Evidence for a functional sidedness to the αβTCR Kuhns, Michael S; Girvin, Andrew T; Klein, Lawrence O ...
Proceedings of the National Academy of Sciences - PNAS,
03/2010, Volume:
107, Issue:
11
Journal Article
Peer reviewed
Open access
The T cell receptor (TCR) and associated CD3γε, δε, and ζζ signaling dimers allow T cells to discriminate between different antigens and respond accordingly, but our knowledge of how these parts fit ...and work together is incomplete. In this study, we provide additional evidence that the CD3 heterodimers congregate on one side of the TCR in both the αβ and γδTCR-CD3 complexes. We also report that the other side of the αβTCR mediates homotypic αβTCR interactions and signaling. Specifically, an erythropoietin receptor-based dimerization assay was used to show that, upon complex assembly, the CD3ε chains of two CD3 heterodimers are arranged side-by-side in both the αβ and γδTCR-CD3 complexes. This system was also used to show that αβTCRs can dimerize in the cell membrane and that mutating the unusual outer strands of the Cα domain impairs this dimerization. Finally, we present data showing that, for CD4 T cells, the mutations that impair αβTCR dimerization also alter ligand-induced calcium mobilization, TCR accumulation at the site of pMHC contact, and polarization toward the site of antigen contact. These data reveal a "functional-sidedness" to the αβTCR constant region, with dimerization occurring on the side of the TCR opposite from where the CD3 heterodimers are located.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
BackgroundChimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T cell proliferation when they are endowed with both activating and costimulatory functions. While ...remission rates have been noticeably elevated in numerous clinical trials targeting CD19, CD22 or BCMA, relapses are common. One of the several underlying relapse mechanisms is antigen escape, which refers to a relapsing tumor that is either negative for the targeted antigen or expresses the latter at a low level. Failure to eliminate antigen-low tumors raises questions about the sensitivity of CARs and the minimum antigen density that is required for effective tumor eradication. Unlike CARs, TCRs engage antigen in an HLA-dependent manner, and they do so with high sensitivity. We hypothesized that a TCR/CD3 complex containing the same heavy and light immunoglobulin chains as a CAR will display increased sensitivity to the target antigen.MethodsWe edited the TRAC locus in human primary T cells to establish a novel antigen receptor structure, termed HLA-independent TCR or HIT receptor, by incorporating into the TCR/CD3 complex the same heavy and light chains as those of a corresponding CAR. We assessed their antigen sensitivity against a panel of cell lines expressing different antigen levels, analyzing their cytotoxicity, cytokine secretion, signaling response and degranulation activity. HIT and CAR T cells were further evaluated for their anti-tumor response using established ALL and AML mouse models.ResultsCD19-TRAC-HIT and CD19-TRAC-CAR T cells lysed wild-type NALM6 (~27,000 CD19 molecules) and NALM6 variants with 100-fold less CD19. As CD19 levels decreased further, CAR T cells no longer killed their target, in contrast to HIT T cells. HIT T cells showed increased expression of IFN-gamma, IL-2 and TNF-alpha upon exposure to NALM6 cells expressing ~20 CD19 molecules per cell, compared to CAR T cells. This increased sensitivity of HIT receptors correlated to their greater signaling response, upon exposure to the low-antigen-density NALM6. Phospho-proteomic analyses further confirmed this increased response of HIT T cells to low antigen levels. Altogether, these results confirm that HIT receptors endow T cells with greater antigen sensitivity than canonical CARs. We further showed that HIT T cells have higher in vivo anti-tumor activity compared to CAR T cells in mice bearing low-antigen-density ALL or AML.ConclusionsHIT receptors consistently afford high antigen sensitivity and mediate tumor recognition beyond what current CARs can provide. HIT receptors open new prospects for targeting cell surface antigens of low abundance.Ethics ApprovalEight- to 12-week-old NOD/SCID/IL-2Rgamma-null (NSG) male mice (Jackson Laboratory) were used under a protocol approved by the MSKCC Institutional Animal Care and Use Committee.
Actin remodeling promotes B cell activation by enabling B cell antigen receptor clustering in the immune synapse. In the current issue of JCB, Droubi et al. (2022. J. Cell ...Biol.https://doi.org/10.1083/jcb.202112018) find that this process is initiated by the lipid phosphatase INPP5B, which shapes synaptic actin architecture by locally depleting phosphatidylinositol 4,5 bisphosphate.
T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that ...regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.
Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence
. Patient-derived CAR T cells have demonstrated remarkable efficacy ...against a range of B-cell malignancies
, and the results of early clinical trials suggest activity in multiple myeloma
. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low
. Unlike the mechanisms that result in complete and permanent antigen loss
, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract
Epithelial transformation and carcinogenesis are characterized by profound alterations in cell mechanics that significantly affect multiple steps of the metastatic cascade. The ability of ...cancer cells to grow in the primary tumor, to locally invade through the confining extracellular matrix, to survive in circulation, and to extravasate into distant vital organs all depend on specific mechanical characteristics. Importantly, recent studies have shown that the mechanical properties of cancer cells also influence their interactions with immune and stromal cells. Here, we discuss the mechanical changes that cancer cells undergo during metastasis, how these changes affect immune and stromal responses, and the implications of these new insights for therapeutic intervention.
Cells have the remarkable ability to sense the mechanical stiffness of their surroundings. This has been studied extensively in the context of cells interacting with planar surfaces, a conceptually ...elegant model that also has application in biomaterial design. However, physiological interfaces are spatially complex, exhibiting topographical features that are described over multiple scales. This report explores mechanosensing of microstructured elastomer surfaces by CD4⁺ T cells, key mediators of the adaptive immune response. We show that T cells form complex interactions with elastomer micropillar arrays, extending processes into spaces between structures and forming local areas of contraction and expansion dictated by the layout of microtubules within this interface. Conversely, cytoskeletal reorganization and intracellular signaling are sensitive to the pillar dimensions and flexibility. Unexpectedly, these measures show different responses to substrate rigidity, suggesting competing processes in overall T cell mechanosensing. The results of this study demonstrate that T cells sense the local rigidity of their environment, leading to strategies for biomaterial design.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Transient,specialized cell–cell interactions play a central role in leukocyte function by enabling specific intercellular communication in the context of a highly dynamic systems level response. The ...dramatic structural changes required for the formation of these contacts are driven by rapid and precise cytoskeletal remodeling events. In recent years, the immunological synapse that forms between a T lymphocyte and its antigen-presenting target cell has emerged as an important model system for understanding immune cell interactions. In this review, we discuss how regulators of the cortical actin cytoskeleton control synaptic architecture and in this way specify T cell function.
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EMUNI, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UL, UM, UPUK, VKSCE, ZAGLJ
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