite the critical role of CD8+ T cells in tumor clearance, their functions are impaired by immunosuppressive cells/cytokines, through inhibitory receptors, and metabolic restrictions in the tumor microenvironment (TME). Targeting these suppressive pathways were shown to promote tumor clearance, yet unknown mechanisms may still exist curtailing the T cell responses. The T cell activation and anti-tumor response are initiated by the T cells receptor (TCR) recognizing antigen peptide presented by major histocompatibility complex (pMHC) molecules on antigen presenting cells (APC). Recent studies have demonstrated that comparing to in solution (or three-dimensional, 3D) kinetic measurements that uses purified TCR molecules, analysis of pMHC interacting with TCRs expressed on native T cells (or two-dimensional, 2D) provides a better prediction of T cell function and is able to capture perturbations of antigen recognition by T cell intrinsic and extrinsic mechanisms. In this study, we examined whether T cell antigen recognition is altered by the TME, and thus contributes to the T cell dysfunction. By testing the OT-I T cells from the murine B16F10 melanoma TME with their cognate antigen pMHC OVA:H2Kb, we showed that the TCR-pMHC 2D affinity is reduced in TME. The presence of tumor modulated TCR mechanosensing of antigen pMHC, converting a typical TCR-pMHC catch bond into slip bonds. The T cells from TME gave a reduced spreading on pMHC coated surface, with a decreased TCR-pMHC tension signal generated by spontaneous T cell pulling on pMHC at force over 4.7pN. The TME altered dynamic response of T cell CD3ζ phosphorylation, and reduced level of calcium flux following in vitro stimulations. Using T cell in vitro activation, in vivo proliferation and ex vivo cytokine production as readouts, we showed that removing the TME restores T cell function that was impaired by this antigen inexperienced mechanism. Further analysis showed that nitration of TCR, which can be caused by presence of MDSCs in TME and induces T cell tolerance induced dysfunction, reduced TCR-pMHC 2D affinity. Presence of immunosuppressive Treg and cytokine TGF-β in TME is known to impair CD8+ T cell activation and function. We showed that in vivo TGF-β inhibition and CD4 depletion in tumor bearing animal partially restored the TME altered TCR-pMHC interaction. To summarize, we found that the impaired TCR-pMHC mechanosensing correlated with a reduced T cell function in TME, while this tumor antigen inexperienced suppression was functionally reversible. We also identified several immunosuppressive factors as the potential mechanisms of TME impairment on T cell antigen recognition.
TCR α chain and β chain bind noncovalently to dimeric subunits CD3δε, CD3γε, and CD3ζζ to form TCR complex. Upon TCRαβ engaging the antigen pMHC, this binding signal is transmitted through TCR-CD3 interaction, phosphorylates the immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3 cytoplamic tails, which triggers the T cell activation. The interactions among TCR, CD3δε and CD3γε ectodomains are weak in 2D affinities, with short-moderate duration of averaged lifetimes, however disrupting these interactions affects TCR complex stability, signaling and significantly reduces T cell function. In this study, we examed how this weak TCR-CD3 extracellular interaction severely impacts T cell function and whether this impact is through regulating T cell antigen recongition. We found that purified TCR proteins bind to a mixture of CD3δε and CD3γε ectodomain proteins at an increased likelihood and increased average lifetime, comparing to TCR-CD3δε or TCR-CD3γε interaction. This proved the existence of cooperativity among TCR-CD3 extracellular domain interactions. The antibody/Fab targeting TCR and CD3 ectodomains can block TCR-CD3 extracellular interaction and reduce T cell functional response. We showed that the TCR-CD3 interaction blocking Fab treatments decreased TCR-pMHC 2D affinity and altered TCR-pMHC interaction force response profile. Together, these results indicate that TCR-CD3 extracellular interactions is enhanced by the cooperativity among the ectodomain interactions, and regulates T cell function through altering TCR mechanosensing.
In this study, we identified impaired T cell antigen recognition as one mechanism of T cell dysfunction in TME. We also identified the cooperativity enhanced TCR-CD3 extracellular interaction as a regulating factor of T cell function by affecting T cell antigen recognition. The results greatly extend our understanding on how the T cell antigen recognition is regulated in physiological and pathological conditions, providing the molecular basis for developing pharmacological approaches to restore/promote or suppress T cell response by regulating T cell antigen recognition.Ph.D
CD28 and TCR In-Situ Biophysical Analyses Inform T Cell Immunity Mechanisms
No full textThis work investigates two receptors on T lymphocytes that shape immunity, the T cell receptor (TCR) and cluster of differentiation 28 (CD28). T cells coordinate adaptive immunity, but how signaling via TCR and CD28 interactions with peptide-major compatibility complex (pMHC) and B7 family ligands on antigen presenting cells govern T cell function and differentiation remains poorly understood. In-situ biophysical measurements on live T cell surfaces suggest both B7 family ligands form monomeric bonds with CD28. This work demonstrated CD28 catch bonds with B7 family ligands. Catch bonds refer to a counter-intuitive phenomenon where force prolongs bond lifetime contrasted with the more intuitive slip bond where force shortens bond lifetime. Although TCR–pMHC catch bonds on splenic T cells characterize a well-established TCR mechanosensing mechanism, the same interaction on hepatic T cells showed slip bonds correlating with a more activated state among liver T cells. We also analyzed both short- and long-term memory effects from the same molecular interactions. Short-term (within seconds) memory analyses found that bond formation increased bond formation likelihood but not dissociation in the immediate future. Long-term (~5 minutes) memory analyses found that splenic T cells became more activated by repeated ligand engagement and receptor tension resulting in TCR–pMHC catch bond elimination. Our sensitive assay also revealed subtle T cell activation by piconewton-level T cell pushing and pulling forces as well as changes in short-term memory. This work suggests biophysical instrumentation employed in-situ can reveal information about dynamic processes mediating important immunological functions. The findings within this work provide insights into mechanistically how co-stimulation works at a single molecule level as well as how signaling overlap between TCR and CD28 influence receptor localization, mechanosensing, and triggering. These insights answer longstanding mechanistic questions about how T cells function and provide foundations for future investigations.Ph.D
T-Cell Receptor-CD3 Signaling Complex Extracellular Interactions Characterized by Genetic Incorporation of Unnatural Amino Acid Photo-Crosslinkers
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Molecular flexibility can influence the stimulatory ability of receptor–ligand interactions at cell–cell junctions
No full textAbstract 4044: The immune phenotype in serial biopsies from metastatic TNBC undergoing chemo-immunotherapy
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