The interaction between signal regulatory protein alpha (SIRPα) and CD47: structure, function, and therapeutic target.

CD47 is a broadly expressed membrane protein that interacts with the myeloid inhibitory immunoreceptor SIRPα (also termed CD172a or SHPS-1). SIRPα is the prototypic member of the SIRP paired receptor family of closely related SIRP proteins. Engagement of SIRPα by CD47 provides a downregulatory signal that inhibits host cell phagocytosis, and CD47 therefore functions as a "don't-eat-me" signal. Here, we discuss recent structural analysis of CD47-SIRPα interactions and implications of this for the function and evolution of SIRPα and paired receptors in general. Furthermore, we review the proposed roles of CD47-SIRPα interactions in phagocytosis, (auto)immunity, and host defense, as well as its potential significance as a therapeutic target in cancer and inflammation and for improving graft survival in xenotransplantation.

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.

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.

Functional anatomy of T cell activation and synapse formation.

T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward.

Lipid switches in the immunological synapse.

Adaptive immune responses comprise the activation of T cells by peptide antigens that are presented by proteins of the Major Histocompatibility Complex (MHC) on the surface of an antigen-presenting cell. As a consequence of the T cell receptor interacting productively with a certain peptide-MHC complex, a specialized cell-cell junction known as the immunological synapse forms and is accompanied by changes in the spatiotemporal patterning and function of intracellular signaling molecules. Key modifications occurring at the cytoplasmic leaflet of the plasma and internal membranes in activated T cells comprise lipid switches that affect the binding and distribution of proteins within or near the lipid bilayer. Here, we describe two major classes of lipid switches that act at this critical water/membrane interface. Phosphoinositides are derived from phosphatidylinositol, an amphiphilic molecule that contains two fatty acid chains and a phosphate group that bridges the glycerol backbone to the carbohydrate inositol. The inositol ring can be variably (de-)phosphorylated by dedicated kinases and phosphatases, thereby creating phosphoinositide signatures that define the composition and properties of signaling molecules, molecular complexes, or whole organelles. Palmitoylation refers to the reversible attachment of the fatty acid palmitate to a substrate protein's cysteine residue. DHHC enzymes, named after the four conserved amino acids in their active site, catalyze this post-translational modification and thereby change the distribution of proteins at, between, and within membranes. T cells utilize these two types of molecular switches to adjust their properties to an activation process that requires changes in motility, transport, secretion, and gene expression.

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.

VAMP7 controls T cell activation by regulating the recruitment and phosphorylation of vesicular Lat at TCR-activation sites.

The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.

Role of lamin A/C on dendritic cell function in antiviral immunity.

Dendritic cells (DCs) play a crucial role in orchestrating immune responses, particularly in promoting IFNγ-producing-CD8 cytotoxic T lymphocytes (CTLs) and IFNγ-producing-CD4 T helper 1 (Th1) cells, which are essential for defending against viral infections. Additionally, the nuclear envelope protein lamin A/C has been implicated in T cell immunity. Nevertheless, the intricate interplay between innate and adaptive immunity in response to viral infections, particularly the role of lamin A/C in DC functions within this context, remains poorly understood. In this study, we demonstrate that mice lacking lamin A/C in myeloid LysM promoter-expressing cells exhibit a reduced capacity to induce Th1 and CD8 CTL responses, leading to impaired clearance of acute primary Vaccinia virus (VACV) infection. Remarkably, in vitro-generated granulocyte macrophage colony-stimulating factor bone marrow-derived DCs (GM-CSF BMDCs) show high levels of lamin A/C. Lamin A/C absence on GM-CSF BMDCs does not affect the expression of costimulatory molecules on the cell membrane but it reduces the cellular ability to form immunological synapses with naïve CD4 T cells. Lamin A/C deletion induces alterations in NFκB nuclear localization, thereby influencing NF-κB-dependent transcription. Furthermore, lamin A/C ablation modifies the gene accessibility of BMDCs, predisposing these cells to mount a less effective antiviral response upon TLR stimulation. This study highlights the critical role of DCs in interacting with CD4 T cells during antiviral responses and proposes some mechanisms through which lamin A/C may modulate DC function via gene accessibility and transcriptional regulation.

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.

Rewired signaling network in T cells expressing the chimeric antigen receptor (CAR).

The chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, nor actin remodeling following CAR activation. However, CAR T cells still require LAT for an optimal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.

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.

Integration of the movement of signaling microclusters with cellular motility in immunological synapses.

Immune synapses form between T cells and antigen-presenting cells (APCs). Increasing evidence suggests synapses must form flexibly to accommodate ongoing motility and displacement of the synapse. Here, time-lapse total internal reflection fluorescence (TIRF) microscopy showed that signaling via the T cell antigen receptor (TCR) occurred during synapse translation. TCR microclusters in motile synapses did not flow directly into supramolecular activating complexes (SMACs) but were directed, independently of myosin II contractility, toward an F-actin-poor 'sink' region. Inward microcluster flow often followed collapse of the leading edge, which suggested that actin depolymerization regulated microcluster flow and the formation of SMACs. The coordination of TCR movement with the translocation of this 'sink' shows how T cells coordinate TCR signaling and microcluster flow in dynamic physiological synapses.

Monitoring T cell-dendritic cell interactions in vivo by intercellular enzymatic labelling.

Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell-cell interactions can be monitored in vivo by intravital microscopy, this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor-ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of 'kiss-and-run' interactions between immune cells 'Labelling Immune Partnerships by SorTagging Intercellular Contacts' (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4+ T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40-CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell-cell interactions both in vitro and in vivo. Given its flexibility for use with different receptor-ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.

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.

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.

A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells.

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell (APC) is driven by the accumulation of diacylglycerol (DAG) at the immunological synapse (IS). The mechanisms that couple DAG to the MTOC are not known. By single-cell photoactivation of the T cell antigen receptor (TCR), we found that three distinct isoforms of protein kinase C (PKC) were recruited by DAG to the IS in two steps. PKC-ɛ and PKC-η accumulated first in a broad region of membrane, whereas PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation and that PKC-ɛ and PKC-η operated redundantly to promote the recruitment of PKC-θ and subsequent polarization responses. Our results establish a previously uncharacterized role for PKC proteins in T cell polarity.

TFH-derived dopamine accelerates productive synapses in germinal centres.

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (TFH) cells in germinal centres. The rapid T-B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human TFH cells contain dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. TFH cells produce high amounts of dopamine and release it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T-B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T-B-cell synapse may be advantageous in the face of infection.

Beyond the CRAC: Diversification of ion signaling in B cells.

Although calcium signaling and the important role of calcium release-activated calcium channels is well recognized in the context of immune cell signaling, there is a vast diversity of ion channels and transporters that regulate the entry of ions beyond calcium, including magnesium, zinc, potassium, sodium, and chloride. These ions play a critical role in numerous metabolic and cellular processes. The importance of ions in human health and disease is illustrated by the identification of primary immunodeficiencies in patients with mutations in genes encoding ion channels and transporters, as well as the immunological defects observed in individuals with nutritional ion deficiencies. Despite progress in identifying the important role of ions in immune cell development and activation, we are still in the early stages of exploring the diversity of ion channels and transporters and mechanistically understanding the role of these ions in immune cell biology. Here, we review the biology of ion signaling in B cells and the identification of critical ion channels and transporters in B-cell development, activation, and differentiation into effector cells. Elucidating the role of ion channels and transporters in immune cell signaling is critical for expanding the repertoire of potential therapeutics for the treatment of immune disorders. Moreover, increased understanding of the role of ions in immune cell function will enhance our understanding of the potentially serious consequences of ion deficiencies in human health and disease.

A role for TASK2 channels in the human immunological synapse.

The immunological synapse is a transient junction that occurs when the plasma membrane of a T cell comes in close contact with an APC after recognizing a peptide from the antigen-MHC. The interaction starts when CRAC channels embedded in the T cell membrane open, flowing calcium ions into the cell. To counterbalance the ion influx and subsequent depolarization, Kv 1.3 and KCa3.1 channels are recruited to the immunological synapse, increasing the extracellular K+ concentration. These processes are crucial as they initiate gene expression that drives T cell activation and proliferation. The T cell-specific function of the K2P channel family member TASK2 channels and their role in autoimmune processes remains unclear. Using mass spectrometry analysis together with epifluorescence and super-resolution single-molecule localization microscopy, we identified TASK2 channels as novel players recruited to the immunological synapse upon stimulation. TASK2 localizes at the immunological synapse, upon stimulation with CD3 antibodies, likely interacting with these molecules. Our findings suggest that, together with Kv 1.3 and KCa3.1 channels, TASK2 channels contribute to the proper functioning of the immunological synapse, and represent an interesting treatment target for T cell-mediated autoimmune disorders.