Effects of High Efficacy Multiple Sclerosis Disease Modifying Drugs on the Immune Synapse: A Systematic Review.

BACKGROUND: Co-signaling and adhesion molecules are important elements for creating immune synapses between T lymphocytes and antigen-presenting cells; they positively or negatively regulate the interaction between a T cell receptor with its cognate antigen, presented by the major histocompatibility complex. OBJECTIVES: We conducted a systematic review on the effects of High Efficacy Disease Modifying Drugs (HEDMDs) for Multiple Sclerosis (MS) on the co-signaling and adhesion molecules that form the immune synapse. METHODS: We searched EMBASE, MEDLINE, and other sources to identify clinical or preclinical reports on the effects of HEDMDs on co-signaling and adhesion molecules that participate in the formation of immune synapses in patients with MS or other autoimmune disorders. We included reports on cladribine tablets, anti- CD20 monoclonal antibodies, S1P modulators, inhibitors of Bruton's Tyrosine Kinase, and natalizumab. RESULTS: In 56 eligible reports among 7340 total publications, limited relevant evidence was uncovered. Not all co-signaling and adhesion molecules have been studied in relation to every HEDMD, with more data being available on the anti-CD20 monoclonal antibodies (that affect CD80, CD86, GITR and TIGIT), cladribine tablets (affecting CD28, CD40, ICAM-1, LFA-1) and the S1P modulators (affecting CD86, ICAM-1 and LFA-1) and less on Natalizumab (affecting CD80, CD86, CD40, LFA-1, VLA-4) and Alemtuzumab (affecting GITR and CTLA-4). CONCLUSION: The puzzle of HEDMD effects on the immune synapse is far from complete. The available evidence suggests that distinguishing differences exist between drugs and are worth pursuing further.

LPA suppresses T cell function by altering the cytoskeleton and disrupting immune synapse formation.

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)­induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

LXR directly regulates glycosphingolipid synthesis and affects human CD4+ T cell function.

The liver X receptor (LXR) is a key transcriptional regulator of cholesterol, fatty acid, and phospholipid metabolism. Dynamic remodeling of immunometabolic pathways, including lipid metabolism, is a crucial step in T cell activation. Here, we explored the role of LXR-regulated metabolic processes in primary human CD4+ T cells and their role in controlling plasma membrane lipids (glycosphingolipids and cholesterol), which strongly influence T cell immune signaling and function. Crucially, we identified the glycosphingolipid biosynthesis enzyme glucosylceramide synthase as a direct transcriptional LXR target. LXR activation by agonist GW3965 or endogenous oxysterol ligands significantly altered the glycosphingolipid:cholesterol balance in the plasma membrane by increasing glycosphingolipid levels and reducing cholesterol. Consequently, LXR activation lowered plasma membrane lipid order (stability), and an LXR antagonist could block this effect. LXR stimulation also reduced lipid order at the immune synapse and accelerated activation of proximal T cell signaling molecules. Ultimately, LXR activation dampened proinflammatory T cell function. Finally, compared with responder T cells, regulatory T cells had a distinct pattern of LXR target gene expression corresponding to reduced lipid order. This suggests LXR-driven lipid metabolism could contribute to functional specialization of these T cell subsets. Overall, we report a mode of action for LXR in T cells involving the regulation of glycosphingolipid and cholesterol metabolism and demonstrate its relevance in modulating T cell function.

Epigenetic modulation of immune synaptic-cytoskeletal networks potentiates γδ T cell-mediated cytotoxicity in lung cancer.

γδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune system and can attack cancer cells in an MHC-unrestricted manner. Trials of adoptive γδ T cell transfer in solid tumors have had limited success. Here, we show that DNA methyltransferase inhibitors (DNMTis) upregulate surface molecules on cancer cells related to γδ T cell activation using quantitative surface proteomics. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded Vδ1-enriched γδ T cells. Mechanistically, DNMTi enhances immune synapse formation and mediates cytoskeletal reorganization via coordinated alterations of DNA methylation and chromatin accessibility. Genetic depletion of adhesion molecules or pharmacological inhibition of actin polymerization abolishes the potentiating effect of DNMTi. Clinically, the DNMTi-associated cytoskeleton signature stratifies lung cancer patients prognostically. These results support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.

One size does not fit all: navigating the multi-dimensional space to optimize T-cell engaging protein therapeutics.

T-cell engaging biologics is a class of novel and promising immune-oncology compounds that leverage the immune system to eradicate cancer. Here, we compared and contrasted a bispecific diabody-Fc format, which displays a relatively short antigen-binding arm distance, with our bispecific IgG platform. By generating diverse panels of antigen-expressing cells where B cell maturation antigen is either tethered to the cell membrane or located to the juxtamembrane region and masked by elongated structural spacer units, we presented a systematic approach to investigate the role of antigen epitope location and molecular formats in immunological synapse formation and cytotoxicity. We demonstrated that diabody-Fc is more potent for antigen epitopes located in the membrane distal region, while bispecific IgG is more efficient for membrane-proximal epitopes. Additionally, we explored other parameters, including receptor density, antigen-binding affinity, and kinetics. Our results show that molecular format and antigen epitope location, which jointly determine the intermembrane distance between target cells and T cells, allow decoupling of cytotoxicity and cytokine release, while antigen-binding affinities appear to be positively correlated with both readouts. Our work offers new insight that could potentially lead to a wider therapeutic window for T-cell engaging biologics in general.

T-Cell Dynamics in Chronic Lymphocytic Leukemia under Different Treatment Modalities.

PURPOSE: Using next-generation sequencing (NGS), we recently documented T-cell oligoclonality in treatment-naïve chronic lymphocytic leukemia (CLL), with evidence indicating T-cell selection by restricted antigens. EXPERIMENTAL DESIGN: Here, we sought to comprehensively assess T-cell repertoire changes during treatment in relation to (i) treatment type [fludarabine-cyclophosphamide-rituximab (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], and (ii) clinical response, by combining NGS immunoprofiling, flow cytometry, and functional bioassays. RESULTS: T-cell clonality significantly increased at (i) 3 months in the FCR and R-ID treatment groups, and (ii) over deepening clinical response in the R-ID group, with a similar trend detected in the IB group. Notably, in constrast to FCR that induced T-cell repertoire reconstitution, B-cell receptor signaling inhibitors (BcRi) preserved pretreatment clones. Extensive comparisons both within CLL as well as against T-cell receptor sequence databases showed little similarity with other entities, but instead revealed major clonotypes shared exclusively by patients with CLL, alluding to selection by conserved CLL-associated antigens. We then evaluated the functional effect of treatments on T cells and found that (i) R-ID upregulated the expression of activation markers in effector memory T cells, and (ii) both BcRi improved antitumor T-cell immune synapse formation, in marked contrast to FCR. CONCLUSIONS: Taken together, our NGS immunoprofiling data suggest that BcRi retain T-cell clones that may have developed against CLL-associated antigens. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response.

Immunomodulatory Agents in Follicular Lymphoma.

New treatment strategies in follicular lymphoma (FL) are driven by a deeper understanding of microenvironmental cues supporting lymphomagenesis, chemoresistance, and immuno-escape. These immune-mediated signaling pathways contribute to initial learnings and clinical successes with lenalidomide, the first, oral, non-chemotherapeutic immunomodulatory drug, combined with anti-CD20 antibodies. This combination of lenalidomide with rituximab showed similar efficacy to chemoimmunotherapy (CIT) in first-line patients requiring therapy, and is approved in relapsed/refractory FL. We review the biology supporting the rationale for adequate inhibitory receptor/ligand pathways targeting the tissue immune microenvironment of FL cells, and potential immunomodulating combinations to replace CIT in the near future.

Adenovirus-mediated specific tumor tagging facilitates CAR-T therapy against antigen-mismatched solid tumors.

Chimeric antigen receptor T (CAR-T) therapy faces at least two major obstacles in solid tumors, including to find specific antigen among the heterogeneous tumor mass and to overcome the inhibitory microenvironment. Developing novel strategies to overcome these difficulties has been the burning issue in immunotherapy. Here we came up with the concept of tagging cancer cells by tumor-targeting adenoviruses (Ad). We constructed recombinant Ads expressing CD19 tag driven by tumor-specific promoters, which could label antigenically different tumors for single anti-CD19 CAR-T recognition. One Ad, namely AdC68-TMC-tCD19 could mediate universal tag expression and functional immunological synapse formation between CAR-T and cancer cells. In premixed mice model, all tagged mice survived after CAR-T infusion and tumor volume were inhibited by 91.78%. Furthermore, we combined the tumor tagging ability with oncolysis and generated the replicative AdC68-Sur-E1A-TMC-tCD19. Oncolytic tagging system could diminish established tumors in vivo and prolong mice survival significantly. Therefore, we suggest the universal oncolytic Ad-tagging system in combination with single target CAR-T cells could be a powerful complement in immunotherapy against antigenically mismatched solid tumors.

Neurokinin-1 Receptor Signaling Is Required for Efficient Ca2+ Flux in T-Cell-Receptor-Activated T Cells.

Efficient Ca2+ flux induced during cognate T cell activation requires signaling the T cell receptor (TCR) and unidentified G-protein-coupled receptors (GPCRs). T cells express the neurokinin-1 receptor (NK1R), a GPCR that mediates Ca2+ flux in excitable and non-excitable cells. However, the role of the NK1R in TCR signaling remains unknown. We show that the NK1R and its agonists, the neuropeptides substance P and hemokinin-1, co-localize within the immune synapse during cognate activation of T cells. Simultaneous TCR and NK1R stimulation is necessary for efficient Ca2+ flux and Ca2+-dependent signaling that sustains the survival of activated T cells and helper 1 (Th1) and Th17 bias. In a model of contact dermatitis, mice with T cells deficient in NK1R or its agonists exhibit impaired cellular immunity, due to high mortality of activated T cells. We demonstrate an effect of the NK1R in T cells that is relevant for immunotherapies based on pro-inflammatory neuropeptides and its receptors.

Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors.

The spatiotemporal distribution of cytokines orchestrates immune responses in vivo, yet the underlying mechanisms remain to be explored. We showed here that the spatial distribution of interleukin-4 (IL4) in invariant natural killer T (iNKT) cells regulated crosstalk between iNKT cells and dendritic cells (DCs) and controlled iNKT cell-mediated T-helper type 1 (Th1) responses. The persistent polarization of IL4 induced by strong lipid antigens, that is, α-galactosylceramide (αGC), caused IL4 accumulation at the immunological synapse (IS), which promoted the activation of the IL4R-STAT6 (signal transducer and activator of transcription 6) pathway and production of IL12 in DCs, which enhanced interferon-γ (IFNγ) production in iNKT cells. Conversely, the nonpolarized secretion of IL4 induced by Th2 lipid antigens with a short or unsaturated chain was incapable of enhancing this iNKT cell-DC crosstalk and thus shifted the immune response to a Th2-type response. The nonpolarized secretion of IL4 in response to Th2 lipid antigens was caused by the degradation of Cdc42 in iNKT cells. Moreover, reduced Cdc42 expression was observed in tumor-infiltrating iNKT cells, which impaired IL4 polarization and disturbed iNKT cell-DC crosstalk in tumors.

Combination lenalidomide-rituximab immunotherapy activates anti-tumour immunity and induces tumour cell death by complementary mechanisms of action in follicular lymphoma.

Chemotherapy plus rituximab has been the mainstay of treatment for follicular lymphoma (FL) for two decades but is associated with immunosuppression and relapse. In phase 2 studies, lenalidomide combined with rituximab (R2 ) has shown clinical synergy in front-line and relapsed/refractory FL. Here, we show that lenalidomide reactivated dysfunctional T and Natural Killer (NK) cells ex vivo from FL patients by enhancing proliferative capacity and T-helper cell type 1 (Th1) cytokine release. In combination with rituximab, lenalidomide improved antibody-dependent cellular cytotoxicity in sensitive and chemo-resistant FL cells, via a cereblon-dependent mechanism. While single-agent lenalidomide and rituximab increased formation of lytic NK cell immunological synapses with primary FL tumour cells, the combination was superior and correlated with enhanced cytotoxicity. Immunophenotyping of FL patient samples from a phase 3 trial revealed that R2 treatment increased circulating T- and NK-cell counts, while R-chemotherapy was associated with reduced cell numbers. Finally, using an in vitro model of myeloid differentiation, we demonstrated that lenalidomide caused a reversible arrest in neutrophil maturation that was distinct from a cytotoxic chemotherapeutic agent, which may help explain the lower rates of neutropenia observed with R2 versus R-chemotherapy. Taken together, we believe these data support a paradigm shift in the treatment of FL - moving from combination immunochemotherapy to chemotherapy-free immunotherapy.

Novel natural killer cell-mediated cancer immunotherapeutic activity of anisomycin against hepatocellular carcinoma cells.

Despite advances in the clinical management of hepatocellular carcinoma (HCC), this form of cancer remains the second leading cause of cancer-related death worldwide. Currently, there are few treatment options for advanced HCC. Therefore, novel treatment strategies for HCC are required. Here, we described the promising antitumour effects of anisomycin, which exerts both direct killing effects and natural killer cell (NK)-mediated immunotherapeutic effects in HCC. To better elucidate the mechanisms through which anisomycin mediates its antitumour effects, we performed a genome-scale transcriptional analysis. We found that anisomycin treatment of HCC differentially modulated a broad range of immune regulation-associated genes. Among these immune regulation-associated genes, we found that lymphocyte function-associated antigen-3 (LFA-3, also called CD58), whose expression was significantly increased in anisomycin-treated HCC cells, was a critical player in NK-mediated immunotherapeutic effects. Furthermore major histocompatibility complex molecules class I (MHC-I) on HCC cells were also significantly regulated by treatment of anisomycin. Those adhesion molecules like CD58, MHC-I, and ICAM4 should be important for immune synapse formation between NK cells and HCC cells to boost NK-mediated immunotherapeutic effects. Notably, this is the first report of NK-dependent immunomodulatory effects of anisomycin suggesting anisomycin as a novel therapeutic drug for treatment of HCC.

The immunological synapse as a pharmacological target.

The development of T cell mediated immunity relies on the assembly of a highly specialized interface between T cell and antigen presenting cell (APC), known as the immunological synapse (IS). IS assembly is triggered when the T cell receptor (TCR) binds to specific peptide antigen presented in association to the major histocompatibility complex (MHC) by the APC, and is followed by the spatiotemporal dynamic redistribution of TCR, integrins, co-stimulatory receptors and signaling molecules, allowing for the fine-tuning and integration of the signals that lead to T cell activation. The knowledge acquired to date about the mechanisms of IS assembly underscores this structure as a robust pharmacological target. The activity of molecules involved in IS assembly and function can be targeted by specific compounds to modulate the immune response in a number of disorders, including cancers and autoimmune diseases, or in transplanted patients. Here, we will review the state-of-the art of the current therapies which exploit the IS to modulate the immune response.

Despite disorganized synapse structure, Th2 cells maintain directional delivery of CD40L to antigen-presenting B cells.

Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody.

Live-Cell Super-resolution Reveals F-Actin and Plasma Membrane Dynamics at the T Cell Synapse.

The cortical actin cytoskeleton has been shown to be critical for the reorganization and heterogeneity of plasma membrane components of many cells, including T cells. Building on previous studies at the T cell immunological synapse, we quantitatively assess the structure and dynamics of this meshwork using live-cell superresolution fluorescence microscopy and spatio-temporal image correlation spectroscopy. We show for the first time, to our knowledge, that not only does the dense actin cortex flow in a retrograde fashion toward the synapse center, but the plasma membrane itself shows similar behavior. Furthermore, using two-color, live-cell superresolution cross-correlation spectroscopy, we demonstrate that the two flows are correlated and, in addition, we show that coupling may extend to the outer leaflet of the plasma membrane by examining the flow of GPI-anchored proteins. Finally, we demonstrate that the actin flow is correlated with a third component, α-actinin, which upon CRISPR knockout led to reduced plasma membrane flow directionality despite increased actin flow velocity. We hypothesize that this apparent cytoskeletal-membrane coupling could provide a mechanism for driving the observed retrograde flow of signaling molecules such as the TCR, Lck, ZAP70, LAT, and SLP76.

Human NK cell development requires CD56-mediated motility and formation of the developmental synapse.

While distinct stages of natural killer (NK) cell development have been defined, the molecular interactions that shape human NK cell maturation are poorly understood. Here we define intercellular interactions between developing NK cells and stromal cells which, through contact-dependent mechanisms, promote the generation of mature, functional human NK cells from CD34(+) precursors. We show that developing NK cells undergo unique, developmental stage-specific sustained and transient interactions with developmentally supportive stromal cells, and that the relative motility of NK cells increases as they move through development in vitro and ex vivo. These interactions include the formation of a synapse between developing NK cells and stromal cells, which we term the developmental synapse. Finally, we identify a role for CD56 in developmental synapse structure, NK cell motility and NK cell development. Thus, we define the developmental synapse leading to human NK cell functional maturation.

The Adhesion G Protein-Coupled Receptor GPR56/ADGRG1 Is an Inhibitory Receptor on Human NK Cells.

Natural killer (NK) cells possess potent cytotoxic mechanisms that need to be tightly controlled. Here, we explored the regulation and function of GPR56/ADGRG1, an adhesion G protein-coupled receptor implicated in developmental processes and expressed distinctively in mature NK cells. Expression of GPR56 was triggered by Hobit (a homolog of Blimp-1 in T cells) and declined upon cell activation. Through studying NK cells from polymicrogyria patients with disease-causing mutations in ADGRG1, encoding GPR56, and NK-92 cells ectopically expressing the receptor, we found that GPR56 negatively regulates immediate effector functions, including production of inflammatory cytokines and cytolytic proteins, degranulation, and target cell killing. GPR56 pursues this activity by associating with the tetraspanin CD81. We conclude that GPR56 inhibits natural cytotoxicity of human NK cells.

Aurora A drives early signalling and vesicle dynamics during T-cell activation.

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3ζ-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.