NFIL3 contributes to cytotoxic T lymphocyte-mediated killing.

Cytotoxic T lymphocytes (CTLs) are key effectors of the adaptive immune system that recognize and eliminate virally infected and cancerous cells. In naive CD8+ T cells, T-cell receptor (TCR) engagement drives a number of transcriptional, translational and proliferation changes over the course of hours and days leading to differentiation into CTLs. To gain a better insight into this mechanism, we compared the transcriptional profiles of naive CD8+ T cells to those of activated CTLs. To find new regulators of CTL function, we performed a selective clustered regularly interspaced short palindromic repeats (CRISPR) screen on upregulated genes and identified nuclear factor IL-3 (NFIL3) as a potential regulator of cytotoxicity. Although NFIL3 has established roles in several immune cells including natural killer, Treg, dendritic and CD4+ T cells, its function in CD8+ CTLs is less well understood. Using CRISPR/Cas9 editing, we found that removing NFIL3 in CTLs resulted in a marked decrease in cytotoxicity. We found that in CTLs lacking NFIL3 TCR-induced extracellular signal-regulated kinase phosphorylation, immune synapse formation and granule release were all intact while cytotoxicity was functionally impaired in vitro. Strikingly, NFIL3 controls the production of cytolytic proteins as well as effector cytokines. Thus, NFIL3 plays a cell intrinsic role in modulating cytolytic mechanisms in CTLs.

Cytotoxic T lymphocytes require transcription for infiltration but not target cell lysis.

Effector cytotoxic T lymphocytes (CTLs) are critical for ridding the body of infected or cancerous cells. CTL T cell receptor (TCR) ligation not only drives the delivery and release of cytolytic granules but also initiates a new wave of transcription. In order to address whether TCR-induced transcriptomic changes impact the ability of CTLs to kill, we asked which genes are expressed immediately after CTLs encounter targets and how CTL responses change when inhibiting transcription. Our data demonstrate that while expression of cytokines/chemokines and transcriptional machinery depend on transcription, cytotoxic protein expression and cytolytic activity are relatively robust to transcription blockade, with CTLs lysing nearby target cells for several hours after actinomycin D treatment. Monitoring CTL movement among target cells after inhibiting transcription demonstrates an infiltration defect that is not rectified by provision of exogenous cytokine/chemokine gradients, indicating a cell-intrinsic transcriptional requirement for infiltration. Together, our results reveal differential molecular control of CTL functions, separating recruitment and infiltration from cytolysis.

Immunodeficiency with susceptibility to lymphoma with complex genotype affecting energy metabolism (FBP1, ACAD9) and vesicle trafficking (RAB27A).

Introduction: Inborn errors of immunity (IEI) are characterized by a dysfunction of the immune system leading to increased susceptibility to infections, impaired immune regulation and cancer. We present a unique consanguineous family with a history of Hodgkin lymphoma, impaired EBV control and a late onset hemophagocytic lymphohistiocytosis (HLH). Methods and results: Overall, family members presented with variable impairment of NK cell and cytotoxic T cell degranulation and cytotoxicity. Exome sequencing identified homozygous variants in RAB27A, FBP1 (Fructose-1,6-bisphosphatase 1) and ACAD9 (Acyl-CoA dehydrogenase family member 9). Variants in RAB27A lead to Griscelli syndrome type 2, hypopigmentation and HLH predisposition. Discussion: Lymphoma is frequently seen in patients with hypomorphic mutations of genes predisposing to HLH. We hypothesize that the variants in FBP1 and ACAD9 might aggravate the clinical and immune phenotype, influence serial killing and lytic granule polarization by CD8 T cells. Understanding of the interplay between the multiple variants identified by whole exome sequencing (WES) is essential for correct interpretation of the immune phenotype and important for critical treatment decisions.

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.

Imaging the T-cell receptor: new approaches, new insights.

T cells recognize pathogenic antigens via the T-cell antigen receptor (TCR). This protein complex binds to antigen fragments on the surface of antigen-presenting cells. To understand how cellular activation can ensue rapidly from molecular recognition, the localization and distribution of the TCR on the surface of the resting T cell are of particular importance. Conflicting results regarding TCR distribution have emerged from recent studies using a range of imaging techniques, including total internal reflection and single-molecule localization microscopy modalities. Here, we review the differing results and the potential biases inherent in differing imaging approaches. In addition, we review studies showing the impact of differing imaging surfaces on T-cell activation.

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.

Arming a killer: mitochondrial regulation of CD8+ T cell cytotoxicity.

While once regarded as ATP factories, mitochondria have taken the spotlight as important regulators of cellular homeostasis. The past two decades have witnessed an intensifying interest in the study of mitochondria in cells of the immune system, with many new and unexpected roles for mitochondria emerging. Immune cells offer intriguing insights as mitochondria appear to play different roles at different stages of T cell development, matching the changing functions of the cells. Here we briefly review the multifaceted roles of mitochondria during T cell differentiation, focusing on CD8+ cytotoxic T lymphocytes (CTLs) and we consider how mitochondrial dysfunction can contribute to CTL exhaustion. In addition, we highlight a newly appreciated role for mitochondria as homeostatic regulators of CTL-mediated killing and explore the emerging literature describing mechanisms linking cytosolic and mitochondrial protein synthesis.

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.

Staggered starts in the race to T cell activation.

How T lymphocytes tune their responses to different strengths of stimulation is a fundamental question in immunology. Recent work using new optogenetic, single-cell genomic, and live-imaging approaches has revealed that stimulation strength controls the rate of individual cell responses within a population. Moreover, these responses have been found to use shared molecular programs, regardless of stimulation strength. However, additional data indicate that stimulation duration or cytokine feedback can impact later gene expression phenotypes of activated cells. In-depth molecular studies have suggested mechanisms by which stimulation strength might modulate the probability of T cell activation. This emerging model allows activating T cells to achieve a wide range of population responses through probabilistic control within individual cells.

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.

Signal strength controls the rate of polarization within CTLs during killing.

Cytotoxic T lymphocytes (CTLs) are key effector cells in the immune response against viruses and cancers, killing targets with high precision. Target cell recognition by CTL triggers rapid polarization of intracellular organelles toward the synapse formed with the target cell, delivering cytolytic granules to the immune synapse. Single amino acid changes within peptides binding MHC class I (pMHCs) are sufficient to modulate the degree of killing, but exactly how this impacts the choreography of centrosome polarization and granule delivery to the target cell remains poorly characterized. Here we use 4D imaging and find that the pathways orchestrating killing within CTL are conserved irrespective of the signal strength. However, the rate of initiation along these pathways varies with signal strength. We find that increased strength of signal leads to an increased proportion of CTLs with prolonged dwell times, initial Ca2+ fluxes, centrosome docking, and granule polarization. Hence, TCR signal strength modulates the rate but not organization of effector CTL responses.

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.

Cytoskeletal control of the secretory immune synapse.

The immune synapse is a very important but often transient site for secretion between immune cells. How secretion is controlled in a coordinated fashion at the synapse is a subject of much investigation. Two key mechanisms are the polarisation of the centrosome and rapid actin dynamics across the immune synapses that form between interacting immune cells. In recent years it has become clear that different immune cells utilise a diversity of immune synapses that modify these mechanisms in order to optimise specialised modes of secretion. Here we describe some of the latest research, focusing on regulation by centrosomal and actin dynamics in a variety of immune cells.

Griscelli Syndrome Type 2 Sine Albinism: Unraveling Differential RAB27A Effector Engagement.

Griscelli syndrome type 2 (GS-2) is an inborn error of immunity characterized by partial albinism and episodes of hemophagocytic lymphohistiocytosis (HLH). It is caused by RAB27A mutations that encode RAB27A, a member of the Rab GTPase family. RAB27A is expressed in many tissues and regulates vesicular transport and organelle dynamics. Occasionally, GS-2 patients with RAB27A mutation display normal pigmentation. The study of such variants provides the opportunity to map distinct binding sites for tissue-specific effectors on RAB27A. Here we present a new case of GS-2 without albinism (GS-2 sine albinism) caused by a novel missense mutation (Val143Ala) in the RAB27A and characterize its functional cellular consequences. Using pertinent animal cell lines, the Val143Ala mutation impairs both the RAB27A-SLP2-A interaction and RAB27A-MUNC13-4 interaction, but it does not affect the RAB27A-melanophilin (MLPH)/SLAC2-A interaction that is crucial for skin and hair pigmentation. We conclude that disruption of the RAB27A-MUNC13-4 interaction in cytotoxic lymphocytes leads to the HLH predisposition of the GS-2 patient with the Val143Ala mutation. Finally, we include a review of GS-2 sine albinism cases reported in the literature, summarizing their genetic and clinical characteristics.

Stimulation strength controls the rate of initiation but not the molecular organisation of TCR-induced signalling.

Millions of naïve T cells with different TCRs may interact with a peptide-MHC ligand, but very few will activate. Remarkably, this fine control is orchestrated using a limited set of intracellular machinery. It remains unclear whether changes in stimulation strength alter the programme of signalling events leading to T cell activation. Using mass cytometry to simultaneously measure multiple signalling pathways during activation of murine CD8+ T cells, we found a programme of distal signalling events that is shared, regardless of the strength of TCR stimulation. Moreover, the relationship between transcription of early response genes Nr4a1 and Irf8 and activation of the ribosomal protein S6 is also conserved across stimuli. Instead, we found that stimulation strength dictates the rate with which cells initiate signalling through this network. These data suggest that TCR-induced signalling results in a coordinated activation program, modulated in rate but not organization by stimulation strength.

Amongst the different types of cells the body uses to protect itself, killer T cells have an unique role: they can detect and neutralize cells that have been become dangerous for the organism ­ for example, cells which are cancerous or hijacked by viruses. In a healthy organism, T cells circulate through the body in an inactivated state. When a disease emerges, receptors at the surface of the cells can detect elements coming from harmful agents; this stimulation then triggers a molecular cascade inside the T cell that leads to activation. This system is relatively simple, pairing a finite number of receptors with a limited set of internal components. At the same time, the activity of T cells is finely regulated, and their activation tightly controlled: they must kill enough cells to stop the illness without causing excess damage. How this is accomplished is still unclear. A T cell can recognize harmful agents that bind its receptors with differing strengths, but how this variability in stimulation strength affects the signaling processes within the cell is still poorly understood. To investigate this question, Ma et al. used an approach called mass cytometry and analyzed the internal processes of mouse killer T cells receiving different strengths of stimulation. This investigation revealed little change in the patterns of signaling in response to signals of different strength. Instead, what differed was the proportion of T cells that became activated, and how fast this process took place: stronger stimulations led to a larger population of killer T cells being activated more rapidly. Overall, this work sheds light on how killer T cells fine-tune their response to illness using only a simple system to control their activation.

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.

Phospholipids: Pulling Back the Actin Curtain for Granule Delivery to the Immune Synapse.

Phosphoinositides, together with the phospholipids phosphatidylserine and phosphatidic acid, are important components of the plasma membrane acting as second messengers that, with diacylglycerol, regulate a diverse range of signaling events converting extracellular changes into cellular responses. Local changes in their distribution and membrane charge on the inner leaflet of the plasma membrane play important roles in immune cell function. Here we discuss their distribution and regulators highlighting the importance of membrane changes across the immune synapse on the cytoskeleton and the impact on the function of cytotoxic T lymphocytes.

PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes.

How cytotoxic T lymphocytes (CTLs) sense T cell receptor (TCR) signaling in order to specialize an area of plasma membrane for granule secretion is not understood. Here, we demonstrate that immune synapse formation led to rapid localized changes in the phosphoinositide composition of the plasma membrane, both reducing phosphoinositide-4-phosphate (PI(4)P), PI(4,5)P2, and PI(3,4,5)P3 and increasing diacylglycerol (DAG) and PI(3,4)P2 within the first 2 min of synapse formation. These changes reduced negative charge across the synapse, triggering the release of electrostatically bound PIP5 kinases that are required to replenish PI(4,5)P2. As PI(4,5)P2 decreased, actin was depleted from the membrane, allowing secretion. Forced localization of PIP5Kß across the synapse prevented actin depletion, blocking both centrosome docking and secretion. Thus, PIP5Ks act as molecular sensors of TCR activation, controlling actin recruitment across the synapse, ensuring exquisite co-ordination between TCR signaling and CTL secretion.

T cell cytolytic capacity is independent of initial stimulation strength.

How cells respond to myriad stimuli with finite signaling machinery is central to immunology. In naive T cells, the inherent effect of ligand strength on activation pathways and endpoints has remained controversial, confounded by environmental fluctuations and intercellular variability within populations. Here we studied how ligand potency affected the activation of CD8+ T cells in vitro, through the use of genome-wide RNA, multi-dimensional protein and functional measurements in single cells. Our data revealed that strong ligands drove more efficient and uniform activation than did weak ligands, but all activated cells were fully cytolytic. Notably, activation followed the same transcriptional pathways regardless of ligand potency. Thus, stimulation strength did not intrinsically dictate the T cell-activation route or phenotype; instead, it controlled how rapidly and simultaneously the cells initiated activation, allowing limited machinery to elicit wide-ranging responses.