The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

Polo-like kinase 1 regulates immune synapse assembly and cytotoxic T cell function by driving microtubule dynamics.

Elimination of virally infected or tumoral cells is mediated by cytotoxic T cells (CTL). Upon antigen recognition, CTLs assemble a specialized signaling and secretory domain at the interface with their target, the immune synapse (IS). During IS formation, CTLs acquire a transient polarity, marked by re-orientation of the centrosome and microtubule cytoskeleton toward the IS, thus directing the transport and delivery of the lytic granules to the target cell. Based on the implication that the kinase Aurora A has a role in CTL function, we hypothesized that its substrate, the mitotic regulator Polo-like kinase 1 (PLK1), might participate in CTL IS assembly. We demonstrate that PLK1 is phosphorylated upon TCR triggering and polarizes to the IS. PLK1 silencing or inhibition results in impaired IS assembly and function, as witnessed by defective synaptic accumulation of T cell receptors (TCRs), as well as compromised centrosome and lytic granule polarization to the IS, resulting in impaired target cell killing. This function is achieved by coupling early signaling to microtubule dynamics, a function pivotal for CTL-mediated cytotoxicity. These results identify PLK1 as a new player in CTL IS assembly and function.

Self-programmed dynamics of T cell receptor condensation.

A common event upon receptor-ligand engagement is the formation of receptor clusters on the cell surface, in which signaling molecules are specifically recruited or excluded to form signaling hubs to regulate cellular events. These clusters are often transient and can be disassembled to terminate signaling. Despite the general relevance of dynamic receptor clustering in cell signaling, the regulatory mechanism underlying the dynamics is still poorly understood. As a major antigen receptor in the immune system, T cell receptors (TCR) form spatiotemporally dynamic clusters to mediate robust yet temporal signaling to induce adaptive immune responses. Here we identify a phase separation mechanism controlling dynamic TCR clustering and signaling. The TCR signaling component CD3ε chain can condensate with Lck kinase through phase separation to form TCR signalosomes for active antigen signaling. Lck-mediated CD3ε phosphorylation, however, switched its binding preference to Csk, a functional suppressor of Lck, to cause the dissolvement of TCR signalosomes. Modulating TCR/Lck condensation by targeting CD3ε interactions with Lck or Csk directly affects T cell activation and function, highlighting the importance of the phase separation mechanism. The self-programmed condensation and dissolvement is thus a built-in mechanism of TCR signaling and might be relevant to other receptors.

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.

Changing the location of proteins on the cell surface is a promising strategy for modulating T cell functions.

Targeting immune receptors on T cells is a common strategy to treat cancer and autoimmunity. Frequently, this is accomplished through monoclonal antibodies targeting the ligand binding sites of stimulatory or inhibitory co-receptors. Blocking ligand binding prevents downstream signalling and modulates specific T cell functions. Since 1985, the FDA has approved over 100 monoclonal antibodies against immune receptors. This therapeutic approach significantly improved the care of patients with numerous immune-related conditions; however, many patients are unresponsive, and some develop immune-related adverse events. One reason for that is the lack of consideration for the localization of these receptors on the cell surface of the immune cells in the context of the immune synapse. In addition to blocking ligand binding, changing the location of these receptors on the cell surface within the different compartments of the immunological synapse could serve as an alternative, efficient, and safer approach to treating these patients. This review discusses the potential therapeutic advantages of altering proteins' localization within the immune synapse and summarizes published work in this field. It also discusses the novel use of bispecific antibodies to induce the clustering of receptors on the cell surface. It presents the rationale for developing novel antibodies, targeting the organization of signalling receptor complexes on the cell surface. This approach offers an innovative and emerging technology to treat cancer patients resistant to current immunotherapies.

CD147 regulates the formation and function of immune synapses.

CD147 is a T cell activation-associated molecule which is closely involved in the formation of the immune synapse (IS). However, the precise role of CD147 in T cell activation and IS formation remains unclear. In the present study, we demonstrated that CD147 translocated to the IS upon T cell activation and was primarily distributed in the peripheral super molecular cluster (p-SMAC). The knock down of CD147 expression in T cells, but not in B cells, impaired IS formation. CD147 participated in IS formation between T cells and different types of antigen-presenting cells (APCs), including macrophages and dendritic cells. Ligation of CD147 with its monoclonal antibody (mAb) HAb18 effectively inhibited T cell activation and IL-2 secretion. CD98, a critical molecule interacting with CD147, was distributed in IS in a CD147-dependent way. Phosphorylation levels of T cell receptor (TCR) related molecules, like ZAP-70, ERK, and cJun, were down-regulated by CD147 ligation, which is crucial for the interaction of CD147 and TCR signaling transduction. CD147 is indispensable for the formation of immune synapses and plays an important role in the regulation of its function.

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response.

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.

The Bardet-Biedl syndrome complex component BBS1 controls T cell polarity during immune synapse assembly.

Components of the intraflagellar transport (IFT) system that regulates the assembly of the primary cilium are co-opted by the non-ciliated T cell to orchestrate polarized endosome recycling and to sustain signaling during immune synapse formation. Here, we investigated the potential role of Bardet-Biedl syndrome 1 protein (BBS1), an essential core component of the BBS complex that cooperates with the IFT system in ciliary protein trafficking, in the assembly of the T cell synapse. We demonstrated that BBS1 allows for centrosome polarization towards the immune synapse. This function is achieved through the clearance of centrosomal F-actin and its positive regulator WASH1 (also known as WASHC1), a process that we demonstrated to be dependent on the proteasome. We show that BBS1 regulates this process by coupling the 19S proteasome regulatory subunit to the microtubule motor dynein for its transport to the centrosome. Our data identify the ciliopathy-related protein BBS1 as a new player in T cell synapse assembly that functions upstream of the IFT system to set the stage for polarized vesicular trafficking and sustained signaling. This article has an associated First Person interview with the first author of the paper.

LFA-1 and kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors.

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4ß1 integrin) and LFA-1 (αLß2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin ß2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin ß1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin ß2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.

Natural killer cell immune synapse formation and cytotoxicity are controlled by tension of the target interface.

Natural killer (NK) cells can kill infected or transformed cells via a lytic immune synapse. Diseased cells may exhibit altered mechanical properties but how this impacts NK cell responsiveness is unknown. We report that human NK cells were stimulated more effectively to secrete granzymes A and B, FasL (also known as FasLG), granulysin and IFNγ, by stiff (142 kPa) compared to soft (1 kPa) planar substrates. To create surrogate spherical targets of defined stiffness, sodium alginate was used to synthesise soft (9 kPa), medium (34 kPa) or stiff (254 kPa) cell-sized beads, coated with antibodies against activating receptor NKp30 (also known as NCR3) and the integrin LFA-1 (also known as ITGAL). Against stiff beads, NK cells showed increased degranulation. Polarisation of the microtubule-organising centre and lytic granules were impaired against soft targets, which instead resulted in the formation of unstable kinapses. Thus, by varying target stiffness to characterise the mechanosensitivity of immune synapses, we identify soft targets as a blind spot in NK cell recognition. This article has an associated First Person interview with the co-first authors of the paper.

CD33 BiTE® molecule-mediated immune synapse formation and subsequent T-cell activation is determined by the expression profile of activating and inhibitory checkpoint molecules on AML cells.

Bispecific T-cell engager (BiTE®) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy.

Synaptic secretion from human natural killer cells is diverse and includes supramolecular attack particles.

Natural killer (NK) cells form immune synapses to ascertain the state of health of cells they encounter. If a target cell triggers NK cell cytotoxicity, lytic granules containing proteins including perforin and granzyme B, are secreted into the synaptic cleft inducing target cell death. Secretion of these proteins also occurs from activated cytotoxic T lymphocytes (CTLs) where they have recently been reported to complex with thrombospondin-1 (TSP-1) in specialized structures termed supramolecular attack particles (SMAPs). Here, using an imaging method to define the position of each NK cell after removal, secretions from individual cells were assessed. NK cell synaptic secretion, triggered by ligation of NKp30 or NKG2D, included vesicles and SMAPs which contained TSP-1, perforin, and granzyme B. Individual NK cells secreted SMAPs, CD63+ vesicles, or both. A similar number of SMAPs were secreted per cell for both NK cells and CTLs, but NK cell SMAPs were larger. These data establish an unexpected diversity in NK cell synaptic secretions.

Is there a place and role for endocytic TCR signaling?

T-lymphocyte activation relies on the cognate recognition by the TCR of the MHC-associated peptide ligand (pMHC) presented at the surface of an antigen-presenting cell (APC). This leads to the dynamic formation of a cognate contact between the T lymphocyte and the APC: the immune synapse (IS). Engagement of the TCR by the pMHC in the synaptic zone induces a cascade of signaling events leading to phosphorylation and dephosphorylation of proteins and lipids, which ultimately shapes the response of T lymphocytes. Although the engagement of the T-cell receptor (TCR) takes place at the plasma membrane, the TCR/CD3 complexes and the signaling molecules involved in transduction of the TCR signal are also present in intracellular membrane pools. These pools, which are both endocytic and exocytic, have tentatively been characterized by several groups including ours. We will herein summarize what is known on the intracellular pools of TCR signaling components. We will discuss their origin and the mechanisms involved in their mobility at the IS. Finally, we will propose several hypotheses concerning the functional role(s) that these intracellular pools might play in T-cell activation. We will also discuss the tools that could be used to test these hypotheses.

T Cell Antigen Recognition and Discrimination by Electrochemiluminescence Imaging.

Adoptive T lymphocyte (T cell) transfer and tumour-specific peptide vaccines are innovative cancer therapies. An accurate assessment of the specific reactivity of T cell receptors (TCRs) to tumour antigens is required because of the high heterogeneity of tumour cells and the immunosuppressive tumour microenvironment. In this study, we report a label-free electrochemiluminescence (ECL) imaging approach for recognising and discriminating between TCRs and tumour-specific antigens by imaging the immune synapses of T cells. Various T cell stimuli, including agonistic antibodies, auxiliary molecules, and tumour-specific antigens, were modified on the electrode's surface to allow for their interaction with T cells bearing different TCRs. The formation of immune synapses activated by specific stimuli produced a negative (shadow) ECL image, from which T cell antigen recognition and discrimination were evaluated by analysing the spreading area and the recognition intensity of T cells. This approach provides an easy way to assess TCR-antigen specificity and screen both of them for immunotherapies.

The role of actin and myosin in antigen extraction by B lymphocytes.

B cells must extract antigens attached to the surface of antigen presenting cells to generate high-affinity antibodies. Antigen extraction requires force, and recent studies have implicated actomyosin-dependent pulling forces generated within the B cell as the major driver of antigen extraction. These actomyosin-dependent pulling forces also serve to test the affinity of the B cell antigen receptor for antigen prior to antigen extraction. Such affinity discrimination is central to the process of antibody affinity maturation. Here we review the evidence that actomyosin-dependent pulling forces generated within the B cell promote affinity discrimination and power antigen extraction. Our take on these critical B cell functions is influenced significantly by the recent identification of formin-generated, myosin-rich, concentric actin arcs in the medial portion of the T cell immune synapse, as B cells appear to contain a similar contractile actomyosin structure.

An Autopsy Case of Wiskott-Aldrich Syndrome Revealing "FDC-Only Lymphoid Follicles" in Lymphoid Tissue: A Morphologic Correlate of Defective Immune Synapse.

Wiskott-Aldrich Syndrome (WAS) is an inherited disorder characterized by the classical triad of eczema, micro-thrombocytopenia, and immune deficiency. This disease affects the hematopoietic cells to a variable extent. The spectrum of clinical and laboratory data for WAS has been well described in the literature though there is a paucity of its histopathologic and immunohistochemical correlates. The current case describes the autopsy findings of this rare entity in an 8-year old male child with specific recognition of altered histology noticed in the lymphoreticular tissues. The predominant morphological finding in lymphoid tissue was atretic hyalinized germinal centers labeled as "the follicular dendritic cell (FDC)-only lymphoid follicles." Immunohistochemistry revealed a reduction in germinal-center B-cells, T-follicular helper cells, attenuated mantle zone, FDC proliferation, and paracortical plasmacytosis. This case highlights the crippled immune cell population in WAS, ultimately leading to the morphology of atretic follicles rich in FDCs.

LFA-1 cluster formation in T-cells depends on L-plastin phosphorylation regulated by P90RSK and PP2A.

The integrin LFA-1 is crucial for T-cell/ APC interactions and sensitive recognition of antigens. Precise nanoscale organization and valency regulation of LFA-1 are mandatory for an appropriate function of the immune system. While the inside-out signals regulating the LFA-1 affinity are well described, the molecular mechanisms controlling LFA-1 avidity are still not fully understood. Here, we show that activation of the actin-bundling protein L-plastin (LPL) through phosphorylation at serine-5 enables the formation of clusters containing LFA-1 in high-affinity conformation. Phosphorylation of LPL is induced by an nPKC-MEK-p90RSK pathway and counter-regulated by the serine-threonine phosphatase PP2A. Interestingly, recruitment of LFA-1 into the T-cell/APC contact zone is not affected by LPL phosphorylation. Instead, for this process, activation of the actin-remodeling protein cofilin through dephosphorylation is essential. Together, this study reveals a dichotomic spatial regulation of LFA-1 clustering and microscale movement in T-cells by two different actin-binding proteins, LPL and cofilin.

Joining Forces for Cancer Treatment: From "TCR versus CAR" to "TCR and CAR".

T cell-based immunotherapy has demonstrated great therapeutic potential in recent decades, on the one hand, by using tumor-infiltrating lymphocytes (TILs) and, on the other hand, by engineering T cells to obtain anti-tumor specificities through the introduction of either engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs). Given the distinct design of both receptors and the type of antigen that is encountered, the requirements for proper antigen engagement and downstream signal transduction by TCRs and CARs differ. Synapse formation and signal transduction of CAR T cells, despite further refinement of CAR T cell designs, still do not fully recapitulate that of TCR T cells and might limit CAR T cell persistence and functionality. Thus, deep knowledge about the molecular differences in CAR and TCR T cell signaling would greatly advance the further optimization of CAR designs and elucidate under which circumstances a combination of both receptors would improve the functionality of T cells for cancer treatment. Herein, we provide a comprehensive review about similarities and differences by directly comparing the architecture, synapse formation and signaling of TCRs and CARs, highlighting the knowns and unknowns. In the second part of the review, we discuss the current status of combining CAR and TCR technologies, encouraging a change in perspective from "TCR versus CAR" to "TCR and CAR".

Distinct actin cytoskeleton behaviour in primary and immortalised T-cells.

Cytoskeletal actin dynamics are crucial for the activation of T-cells. Immortalised Jurkat T-cells have been the model system of choice to examine and correlate the dynamics of the actin cytoskeleton and the immunological synapse leading to T-cell activation. However, it has remained unclear whether immortalised cellular systems, such as Jurkat T-cells can recapitulate the cytoskeletal behaviour of primary T-cells. Studies delineating the cytoskeletal behaviour of Jurkat T-cells in comparison to primary T-cells are lacking. Here, we employ live-cell super-resolution microscopy to investigate the cytoskeletal actin organisation and dynamics of living primary and immortalised Jurkat T-cells at the appropriate spatiotemporal resolution. Under comparable activation conditions, we found differences in the architectural organisation and dynamics of Jurkat and primary mouse and human T-cells. Although the three main actin network architectures in Jurkat T-cells were reminiscent of primary T-cells, there were differences in the organisation and molecular mechanisms underlying these networks. Our results highlight mechanistic distinctions in the T-cell model system most utilised to study cytoskeletal actin dynamics.

Dark-Side of Exosomes.

Exosomes are nanoscale extracellular vesicles that can transport cargos of proteins, lipids, DNA, various RNA species and microRNAs (miRNAs). Exosomes can enter cells and deliver their contents to recipient cell. Owing to their cargo exosomes can transfer different molecules to the target cells and change the phenotype of these cells. The fate of the contents of an exosome depends on its target destination. Various mechanisms for exosome uptake by target cells have been proposed, but the mechanisms responsible for exosomes internalization into cells are still debated. Exosomes exposed cells produce labeled protein kinases, which are expressed by other cells. This means that these kinases are internalized by exosomes, and transported into the cytoplasm of recipient cells. Many studies have confirmed that exosomes are not only secreted by living cells, but also internalized or accumulated by the other cells. The "next cell hypothesis" supports the notion that exosomes constitute communication vehicles between neighboring cells. By this mechanism, exosomes participate in the development of diabetes and its associated complications, critically contribute to the spreading of neuronal damage in Alzheimer's disease, and non-proteolysed form of Fas ligand (mFasL)-bearing exosomes trigger the apoptosis of T lymphocytes. Furthermore, exosomes derived from human B lymphocytes induce antigen-specific major histocompatibility complex (MHC) class II-restricted T cell responses. Interestingly, exosomes secreted by cancer cells have been demonstrated to express tumor antigens, as well as immune suppressive molecules. This process is defined as "exosome-immune suppression" concept. The interplay via the exchange of exosomes between cancer cells and between cancer cells and the tumor stroma promote the transfer of oncogenes and onco-miRNAs from one cell to other. Circulating exosomes that are released from hypertrophic adipocytes are effective in obesity-related complications. On the other hand, the "inflammasome-induced" exosomes can activate inflammatory responses in recipient cells. In this chapter protein kinases-related checkpoints are emphasized considering the regulation of exosome biogenesis, secretory traffic, and their impacts on cell death, tumor growth, immune system, and obesity.