Cell Immunological Synapse Formation Research Articles

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Lipid mediators in the regulation of innate and adaptive immunity.

Lipid mediators in the regulation of innate and adaptive immunity.

Soluble CD83 Regulates Dendritic Cell-T Cell Immunological Synapse Formation by Disrupting Rab1a-Mediated F-Actin Rearrangement.

Dendritic cell–T cell (DC-T) contacts play an important role in T cell activation, clone generation, and development. Regulating the cytoskeletal protein rearrangement of DCs can modulate DC-T contact and affect T cell activation. However, inhibitory factors on cytoskeletal regulation in DCs remain poorly known. We showed that a soluble form of CD83 (sCD83) inhibited T cell activation by decreasing DC-T contact and synapse formation between DC and T cells. This negative effect of sCD83 on DCs was mediated by disruption of F-actin rearrangements, leading to alter expression and localization of major histocompatibility complex class II (MHC-II) and immunological synapse formation between DC and T cells. Furthermore, sCD83 was found to decrease GTP-binding activity of Rab1a, which further decreased colocalization and expression of LRRK2 and F-actin rearrangements in DCs, leading to the loss of MHC-II at DC-T synapses and reduced DC-T synapse formation. Further, sCD83-treated DCs alleviated symptoms of experimental autoimmune uveitis in mice and decreased the number of T cells in the eyes and lymph nodes of these animals. Our findings demonstrate a novel signaling pathway of sCD83 on regulating DC-T contact, which may be harnessed to develop new immunosuppressive therapeutics for autoimmune disease.

762. A Quantitative Imaging Tool Box for the Functional Analysis of Chimeric Antigen Receptor - T Cell Immune Synapse Helps Identify Novel Structural and Functional Features of CAR T Cells

Introduction: The cytolytic immunological synapse (IS) in T cells is a discrete structural entity formed after the ligation of specific activation receptors and leading to the destruction of a cancerous cell through the targeted release of the contents of T cell lytic granules. Although the “native” T cell IS formation where the TCR binds to antigenic peptide-MHC complex has been intensely studied, the “engineered” CAR T cell synapse and its variations based on single chain variable fragment (scFv) design and co-stimulatory domain arrangements, still remains a black box. Our work focuses on the quantitative evaluation of the CAR T cell lytic IS using highly quantitative high and super resolution imaging techniques. Specifically, by combining quantitative interrogations of CAR synapse formation kinetics and dynamics at the cellular level, we are able to obtain an unprecedented level of insight into the effectiveness of therapy cells relative to the traditional measures of cytotoxicity and effector assays like chromium release, ELISPOT and flow cytometry. Super-resolution microscopy enables us to further “see” subtle variations in therapy cell quality at a near molecular level and can be used as a very precise functional readout of CAR T cell rational design. Methods: High and super resolution fluorescence microscopy platforms are used to image CAR T cell-target conjugates in the x, y and z dimensions. Imaging parameters include direct evaluation of CAR engagement at the IS by measuring mean fluorescence intensity (MFI) and volume of synaptic aggregates of CAR specific ligands as well as their relative distribution at the synapse. Results: We report here the quantitative measurement and comparison of single and dual CAR targeted ligand aggregation at the IS, lytic machinery re-arrangement and time to target cell killing. Our studies using this predictive imaging tool box establishes the superiority of T cells expressing a bispecific (tandem) CAR to those expressing one or two monospecific CARs by the presence of significantly increased accumulation of F-Actin (> five fold), greater convergence and polarization of the lytic granule machinery. Dual engagement of the targeted ligands by a tandem CAR also significantly offsets antigen escape variants in tumors predicting reduced tumor relapse. Furthermore, application of our imaging tool box reveals that tonic signaling of CARs containing a 4-1BB co-stimulatory domain can result in an almost three fold upregulation of Fas on plasma membrane and co-localization with Fas L leading to enhanced apoptosis and lower persistence of CAR T cells. Conclusions: Using our quantitative imaging tool box we are able to define a specific list of parameters to measure CAR engagement and lytic function at the IS. These imaging parameters can be directly applied to interrogate and predict functionality outcomes upon CAR design variations. By doing this we hope to provide crucial early insight in CAR T cell design that can guide improvements in the efficacy, safety and, ultimately, clinical effectiveness of CAR T cell therapies.

UNC-45A Is a Nonmuscle Myosin IIA Chaperone Required for NK Cell Cytotoxicity via Control of Lytic Granule Secretion.

NK cell's killing is a tightly regulated process under the control of specific cytoskeletal proteins. This includes Wiskott-Aldrich syndrome protein, Wiskott-Aldrich syndrome protein-interacting protein, cofilin, Munc13-4, and nonmuscle myosin IIA (NMIIA). These proteins play a key role in controlling NK-mediated cytotoxicity either via regulating the attachment of lytic granules to the actin-based cytoskeleton or via promoting the cytoskeletal reorganization that is requisite for lytic granule release. UNC-45A is a highly conserved member of the UNC-45/CRO1/She4p family of proteins that act as chaperones for both conventional and nonconventional myosin. Although we and others have shown that in lower organisms and in mammalian cells NMIIA-associated functions, such as cytokinesis, cell motility, and organelle trafficking, are dependent upon the presence of UNC-45A, its role in NK-mediated functions is largely unknown. In this article, we describe UNC-45A as a key regulator of NK-mediated cell toxicity. Specifically we show that, in human NK cells, UNC-45A localize at the NK cell immunological synapse of activated NK cells and is part of the multiprotein complex formed during NK cell activation. Furthermore, we show that UNC-45A is disposable for NK cell immunological synapse formation and lytic granules reorientation but crucial for lytic granule exocytosis. Lastly, loss of UNC-45A leads to reduced NMIIA binding to actin, suggesting that UNC-45A is a crucial component in regulating human NK cell cytoskeletal dynamics via promoting the formation of actomyosin complexes.

Functional Screening Studies Identify Combinational Activity of PD-L1 and CD200 In Mediating Dysfunctional T Cell Immunological Synapse Formation In Chronic Lymphocytic Leukemia

AbstractAbstract 696The ability of cancer cells to modulate the immune microenvironment is now recognized as an important hallmark of disease pathophysiology. Identifying the molecular mechanisms of cancer immune suppression in the laboratory is key to the design of more effective immunotherapeutic treatment strategies. We previously demonstrated that chronic lymphocytic leukemia (CLL) cells induce alterations in global gene expression profiles in patient CD4 and CD8 T cells, and a profound T cell immunological synapse formation defect that can be reversed with lenalidomide (J Clin Invest. 2005;115(7):1797-1805, and 2008;118(7):2427-2437). Here we used small interfering RNA (siRNA) with a 2-part functional screen to identify key CLL cell molecules inducing T cell immune suppression. siRNA treated tumor cells were cocultured in direct contact with healthy allogeneic T cells for 24 hours, T cells purified from coculture and used in cell conjugation immune synapse assays with superantigen-pulsed third party B cells as antigen-presenting cells (APCs). Confocal microscopy and image analysis software was used to quantify the mean area of T cell F-actin immune synapse formation events from each experimental cell population. Treatment of the CLL cell line MEC-1 with either TNFα, TGFβ, IL-10, or IL-6 siRNA identified no gain in subsequent CD3 T cell immune synapse function compared to control non-targeting siRNA or untreated CLL cells. However, CD200 or programmed death 1 (PD1) ligand 1 (PD-L1, CD274) siRNA treatment significantly enhanced (P < .01) subsequent T cell synapse formation events with APCs (comparable to positive control experiments blocking tumor cell:T cell direct contact with ICAM-1 siRNA, or primary coculture of T cells with allogeneic healthy donor B cells). Primary CLL patient cells (n=10) were treated with individual or pooled neutralizing antibodies, or siRNA, targeting PD-L1, CD200, or cytokines. This analysis revealed that counteracting the combined activity of PD-L1, CD200 and TGFβ exhibited the most pronounced repair of subsequent T cell synapse function compared to control treated tumor cells (P < .01). These data suggest that CLL-released cytokines such as TGFβ contribute to, but are not essential for the T cell synapse defect. We also identified that blocking the T cell receptors PD-1, CD200-R and TGFβ-R1 with neutralizing antibodies prevents CLL inhibitory signaling (P < .01) compared to isotype control IgG treated T cells in contact with tumor cells. We further show that knock-down of PD-L1, CD200 and TGFβ on ex vivo CLL cells prevents inhibitory CD4 and CD8 T cell synapse function compared to control siRNA (P < .01) using the Eμ-TCL1 mouse model of CLL. The addition of lenalidomide (1μM) in ex vivo CLL cell:T cell coculture assays significantly increased (P < .01) subsequent T cell synapse function compared to untreated vehicle control experiments. Flow cytometric analysis identified that lenalidomide down-regulates both CLL expressed PD-L1 and CD200 ligands, and T cell cognate receptor PD1 and CD200R expression during intercellular contact interactions. Moreover, subsequent effector T cell killing function was significantly enhanced (P < .05) following antibody blockade of CLL cell PD-L1 and CD200 with or without lenalidomide treatment during primary coculture with CD8 T cells. We are currently investigating the expression and activity of PD-L1, CD200, and other co-inhibitory molecules in CLL and other haematological and solid malignancies, using patient tissue microarray analysis and confocal co-localization analysis. This work is identifying common inhibitory ligands utilized by tumor cells to suppress T cell synapse function. These results provide important mechanistic insight into immune suppression in CLL and the action of lenalidomide, and identify co-inhibitory ligands as potential immunotherapeutic targets to repair T cell function. Disclosures:Gribben:Roche: Consultancy; Celgene: Consultancy; GSK: Honoraria; Napp: Honoraria.

Impaired Natural Killer Cells Immune Synapse Formation in Acute Myeloid Leukemia.

Abstract 2663Poster Board II-639Natural killer (NK) cells are an important component of the innate immune surveillance of tumor cells. Defective NK cell function has been correlated with poor prognosis in acute myeloid leukemia (AML). It is well established that NK cell-mediated cytolytic activity is significantly diminished in AML patients; the mechanisms of this hypo-function are not well understood. Identifying mechanisms of tumor-induced immune suppression of lymphocytes function will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired NK cell responses in AML and demonstrate impaired NK cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between CD56+ CD3- NK cells and autologous AML blasts. We identified a significant reduction in formation of the NK cell immune synapse (NKIS) (p<0.001) from AML patients compared healthy donors (> 70% reduction). This defect was induced by direct tumor contact since NK cell defects were induced in healthy NK cells when they were co-cultured (in direct contact) for 48 hr with allogeneic AML blasts, but not with healthy allogeneic monocytes (P < 0.01). In control transwell co-culture experiments, where the NK cells and AML blast were not in direct contact, we did not observe the induced defect. We examined the molecular nature of the AML blast induced defect by quantifying recruitment of a number of these NK cell adhesion and cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with AML blasts, healthy NK cells showed significantly reduced recruitment of integrin LFA-1, CD2, Lck, WASP, and tyrosine-phosphorylated protein to the NK-AML target interactions synapse (P < 0.001). These studies demonstrate a role for the tumor induced immune suppression of NK cells and will aid in the development of immunotherapeutic approaches targeting AML. Disclosures:No relevant conflicts of interest to declare.

Lenalidomide Repairs Suppressed T Cell Immunological Synapse Formation in Follicular Lymphoma

Identifying mechanisms of tumor-induced immune suppression will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired T cell responses in follicular lymphoma (FL) and demonstrate impaired T cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between tumor-infiltrating CD4 and CD8 T cells and autologous FL tumor cells with and without superantigen as antigen-presenting cells (APCs). We identified a significant reduction in formation of the T cell immune synapse in both CD4 (p<0.01) and CD8 T cells (p<0.01) from FL patients compared to age-matched healthy donors (> 50% reduction). Comparable immunological defects were also identified in CD4 and CD8 T cells from transformed-FL (t-FL) patients. This defect was induced by tumor contact since T cell defects were induced in healthy T cells when they were were co-cultured for 48 hr with either allogeneic FL or t-FL cells, but not with healthy allogeneic B cells (P < 0.01). Of interest there was enhanced suppression of CD4 T cell synapses following FL co-culture assays and CD8 T cells in the t-FL setting. Since previous gene expression profile studies (Dave et al. NEJM, 2003) demonstrated prognostic significance of altered expression of immune signature genes, we examined the molecular nature of the FL-induced T cell defect by quantifying recruitment of a number of these T cell cytoskeletal signaling proteins to the immune synapse. Following primary co-culture with FL cells, previously healthy T cells showed suppressed recruitment of integrin LFA-1, Lck, Itk, Rab27A and filamin-A to the synapse in subsequent T cell:APC interactions (P < 0.01). We further demonstrate that the immune synapse defects were repaired in part by treatment of the cells with the immunomodulatory drug lenalidomide. Of note, treatment of both FL B cells and autologous T cells with lenalidomide (1 μM for 24h) was required to enhance formation of the F-actin synapse and recruitment of tyrosine-phosphorylated protein and filamin-A irrespective of the presence of exogenous antigen (P < 0.01). We validated the altered expression of a number of these molecules including Itk, Rab27A and filamin-A at the protein level in FL tissue microarrays (TMA). Of note, elevated expression of intrafollicular Rab27A, that mediates targeted secretion of CD8 T cell cytolytic granules, was found in samples from a long-survival group (FL patients who lived more than 15 years). These results provide mechanistic insight into the demonstrated activity of lenalidomide in relapsed or refractory aggressive FL (Wiernik et al. JCO, 2008). These studies demonstrate the role of the tumor in the observed T cell defects in FL, and the molecular basis of these defects and suggest that immunotherapeutic approaches including the use lenalidomide offer the exciting prospect of repairing T cell suppression in B cell malignancies to enhance immune-mediated immunological responses against lymphoma cells.

Abeta-induced meningoencephalitis is IFN-gamma-dependent and is associated with T cell-dependent clearance of Abeta in a mouse model of Alzheimer's disease.

Vaccination against amyloid beta-peptide (Abeta) has been shown to be successful in reducing Abeta burden and neurotoxicity in mouse models of Alzheimer's disease (AD). However, although Abeta immunization did not show T cell infiltrates in the brain of these mice, an Abeta vaccination trial resulted in meningoencephalitis in 6% of patients with AD. Here, we explore the characteristics and specificity of Abeta-induced, T cell-mediated encephalitis in a mouse model of the disease. We demonstrate that a strong Abeta-specific T cell response is critically dependent on the immunizing T cell epitope and that epitopes differ depending on MHC genetic background. Moreover, we show that a single immunization with the dominant T cell epitope Abeta10-24 induced transient meningoencephalitis only in amyloid precursor protein (APP)-transgenic (Tg) mice expressing limited amounts of IFN-gamma under an myelin basic protein (MBP) promoter. Furthermore, immune infiltrates were targeted primarily to sites of Abeta plaques in the brain and were associated with clearance of Abeta. Immune infiltrates were not targeted to the spinal cord, consistent with what was observed in AD patients vaccinated with Abeta. Using primary cultures of microglia, we show that IFN-gamma enhanced clearance of Abeta, microglia, and T cell motility, and microglia-T cell immunological synapse formation. Our study demonstrates that limited expression of IFN-gamma in the brain, as observed during normal brain aging, is essential to promote T cell-mediated immune infiltrates after Abeta immunization and provides a model to investigate both the beneficial and detrimental effects of Abeta-specific T cells.