Claudin 18 turns up the heat in cancer.

A major barrier to antitumor immunity in solid tumors is T cell exclusion. In this issue of Immunity, De Sanctis et al.1 elucidate how CLDN18 on pancreatic and lung cancer cells enhances infiltration, immunological synapse formation, and activation of cytotoxic T lymphocytes.

Immunological synapse formation between T regulatory cells and cancer-associated fibroblasts promotes tumour development.

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA+ CAFs can form immunological synapses with Foxp3+ regulatory T cells (Tregs) in tumours. Notably, α-SMA+ CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA+ CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA+ CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA+ CAFs and Tregs in an autophagy-dependent manner.

Tumor cells impair immunological synapse formation via central nervous system-enriched metabolite.

Tumors employ various strategies to evade immune surveillance. Central nervous system (CNS) has multiple features to restrain immune response. Whether tumors and CNS share similar programs of immunosuppression is elusive. Here, we analyze multi-omics data of tumors from HER2+ breast cancer patients receiving trastuzumab and anti-PD-L1 antibody and find that CNS-enriched N-acetyltransferase 8-like (NAT8L) and its metabolite N-acetylaspartate (NAA) are overexpressed in resistant tumors. In CNS, NAA is released during brain inflammation. NAT8L attenuates brain inflammation and impairs anti-tumor immunity by inhibiting cytotoxicity of natural killer (NK) cells and CD8+ T cells via NAA. NAA disrupts the formation of immunological synapse by promoting PCAF-induced acetylation of lamin A-K542, which inhibits the integration between lamin A and SUN2 and impairs polarization of lytic granules. We uncover that tumor cells mimic the anti-inflammatory mechanism of CNS to evade anti-tumor immunity and NAT8L is a potential target to enhance efficacy of anti-cancer agents.

Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

EFHD2 regulates T cell receptor signaling and modulates T helper cell activation in early sepsis.

EFHD2 (EF-hand domain family, member D2) has been identified as a calcium-binding protein with immunomodulatory effects. In this study, we characterized the phenotype of Efhd2-deficient mice in sepsis and examined the biological functions of EFHD2 in peripheral T cell activation and T helper (Th) cell differentiation. Increased levels of EFHD2 expression accompanied peripheral CD4+ T cell activation in the early stages of sepsis. Transcriptomic analysis indicated that immune response activation was impaired in Efhd2-deficient CD4+ T cells. Further, Efhd2-deficient CD4+ T cells isolated from the spleen of septic mice showed impaired T cell receptor (TCR)-induced Th differentiation, especially Th1 and Th17 differentiation. In vitro data also showed that Efhd2-deficient CD4+ T cells exhibit impaired Th1 and Th17 differentiation. In the CD4+ T cells and macrophages co-culture model for antigen presentation, the deficiency of Efhd2 in CD4+ T cells resulted in impaired formation of immunological synapses. In addition, Efhd2-deficient CD4+ T cells exhibited reduced levels of phospho-LCK and phospho-ZAP70, and downstream transcription factors including Nfat, Nfκb and Nur77 following TCR engagement. In summary, EFHD2 may promote TCR-mediated T cell activation subsequent Th1 and Th17 differentiation in the early stages of sepsis by regulating the intensity of TCR complex formation.

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation.

ABSTRACT: The inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain-containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

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.

Imaging PIP2 and BCR microclusters in B cell immunological synapse.

The humoral immune response is dependent on B cell activation and differentiation, which is typically triggered by the formation of immunological synapses at the interface between B cells and the antigen presenting surfaces. However, due to the highly dynamic and transient feature of immunological synapses, it has been difficult to capture and investigate the molecular events that occur within them. The planar lipids bilayer (PLB) supported antigen presenting surface combined with high-resolution high-speed total internal reflection fluorescence microscope (TIRFM) live cell imaging system has been proved to be a powerful tool that allows us to visualize the dynamic events in immunological synapse. In addition, the phospholipid phosphatidylinositol-(4,5)-biphosphate (PIP2) plays a unique role in B cell activation, and it is difficult to investigate the synaptic dynamics of PIP2 molecules. Hence, we describe here the general procedures for the utilization of a PLB based antigen presenting system combining TIRFM based imaging methods to visualize the spatial-temporal co-distribution of PIP2 and BCR microcluster within the B cell immunological synapse.

Activation kinetics of regulatory molecules during immunological synapse in T cells.

T cell activation through TCR stimulation leads to the formation of the immunological synapse (IS), a specialized adhesion organized between T lymphocytes and antigen presenting cells (APCs) in which a dynamic interaction among signaling molecules, the cytoskeleton and intracellular organelles achieves proper antigen-mediated stimulation and effector function. The kinetics of molecular reactions at the IS is essential to determine the quality of the response to the antigen stimulation. Herein, we describe methods based on biochemistry, flow cytometry and imaging in live and fixed cells to study the activation state and dynamics of regulatory molecules at the IS in the Jurkat T cell line CH7C17 and primary human and mouse CD4+ T lymphocytes stimulated by antigen presented by Raji and HOM2 B cell lines and human and mouse dendritic cells.

Correlative light and electron microscopy to explore the lytic immunological synapse between natural killer cells and cancer cells.

Cytotoxic lymphocytes, such as natural killer (NK) cells and cytotoxic T cells, can recognize and kill tumor cells by establishing a highly specialized cell-cell contact called the immunological synapse. The formation and lytic activity of the immunological synapse are accompanied by local changes in the organization, dynamics and molecular composition of the cell membrane, as well as the polarization of various cellular components, such as the cytoskeleton, vesicles and organelles. Characterization and understanding of the molecular and cellular processes underlying immunological synapse formation and activity requires the combination of complementary types of information provided by different imaging modalities, the correlation of which can be difficult. Correlative light and electron microscopy (CLEM) allows for the accurate correlation of functional information provided by fluorescent light microscopy with ultrastructural features provided by high-resolution electron microscopy. In this chapter, we present a detailed protocol describing each step to generate cell-cell conjugates between NK cells and cancer cells, and to analyze these conjugates by CLEM using separate confocal laser-scanning and transmission electron microscopes.

Building the synapse engine to drive B lymphocyte function.

B cell receptor (BCR)-mediated antigen-specific recognition activates B lymphocytes and drives the humoral immune response. This enables the generation of antibody-producing plasma cells, the effector arm of the B cell immune response, and of memory B cells, which confer protection against additional encounters with antigen. B cells search for cognate antigen in the complex cellular microarchitecture of secondary lymphoid organs, where antigens are captured and exposed on the surface of different immune cells. While scanning the cell network, the BCR can be stimulated by a specific antigen and elicit the establishment of the immune synapse with the antigen-presenting cell. At the immune synapse, an integrin-enriched supramolecular domain is assembled at the periphery of the B cell contact with the antigen-presenting cell, ensuring a stable and long-lasting interaction. The coordinated action of the actomyosin cytoskeleton and the microtubule network in the inner B cell space provides a structural framework that integrates signaling events and antigen uptake through the generation of traction forces and organelle polarization. Accordingly, the B cell immune synapse can be envisioned as a temporal engine that drives the molecular mechanisms needed for successful B cell activation. Here, I review different aspects of the B cell synapse engine and provide insights into other aspects poorly known or virtually unexplored.

Detection of Telomere Transfer at Immunological Synapse.

Eukaryotes solve the DNA-end replication problem synthesizing hexameric chromosome ends known as telomeres. Recent studies have uncovered unexpected functions of telomeres in linking synaptic signaling and vesicle transport, with at least one pathway directly involved in transferring telomeres through the immune synapse. These emerging forms of cellular communication may originate a new class of antiaging interventions based on telomere transplants.

Assessment of membrane lipid state at the natural killer cell immunological synapse.

The plasma membrane is a fluid structure that protects cells as one of their first barriers and actively participates in numerous biological processes in many ways including through distinct membrane sub-regions. For immunological cells, highly organized sub-compartments of plasma membranes are vital for them to sense and react to environmental changes. This includes a varying spectrum of lipid ordering in the plasma membrane which signifies or enables cellular functions. Thus, comprehensive analyses of the plasma membrane can facilitate understanding of important cell biological elements which include insights into immune cells. Here, we describe two methods that can be used to assess membrane lipid state at the natural killer cell immunological synapse via high-resolution live cell imaging techniques.

Learning from TCR Signaling and Immunological Synapse Assembly to Build New Chimeric Antigen Receptors (CARs).

Chimeric antigen receptor (CAR) T cell immunotherapy is a revolutionary pillar in cancer treatment. Clinical experience has shown remarkable successes in the treatment of certain hematological malignancies but only limited efficacy against B cell chronic lymphocytic leukemia (CLL) and other cancer types, especially solid tumors. A wide range of engineering strategies have been employed to overcome the limitations of CAR T cell therapy. However, it has become increasingly clear that CARs have unique, unexpected features; hence, a deep understanding of how CARs signal and trigger the formation of a non-conventional immunological synapse (IS), the signaling platform required for T cell activation and execution of effector functions, would lead a shift from empirical testing to the rational design of new CAR constructs. Here, we review current knowledge of CARs, focusing on their structure, signaling and role in CAR T cell IS assembly. We, moreover, discuss the molecular features accounting for poor responses in CLL patients treated with anti-CD19 CAR T cells and propose CLL as a paradigm for diseases connected to IS dysfunctions that could significantly benefit from the development of novel CARs to generate a productive anti-tumor response.

Ganglioside-Functionalized Nanoparticles for Chimeric Antigen Receptor T-Cell Activation at the Immunological Synapse.

Chimeric Antigen Receptor (CAR) T cell therapy has proven to be an effective strategy against hematological malignancies but persistence and activity against solid tumors must be further improved. One emerging strategy for enhancing efficacy is based on directing CAR T cells to antigen presenting cells (APCs). Activation of CAR T cells at the immunological synapse (IS) formed between APC and T cell is thought to promote strong, persistent antigen-specific T cell-mediated immune responses but requires integration of CAR ligands into the APC/T-cell interface. Here, we demonstrate that CAR ligand functionalized, lipid-coated, biodegradable polymer nanoparticles (NPs) that contain the ganglioside GM3 (GM3-NPs) bind to CD169 (Siglec-1)-expressing APCs and localize to the cell contact site between APCs and CAR T cells upon initiation of cell conjugates. The CD169+ APC/CAR T-cell interface is characterized by a strong optical colocalization of GM3-NPs and CARs, enrichment of F-actin, and recruitment of ZAP-70, indicative of integration of GM3-NPs into a functional IS. Ligands associated with GM3-NPs localized to the APC/T-cell contact site remain accessible to CARs and result in robust T-cell activation. Overall, this work identifies GM3-NPs as a potential antigen delivery platform for active targeting of CD169 expressing APCs and enhancement of CAR T-cell activation at the NP-containing IS.

CD28 and chemokine receptors: Signalling amplifiers at the immunological synapse.

T cells are master regulators of the immune response tuning, among others, B cells, macrophages and NK cells. To exert their functions requiring high sensibility and specificity, T cells need to integrate different stimuli from the surrounding microenvironment. A finely tuned signalling compartmentalization orchestrated in dynamic platforms is an essential requirement for the proper and efficient response of these cells to distinct triggers. During years, several studies have depicted the pivotal role of the cytoskeleton and lipid microdomains in controlling signalling compartmentalization during T cell activation and functions. Here, we discuss mechanisms responsible for signalling amplification and compartmentalization in T cell activation, focusing on the role of CD28, chemokine receptors and the actin cytoskeleton. We also take into account the detrimental effect of mutations carried by distinct signalling proteins giving rise to syndromes characterized by defects in T cell functionality.

Phollow the phosphoinositol: Actin dynamics at the B cell immune synapse.

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.

Impairment of cytokine production following immunological synapse formation in patients with Wiskott-Aldrich syndrome and leukocyte adhesion deficiency type 1.

T cells following immunological synapse (IS) formation with antigen-presenting cells produce multiple cytokines through T cell receptor, integrin, and costimulatory signaling. Here, we investigated the cytokine profiles following IS formation in response to staphylococcal superantigen exposure in three adolescent patients with classical Wiskott-Aldrich syndrome (WAS) and in one patient with leukocyte adhesion deficiency (LAD) type 1. All WAS patients showed lower Th1 and Th2-skewed cytokine production; similar results were observed in the flow cytometric analysis of IFNγ- and IL-4-producing T cells. The patient with LAD type 1 with somatic mosaicism in 2% of CD8+ T cells showed lower Th1 and Th2 cytokine production than healthy controls. The patients with WAS were susceptible to infections and atopic manifestations, and the patients with LAD type 1 showed cold abscess on their skin, our findings using patient samples provide clinical insights into the mechanisms underlying immunodeficiency related to the symptoms of each disease.

The Expanding Arsenal of Cytotoxic T Cells.

Cytotoxic T cells (CTLs) are the main cellular mediators of the adaptive immune defenses against intracellular pathogens and malignant cells. Upon recognition of specific antigen on their cellular target, CTLs assemble an immunological synapse where they mobilise their killing machinery that is released into the synaptic cleft to orchestrate the demise of their cell target. The arsenal of CTLs is stored in lysosome-like organelles that undergo exocytosis in response to signals triggered by the T cell antigen receptor following antigen recognition. These organelles include lytic granules carrying a cargo of cytotoxic proteins packed on a proteoglycan scaffold, multivesicular bodies carrying the death receptor ligand FasL, and the recently discovered supramolecular attack particles that carry a core of cytotoxic proteins encased in a non-membranous glycoprotein shell. Here we will briefly review the main features of these killing entities and discuss their interrelationship and interplay in CTL-mediated killing.