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There is now a CONTENT FREEZE for Mercury while we switch to a new platform. It began on Friday, March 10 at 6pm and will end on Wednesday, March 15 at noon. No new content can be created during this time, but all material in the system as of the beginning of the freeze will be migrated to the new platform, including users and groups. Functionally the new site is identical to the old one. webteam@gatech.edu
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Advisor:
Dr. Cheng Zhu (Georgia Institute of Technology)
Committee:
Dr. Susan Thomas (Georgia Institute of Technology)
Dr. Michelle Krogsgaard (New York University)
Dr. Mandy Ford (Emory University)
Dr. Gabe Kwong (Georgia Institute of Technology)
Regulation of T-cell antigen recognition by melanoma tumor microenvironment and TCR-CD3 ectodomain interaction
Despite 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.
