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.

Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy.

Tumor-specific T cells are frequently exhausted by chronic antigenic stimulation. We here report on a human antigen-specific ex vivo model to explore new therapeutic options for T cell immunotherapies. T cells generated with this model resemble tumor-infiltrating exhausted T cells on a phenotypic and transcriptional level. Using a targeted pooled CRISPR-Cas9 screen and individual gene knockout validation experiments, we uncover sorting nexin-9 (SNX9) as a mediator of T cell exhaustion. Upon TCR/CD28 stimulation, deletion of SNX9 in CD8 T cells decreases PLCγ1, Ca2+, and NFATc2-mediated T cell signaling and reduces expression of NR4A1/3 and TOX. SNX9 knockout enhances memory differentiation and IFNγ secretion of adoptively transferred T cells and results in improved anti-tumor efficacy of human chimeric antigen receptor T cells in vivo. Our findings highlight that targeting SNX9 is a strategy to prevent T cell exhaustion and enhance anti-tumor immunity.

Super-killer CTLs are generated by single gene deletion of Bach2.

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8+ T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient splenic CD8+ T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild-type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naïve T cells, but even within separated subsets, BACH2-deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2-deficient compared to wild-type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super-killer.

Mitochondrial translation is required for sustained killing by cytotoxic T cells.

T cell receptor activation of naïve CD8+ T lymphocytes initiates their maturation into effector cytotoxic T lymphocytes (CTLs), which can kill cancer and virally infected cells. Although CTLs show an increased reliance on glycolysis upon acquisition of effector function, we found an essential requirement for mitochondria in target cell­killing. Acute mitochondrial depletion in USP30 (ubiquitin carboxyl-terminal hydrolase 30)­deficient CTLs markedly diminished killing capacity, although motility, signaling, and secretion were all intact. Unexpectedly, the mitochondrial requirement was linked to mitochondrial translation, inhibition of which impaired CTL killing. Impaired mitochondrial translation triggered attenuated cytosolic translation, precluded replenishment of secreted killing effectors, and reduced the capacity of CTLs to carry out sustained killing. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing.

Teasing out function from morphology: Similarities between primary cilia and immune synapses.

Immune synapses are formed between immune cells to facilitate communication and coordinate the immune response. The reorganization of receptors involved in recognition and signaling creates a transient area of plasma membrane specialized in signaling and polarized secretion. Studies on the formation of the immune synapse between cytotoxic T lymphocytes (CTLs) and their targets uncovered a critical role for centrosome polarization in CTL function and suggested a striking parallel between the synapse and primary cilium. Since these initial observations, a plethora of further morphological, functional, and molecular similarities have been identified between these two fascinating structures. In this review, we describe how advances in imaging and molecular techniques have revealed additional parallels as well as functionally significant differences and discuss how comparative studies continue to shed light on the molecular mechanisms underlying the functions of both the immune synapse and primary cilium.

Distinctive phenotypes and functions of innate lymphoid cells in human decidua during early pregnancy.

During early pregnancy, decidual innate lymphoid cells (dILCs) interact with surrounding maternal cells and invading fetal extravillous trophoblasts (EVT). Here, using mass cytometry, we characterise five main dILC subsets: decidual NK cells (dNK)1-3, ILC3s and proliferating NK cells. Following stimulation, dNK2 and dNK3 produce more chemokines than dNK1 including XCL1 which can act on both maternal dendritic cells and fetal EVT. In contrast, dNK1 express receptors including Killer-cell Immunoglobulin-like Receptors (KIR), indicating they respond to HLA class I ligands on EVT. Decidual NK have distinctive organisation and content of granules compared with peripheral blood NK cells. Acquisition of KIR correlates with higher granzyme B levels and increased chemokine production in response to KIR activation, suggesting a link between increased granule content and dNK1 responsiveness. Our analysis shows that dILCs are unique and provide specialised functions dedicated to achieving placental development and successful reproduction.

Loss of ARPC1B impairs cytotoxic T lymphocyte maintenance and cytolytic activity.

CD8 cytotoxic T lymphocytes (CTLs) rely on rapid reorganization of the branched F-actin network to drive the polarized secretion of lytic granules, initiating target cell death during the adaptive immune response. Branched F-actin is generated by the nucleation factor actin-related protein 2/3 (Arp2/3) complex. Patients with mutations in the actin-related protein complex 1B (ARPC1B) subunit of Arp2/3 show combined immunodeficiency, with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell count. Here, we show that loss of ARPC1B led to loss of CTL cytotoxicity, with the defect arising at 2 different levels. First, ARPC1B is required for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, we found that ARPC1B is indispensable for the maintenance of TCR, CD8, and GLUT1 membrane proteins at the plasma membrane of CTLs, as recycling via the retromer and WASH complexes was impaired in the absence of ARPC1B. Loss of TCR, CD8, and GLUT1 gave rise to defects in T cell signaling and proliferation upon antigen stimulation of ARPC1B-deficient CTLs, leading to a progressive loss of CD8+ T cells. This triggered an activation-induced immunodeficiency of CTL activity in ARPC1B-deficient patients, which could explain the susceptibility to severe and prolonged viral infections.

Inducible T Cell Kinase Regulates the Acquisition of Cytolytic Capacity and Degranulation in CD8+ CTLs.

Patients with mutations in inducible T cell kinase (ITK) are susceptible to viral infections, particularly EBV, suggesting that these patients have defective function of CD8+ CTLs. In this study, we evaluated the effects of ITK deficiency on cytolysis in murine CTLs deficient in ITK, and both human and murine cells treated with an ITK inhibitor. We find that ITK deficiency leads to a global defect in the cytolysis of multiple targets. The absence of ITK both affected CTL expansion and delayed the expression of cytolytic effectors during activation. Furthermore, absence of ITK led to a previously unappreciated intrinsic defect in degranulation. Nonetheless, these defects could be overcome by early or prolonged exposure to IL-2, or by addition of IL-12 to cultures, revealing that cytokine signaling could restore the acquisition of effector function in ITK-deficient CD8+ T cells. Our results provide new insight into the effect of ITK and suboptimal TCR signaling on CD8+ T cell function, and how these may contribute to phenotypes associated with ITK deficiency.

The cytotoxic T lymphocyte immune synapse at a glance.

The immune synapse provides an important structure for communication with immune cells. Studies on immune synapses formed by cytotoxic T lymphocytes (CTLs) highlight the dynamic changes and specialised mechanisms required to facilitate focal signalling and polarised secretion in immune cells. In this Cell Science at a Glance article and the accompanying poster, we illustrate the different steps that reveal the specialised mechanisms used to focus secretion at the CTL immune synapse and allow CTLs to be such efficient and precise serial killers.

Mother Centriole Distal Appendages Mediate Centrosome Docking at the Immunological Synapse and Reveal Mechanistic Parallels with Ciliogenesis.

Cytotoxic T lymphocytes (CTLs) are highly effective serial killers capable of destroying virally infected and cancerous targets by polarized release from secretory lysosomes. Upon target contact, the CTL centrosome rapidly moves to the immunological synapse, focusing microtubule-directed release at this point [1-3]. Striking similarities have been noted between centrosome polarization at the synapse and basal body docking during ciliogenesis [1, 4-8], suggesting that CTL centrosomes might dock with the plasma membrane during killing, in a manner analogous to primary cilia formation [1, 4]. However, questions remain regarding the extent and function of centrosome polarization at the synapse, and recent reports have challenged its role [9, 10]. Here, we use high-resolution transmission electron microscopy (TEM) tomography analysis to show that, as in ciliogenesis, the distal appendages of the CTL mother centriole contact the plasma membrane directly during synapse formation. This is functionally important as small interfering RNA (siRNA) targeting of the distal appendage protein, Cep83, required for membrane contact during ciliogenesis [11], impairs CTL secretion. Furthermore, the regulatory proteins CP110 and Cep97, which must dissociate from the mother centriole to allow cilia formation [12], remain associated with the mother centriole in CTLs, and neither axoneme nor transition zone ciliary structures form. Moreover, complete centrosome docking can occur in proliferating CTLs with multiple centriole pairs. Thus, in CTLs, centrosomes dock transiently with the membrane, within the cell cycle and without progression into ciliogenesis. We propose that this transient centrosome docking without cilia formation is important for CTLs to deliver rapid, repeated polarized secretion directed by the centrosome.

Actin depletion initiates events leading to granule secretion at the immunological synapse.

Cytotoxic T lymphocytes (CTLs) use polarized secretion to rapidly destroy virally infected and tumor cells. To understand the temporal relationships between key events leading to secretion, we used high-resolution 4D imaging. CTLs approached targets with actin-rich projections at the leading edge, creating an initially actin-enriched contact with rearward-flowing actin. Within 1 min, cortical actin reduced across the synapse, T cell receptors (TCRs) clustered centrally to form the central supramolecular activation cluster (cSMAC), and centrosome polarization began. Granules clustered around the moving centrosome within 2.5 min and reached the synapse after 6 min. TCR-bearing intracellular vesicles were delivered to the cSMAC as the centrosome docked. We found that the centrosome and granules were delivered to an area of membrane with reduced cortical actin density and phospholipid PIP2. These data resolve the temporal order of events during synapse maturation in 4D and reveal a critical role for actin depletion in regulating secretion.

Communication, the centrosome and the immunological synapse.

Recent findings on the behaviour of the centrosome at the immunological synapse suggest a critical role for centrosome polarization in controlling the communication between immune cells required to generate an effective immune response. The features observed at the immunological synapse show parallels to centrosome (basal body) polarization seen in cilia and flagella, and the cellular communication that is now known to occur at all of these sites.

Distinct structural and catalytic roles for Zap70 in formation of the immunological synapse in CTL.

T cell receptor (TCR) activation leads to a dramatic reorganisation of both membranes and receptors as the immunological synapse forms. Using a genetic model to rapidly inhibit Zap70 catalytic activity we examined synapse formation between cytotoxic T lymphocytes and their targets. In the absence of Zap70 catalytic activity Vav-1 activation occurs and synapse formation is arrested at a stage with actin and integrin rich interdigitations forming the interface between the two cells. The membranes at the synapse are unable to flatten to provide extended contact, and Lck does not cluster to form the central supramolecular activation cluster (cSMAC). Centrosome polarisation is initiated but aborts before reaching the synapse and the granules do not polarise. Our findings reveal distinct roles for Zap70 as a structural protein regulating integrin-mediated control of actin vs its catalytic activity that regulates TCR-mediated control of actin and membrane remodelling during formation of the immunological synapse. DOI: http://dx.doi.org/10.7554/eLife.01310.001.

Cytotoxic T lymphocyte effector function is independent of nucleus-centrosome dissociation.

Cytotoxic T lymphocytes (CTLs) kill tumorigenic and virally infected cells by targeted secretion of lytic granule contents. The precise point at which secretion occurs is directed by the centrosome docking at the immunological synapse (IS). The centrosome is highly dynamic in CTLs, lagging behind the nucleus in the uropod of migrating CTLs, but translocating across the entire length of the cell to dock at the IS when a target cell is recognized. While in most cell types, the centrosome is always closely associated with the nuclear membrane, in CTLs, it often appears to be dissociated from the nucleus, both in migrating cells and when forming an IS. We asked whether this dissociation is required for CTL killing, by expressing GFP-BICD2-NT-nesprin-3, which tethers the centrosome to the nucleus irreversibly. Immunofluorescence microscopy revealed that the centrosome polarized successfully to the central supramolecular activation complex (cSMAC) of the synapse in GFP-BICD2-NT-nesprin-3-expressing CTLs, with the centrosome and nucleus migrating together to the IS. CTLs in which the centrosome was "glued" to the nucleus were able to dock and release granules at the IS as effectively as mock-treated cells. These data demonstrate that CTL cytotoxicity is independent of centrosomal dissociation from the nuclear envelope.

Centriole polarisation to the immunological synapse directs secretion from cytolytic cells of both the innate and adaptive immune systems.

BACKGROUND: Cytolytic cells of the immune system destroy pathogen-infected cells by polarised exocytosis of secretory lysosomes containing the pore-forming protein perforin. Precise delivery of this lethal hit is essential to ensuring that only the target cell is destroyed. In cytotoxic T lymphocytes (CTLs), this is accomplished by an unusual movement of the centrosome to contact the plasma membrane at the centre of the immunological synapse formed between killer and target cells. Secretory lysosomes are directed towards the centrosome along microtubules and delivered precisely to the point of target cell recognition within the immunological synapse, identified by the centrosome. We asked whether this mechanism of directing secretory lysosome release is unique to CTL or whether natural killer (NK) and invariant NKT (iNKT) cytolytic cells of the innate immune system use a similar mechanism to focus perforin-bearing lysosome release. RESULTS: NK cells were conjugated with B-cell targets lacking major histocompatibility complex class I 721.221 cells, and iNKT cells were conjugated with glycolipid-pulsed CD1-bearing targets, then prepared for thin-section electron microscopy. High-resolution electron micrographs of the immunological synapse formed between NK and iNKT cytolytic cells with their targets revealed that in both NK and iNKT cells, the centrioles could be found associated (or 'docked') with the plasma membrane within the immunological synapse. Secretory clefts were visible within the synapses formed by both NK and iNKT cells, and secretory lysosomes were polarised along microtubules leading towards the docked centrosome. The Golgi apparatus and recycling endosomes were also polarised towards the centrosome at the plasma membrane within the synapse. CONCLUSIONS: These results reveal that, like CTLs of the adaptive immune system, the centrosomes of NK and iNKT cells (cytolytic cells of the innate immune system) direct secretory lysosomes to the immunological synapse. Morphologically, the overall structure of the immunological synapses formed by NK and iNKT cells are very similar to those formed by CTLs, with both exocytic and endocytic organelles polarised towards the centrosome at the plasma membrane, which forms a focal point for exocytosis and endocytosis within the immunological synapse. We conclude that centrosomal polarisation provides a rapid, responsive and precise mechanism for secretory lysosome delivery to the immunological synapse in CTLs, NK cells and iNKT cells.

Centrosome docking at the immunological synapse is controlled by Lck signaling.

Docking of the centrosome at the plasma membrane directs lytic granules to the immunological synapse. To identify signals controlling centrosome docking at the synapse, we have studied cytotoxic T lymphocytes (CTLs) in which expression of the T cell receptor-activated tyrosine kinase Lck is ablated. In the absence of Lck, the centrosome is able to translocate around the nucleus toward the immunological synapse but is unable to dock at the plasma membrane. Lytic granules fail to polarize and release their contents, and target cells are not killed. In CTLs deficient in both Lck and the related tyrosine kinase Fyn, centrosome translocation is impaired, and the centrosome remains on the distal side of the nucleus relative to the synapse. These results show that repositioning of the centrosome in CTLs involves at least two distinct steps, with Lck signaling required for the centrosome to dock at the plasma membrane.

The immunological synapse: a focal point for endocytosis and exocytosis.

There are many different cells in the immune system. To mount an effective immune response, they need to communicate with each other. One way in which this is done is by the formation of immunological synapses between cells. Recent developments show that the immune synapse serves as a focal point for exocytosis and endocytosis, directed by centrosomal docking at the plasma membrane. In this respect, formation of the immunological synapse bears striking similarities to cilia formation and cytokinesis. These intriguing observations suggest that the centrosome may play a conserved role in designating a specialized area of membrane for localized endocytosis and exocytosis.

The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.

Killing by cytotoxic T lymphocytes (CTLs) is mediated by the secretion of lytic granules. The centrosome plays a key role in granule delivery, polarizing to the central supramolecular activation complex (cSMAC) within the immunological synapse upon T cell receptor (TCR) activation. Although stronger TCR signals lead to increased target cell death than do weaker signals, it is not known how the strength of TCR signal controls polarization of the centrosome and lytic granules. By using TCR transgenic OT-I CTLs, we showed that both high- and low-avidity interactions led to centrosome polarization to the cSMAC. However, only high-avidity interactions, which induced a higher threshold of intracellular signaling, gave rise to granule recruitment to the polarized centrosome at the synapse. By controlling centrosome and granule polarization independently, the centrosome is able to respond rapidly to weak signals so that CTLs are poised and ready for the trigger for granule delivery.

Secretory mechanisms in cell-mediated cytotoxicity.

Cytotoxic T lymphocytes (CTLs) play a critical role in the immune system; they are able to recognize and destroy virally infected and tumorigenic cells. Specific recognition of MHC class I-peptide complexes by the T cell receptor (TcR) results in precise delivery of lytic granules to the target cell, sparing neighboring cells and the CTL itself. Over the past 10 years various studies have eludicated the mechanisms that lead to the rapid polarization of the secretory apparatus in CTLs. These studies highlight similarities and differences between polarity and secretory mechanisms seen in other cell types and developmental systems. This review focuses on recent advances in our understanding of the molecular basis of polarized secretion from CTLs and the novel mechanism used by these cells to deliver their lethal hit.

Centrosome polarization delivers secretory granules to the immunological synapse.

Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes--termed 'lytic granules'--at the immunological synapse formed between the CTL and the target. On contact with the target cell, the microtubule organizing centre of the CTL polarizes towards the target and granules move along microtubules in a minus-end direction towards the polarized microtubule organizing centre. However, the final steps of secretion have remained unclear. Here we show that CTLs do not require actin or plus-end microtubule motors for secretion, but instead the centrosome moves to and contacts the plasma membrane at the central supramolecular activation cluster of the immunological synapse. Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane. These data show that CTLs use a previously unreported mechanism for delivering secretory granules to the immunological synapse, with granule secretion controlled by centrosome delivery to the plasma membrane.