Living in Syn: T Cell Antigen Identification Based on Synapse Sequencing.

The pivotal role of T cell responses has been well studied in both protective and destructive scenarios. T cells recognize peptide epitopes presented on Human Leukocyte Antigens (HLA) through their surface T cell receptors (TCR). Advances in single-cell RNA sequencing have identified millions of TCRs, but only a minuscule fraction of them have known epitopes. Recently, cell-based T cell antigen discovery platforms have emerged onto the landscape. Here, Jin and colleagues, report a novel antigen discovery platform called Tsyn-seq that relies on sequencing TCR-peptide-HLA-induced synapses for genome-wide epitope screening. See related article by Jin et al., p. 530 (3).

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.

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.

Tsyn-Seq: a T-cell Synapse-Based Antigen Identification Platform.

Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.

Ectocytosis renders T cell receptor signaling self-limiting at the immune synapse.

Cytotoxic T lymphocytes (CTLs) kill virus-infected and cancer cells through T cell receptor (TCR) recognition. How CTLs terminate signaling and disengage to allow serial killing has remained a mystery. TCR activation triggers membrane specialization within the immune synapse, including the production of diacylglycerol (DAG), a lipid that can induce negative membrane curvature. We found that activated TCRs were shed into DAG-enriched ectosomes at the immune synapse rather than internalized through endocytosis, suggesting that DAG may contribute to the outward budding required for ectocytosis. Budding ectosomes were endocytosed directly by target cells, thereby terminating TCR signaling and simultaneously disengaging the CTL from the target cell to allow serial killing. Thus, ectocytosis renders TCR signaling self-limiting.

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.

Immune Synapse Residency of Orai1 Alters Ca2+ Response of T Cells.

CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Zap70 Regulates TCR-Mediated Zip6 Activation at the Immunological Synapse.

The essential microelement zinc plays immunoregulatory roles via its ability to influence signaling pathways. Zinc deficiency impairs overall immune function and resultantly increases susceptibility to infection. Thus, zinc is considered as an immune-boosting supplement for populations with hypozincemia at high-risk for infection. Besides its role as a structural cofactor of many proteins, zinc also acts as an intracellular messenger in immune cell signaling. T-cell activation instructs zinc influx from extracellular and subcellular sources through the Zip6 and Zip8 zinc transporters, respectively. Increased cytoplasmic zinc participates in the regulation of T-cell responses by modifying activation signaling. However, the mechanism underlying the activation-dependent movement of zinc ions by Zip transporters in T cells remains elusive. Here, we demonstrate that Zip6, one of the most abundantly expressed Zip transporters in T cells, is mainly localized to lipid rafts in human T cells and is recruited into the immunological synapse in response to TCR stimulation. This was demonstrated through confocal imaging of the interaction between CD4+ T cells and antigen-presenting cells. Further, immunoprecipitation assays show that TCR triggering induces tyrosine phosphorylation of Zip6, which has at least three putative tyrosine motifs in its long cytoplasmic region, and this phosphorylation is coupled with its physical interaction with Zap70. Silencing Zip6 reduces zinc influx from extracellular sources and suppresses T-cell responses, suggesting an interaction between Zip6-mediated zinc influx and TCR activation. These results provide new insights into the mechanism through which Zip6-mediated zinc influx occurs in a TCR activation-dependent manner in human CD4+ T cells.

Mechanosensation and Mechanotransduction in Natural Killer Cells.

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where ß actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

Microglia as hackers of the matrix: sculpting synapses and the extracellular space.

Microglia shape the synaptic environment in health and disease, but synapses do not exist in a vacuum. Instead, pre- and postsynaptic terminals are surrounded by extracellular matrix (ECM), which together with glia comprise the four elements of the contemporary tetrapartite synapse model. While research in this area is still just beginning, accumulating evidence points toward a novel role for microglia in regulating the ECM during normal brain homeostasis, and such processes may, in turn, become dysfunctional in disease. As it relates to synapses, microglia are reported to modify the perisynaptic matrix, which is the diffuse matrix that surrounds dendritic and axonal terminals, as well as perineuronal nets (PNNs), specialized reticular formations of compact ECM that enwrap neuronal subsets and stabilize proximal synapses. The interconnected relationship between synapses and the ECM in which they are embedded suggests that alterations in one structure necessarily affect the dynamics of the other, and microglia may need to sculpt the matrix to modify the synapses within. Here, we provide an overview of the microglial regulation of synapses, perisynaptic matrix, and PNNs, propose candidate mechanisms by which these structures may be modified, and present the implications of such modifications in normal brain homeostasis and in disease.

CD97 stabilises the immunological synapse between dendritic cells and T cells and is targeted for degradation by the Salmonella effector SteD.

The Salmonella enterica effector SteD depletes mature MHC class II (mMHCII) molecules from the surface of infected antigen-presenting cells through ubiquitination of the cytoplasmic tail of the mMHCII ß chain. This requires the Nedd4 family HECT E3 ubiquitin ligase Wwp2 and a tumor-suppressing transmembrane protein adaptor Tmem127. Here, through a proteomic screen of dendritic cells, we found that SteD targets the plasma membrane protein CD97 for degradation by a similar mechanism. SteD enhanced ubiquitination of CD97 on K555 and mutation of this residue eliminated the effect of SteD on CD97 surface levels. We showed that CD97 localises to and stabilises the immunological synapse between dendritic cells and T cells. Removal of CD97 by SteD inhibited dendritic cell-T cell interactions and reduced T cell activation, independently of its effect on MHCII. Therefore, SteD suppresses T cell immunity by two distinct processes.

CD58 Immunobiology at a Glance.

The glycoprotein CD58, also known as lymphocyte-function antigen 3 (LFA-3), is a costimulatory receptor distributed on a broad range of human tissue cells. Its natural ligand CD2 is primarily expressed on the surface of T/NK cells. The CD2-CD58 interaction is an important component of the immunological synapse (IS) that induces activation and proliferation of T/NK cells and triggers a series of intracellular signaling in T/NK cells and target cells, respectively, in addition to promoting cell adhesion and recognition. Furthermore, a soluble form of CD58 (sCD58) is also present in cellular supernatant in vitro and in local tissues in vivo. The sCD58 is involved in T/NK cell-mediated immune responses as an immunosuppressive factor by affecting CD2-CD58 interaction. Altered accumulation of sCD58 may lead to immunosuppression of T/NK cells in the tumor microenvironment, allowing sCD58 as a novel immunotherapeutic target. Recently, the crucial roles of costimulatory molecule CD58 in immunomodulation seem to be reattracting the interests of investigators. In particular, the CD2-CD58 interaction is involved in the regulation of antiviral responses, inflammatory responses in autoimmune diseases, immune rejection of transplantation, and immune evasion of tumor cells. In this review, we provide a comprehensive summary of CD58 immunobiology.

Intrinsic Resistance of Chronic Lymphocytic Leukemia Cells to NK Cell-Mediated Lysis Can Be Overcome In Vitro by Pharmacological Inhibition of Cdc42-Induced Actin Cytoskeleton Remodeling.

Natural killer (NK) cells are innate effector lymphocytes with strong antitumor effects against hematologic malignancies such as chronic lymphocytic leukemia (CLL). However, NK cells fail to control CLL progression on the long term. For effective lysis of their targets, NK cells use a specific cell-cell interface, known as the immunological synapse (IS), whose assembly and effector function critically rely on dynamic cytoskeletal changes in NK cells. Here we explored the role of CLL cell actin cytoskeleton during NK cell attack. We found that CLL cells can undergo fast actin cytoskeleton remodeling which is characterized by a NK cell contact-induced accumulation of actin filaments at the IS. Such polarization of the actin cytoskeleton was strongly associated with resistance against NK cell-mediated cytotoxicity and reduced amounts of the cell-death inducing molecule granzyme B in target CLL cells. Selective pharmacological targeting of the key actin regulator Cdc42 abrogated the capacity of CLL cells to reorganize their actin cytoskeleton during NK cell attack, increased levels of transferred granzyme B and restored CLL cell susceptibility to NK cell cytotoxicity. This resistance mechanism was confirmed in primary CLL cells from patients. In addition, pharmacological inhibition of actin dynamics in combination with blocking antibodies increased conjugation frequency and improved CLL cell elimination by NK cells. Together our results highlight the critical role of CLL cell actin cytoskeleton in driving resistance against NK cell cytotoxicity and provide new potential therapeutic point of intervention to target CLL immune escape.

Optimization of T Cell Redirecting Strategies: Obtaining Inspirations From Natural Process of T Cell Activation.

Chimeric antigen receptors (CARs) or bispecific antibodies (bsAbs) redirected T cell against tumors is one of the most promising immunotherapy approaches. However, insufficient clinical outcomes are still observed in treatments of both solid and non-solid tumors. Limited efficacy and poor persistence are two major challenges in redirected T cell therapies. The immunological synapse (IS) is a vital component during the T cell response, which largely determines the clinical outcomes of T cell-based therapies. Here, we review the structural and signaling characteristics of IS formed by natural T cells and redirected T cells. Furthermore, inspired by the elaborate natural T cell receptor-mediated IS, we provide potential strategies for higher efficacy and longer persistence of redirected T cells.

Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells.

One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.

Acquisition of optimal TFH cell function is defined by specific molecular, positional, and TCR dynamic signatures.

The development of follicular helper CD4 T (TFH) cells is a dynamic process resulting in a heterogenous pool of TFH subsets. However, the cellular and molecular determinants of this heterogeneity and the possible mechanistic links between them is not clear. We found that human TFH differentiation is associated with significant changes in phenotypic, chemokine, functional, metabolic and transcriptional profile. Furthermore, this differentiation was associated with distinct positioning to follicular proliferating B cells. Single-cell T cell receptor (TCR) clonotype analysis indicated the transitioning toward PD-1hiCD57hi phenotype. Furthermore, the differentiation of TFH cells was associated with significant reduction in TCR level and drastic changes in immunological synapse formation. TFH synapse lacks a tight cSMAC (central supra molecular activation Cluster) but displays the TCR in peripheral microclusters, which are potentially advantageous in the ability of germinal center (GC) B cells to receive necessary help. Our data reveal significant aspects of human TFH heterogeneity and suggest that the PD-1hiCD57hi TFH cells, in particular, are endowed with distinctive programming and spatial positioning for optimal GC B cell help.

Functional heterogeneity of cytotoxic T cells and tumor resistance to cytotoxic hits limit anti-tumor activity in vivo.

Cytotoxic T cells (CTLs) can eliminate tumor cells through the delivery of lethal hits, but the actual efficiency of this process in the tumor microenvironment is unclear. Here, we visualized the capacity of single CTLs to attack tumor cells in vitro and in vivo using genetically encoded reporters that monitor cell damage and apoptosis. Using two distinct malignant B-cell lines, we found that the majority of cytotoxic hits delivered by CTLs in vitro were sublethal despite proper immunological synapse formation, and associated with reversible calcium elevation and membrane damage in the targets. Through intravital imaging in the bone marrow, we established that the majority of CTL interactions with lymphoma B cells were either unproductive or sublethal. Functional heterogeneity of CTLs contributed to diverse outcomes during CTL-tumor contacts in vivo. In the therapeutic settings of anti-CD19 CAR T cells, the majority of CAR T cell-tumor interactions were also not associated with lethal hit delivery. Thus, differences in CTL lytic potential together with tumor cell resistance to cytotoxic hits represent two important bottlenecks for anti-tumor responses in vivo.

Biomechanics of T Cell Dysfunctions in Chronic Diseases.

Understanding the mechanisms behind T cell dysfunctions during chronic diseases is critical in developing effective immunotherapies. As demonstrated by several animal models and human studies, T cell dysfunctions are induced during chronic diseases, spanning from infections to cancer. Although factors governing the onset and the extent of the functional impairment of T cells can differ during infections and cancer, most dysfunctional phenotypes share common phenotypic traits in their immune receptor and biophysical landscape. Through the latest developments in biophysical techniques applied to explore cell membrane and receptor-ligand dynamics, we are able to dissect and gain further insights into the driving mechanisms behind T cell dysfunctions. These insights may prove useful in developing immunotherapies aimed at reinvigorating our immune system to fight off infections and malignancies more effectively. The recent success with checkpoint inhibitors in treating cancer opens new avenues to develop more effective, targeted immunotherapies. Here, we highlight the studies focused on the transformation of the biophysical landscape during infections and cancer, and how T cell biomechanics shaped the immunopathology associated with chronic diseases.