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.

Evolutionary design of explainable algorithms for biomedical image segmentation.

An unresolved issue in contemporary biomedicine is the overwhelming number and diversity of complex images that require annotation, analysis and interpretation. Recent advances in Deep Learning have revolutionized the field of computer vision, creating algorithms that compete with human experts in image segmentation tasks. However, these frameworks require large human-annotated datasets for training and the resulting "black box" models are difficult to interpret. In this study, we introduce Kartezio, a modular Cartesian Genetic Programming-based computational strategy that generates fully transparent and easily interpretable image processing pipelines by iteratively assembling and parameterizing computer vision functions. The pipelines thus generated exhibit comparable precision to state-of-the-art Deep Learning approaches on instance segmentation tasks, while requiring drastically smaller training datasets. This Few-Shot Learning method confers tremendous flexibility, speed, and functionality to this approach. We then deploy Kartezio to solve a series of semantic and instance segmentation problems, and demonstrate its utility across diverse images ranging from multiplexed tissue histopathology images to high resolution microscopy images. While the flexibility, robustness and practical utility of Kartezio make this fully explicable evolutionary designer a potential game-changer in the field of biomedical image processing, Kartezio remains complementary and potentially auxiliary to mainstream Deep Learning approaches.

Measuring CTL Lytic Granule Secretion and Target Cell Membrane Repair by Fluorescent Lipophilic Dye Uptake at the Lytic Synapse.

CD8+ cytotoxic T lymphocytes (CTL) play a key role in anti-tumor immune response. They are therefore at the heart of current immunotherapy protocols against cancer. Despite current strategies to potentiate CTL responses, cancer cells can resist CTL attack, thus limiting the efficacy of immunotherapies. To optimize immunotherapy, it is urgent to develop rapid assays allowing to assess CTL-cancer cell confrontation at the lytic synapse.In this chapter, we describe a flow cytometry-based method to simultaneously assess the extent of CTL activation and of tumor cell reparative membrane turnover in CTL/target cell conjugates. Such a method can be performed using a limited number of cells. It can therefore be employed in clinical settings when only a few patient-derived cells might be available.

Resisting T cell attack: tumor-cell-intrinsic defense and reparation mechanisms.

Cytotoxic T lymphocytes (CTLs) are antigen-specific killer cells equipped to identify and eliminate host cells that have been altered through infection or transformation. Both chimeric antigen-receptor (CAR) T cell therapies and immune checkpoint blockade (ICB) therapies are based on successful elimination of tumor cells by cytotoxic effectors. In this opinion article, we outline cell-intrinsic mechanisms by which tumor cells defend against CTLs, highlighting pathways that confer resistance and proposing opportunities for combination therapies. We discuss how exogenous killing entities [e.g., supramolecular attack particles (SMAPs)] offer a novel strategy to circumvent cellular resistance mechanisms. Our opinion article highlights the importance of identifying, quantifying, and targeting tumor defense mechanisms at the interface between tumor cells and CTLs as a critical consideration in the development of immunotherapy approaches.

Learning from TCR Signaling and Immunological Synapse Assembly to Build New Chimeric Antigen Receptors (CARs).

Chimeric antigen receptor (CAR) T cell immunotherapy is a revolutionary pillar in cancer treatment. Clinical experience has shown remarkable successes in the treatment of certain hematological malignancies but only limited efficacy against B cell chronic lymphocytic leukemia (CLL) and other cancer types, especially solid tumors. A wide range of engineering strategies have been employed to overcome the limitations of CAR T cell therapy. However, it has become increasingly clear that CARs have unique, unexpected features; hence, a deep understanding of how CARs signal and trigger the formation of a non-conventional immunological synapse (IS), the signaling platform required for T cell activation and execution of effector functions, would lead a shift from empirical testing to the rational design of new CAR constructs. Here, we review current knowledge of CARs, focusing on their structure, signaling and role in CAR T cell IS assembly. We, moreover, discuss the molecular features accounting for poor responses in CLL patients treated with anti-CD19 CAR T cells and propose CLL as a paradigm for diseases connected to IS dysfunctions that could significantly benefit from the development of novel CARs to generate a productive anti-tumor response.

Greek Fire, Poison Arrows, and Scorpion Bombs: How Tumor Cells Defend Against the Siege Weapons of Cytotoxic T Lymphocytes.

CD8+ cytotoxic T lymphocytes (CTLs) are the main cellular effectors of the adaptive immune response against cancer cells, which in turn have evolved sophisticated cellular defense mechanisms to withstand CTL attack. Herein we provide a critical review of the pertinent literature on early and late attack/defense events taking place at the CTL/target cell lytic synapse. We examine the earliest steps of CTL-mediated cytotoxicity ("the poison arrows") elicited within seconds of CTL/target cell encounter, which face commensurately rapid synaptic repair mechanisms on the tumor cell side, providing the first formidable barrier to CTL attack. We examine how breach of this first defensive barrier unleashes the inextinguishable "Greek fire" in the form of granzymes whose broad cytotoxic potential is linked to activation of cell death executioners, injury of vital organelles, and destruction of intracellular homeostasis. Herein tumor cells deploy slower but no less sophisticated defensive mechanisms in the form of enhanced autophagy, increased reparative capacity, and dysregulation of cell death pathways. We discuss how the newly discovered supra-molecular attack particles (SMAPs, the "scorpion bombs"), seek to overcome the robust defensive mechanisms that confer tumor cell resistance. Finally, we discuss the implications of the aforementioned attack/defense mechanisms on the induction of regulated cell death (RCD), and how different contemporary RCD modalities (including apoptosis, pyroptosis, and ferroptosis) may have profound implications for immunotherapy. Thus, we propose that understanding and targeting multiple steps of the attack/defense process will be instrumental to enhance the efficacy of CTL anti-tumor activity and meet the outstanding challenges in clinical immunotherapy.

Phenotypic and Histological Distribution Analysis Identify Mast Cell Heterogeneity in Non-Small Cell Lung Cancer.

Mast cells (MCs) are multifaceted innate immune cells often present in the tumor microenvironment (TME). However, MCs have been only barely characterized in studies focusing on global immune infiltrate phenotyping. Consequently, their role in cancer is still poorly understood. Furthermore, their prognosis value is confusing since MCs have been associated with good and bad (or both) prognosis depending on the cancer type. In this pilot study performed on a surgical cohort of 48 patients with Non-Small Cell Lung Cancer (NSCLC), we characterized MC population within the TME and in matching non-lesional lung areas, by multicolor flow cytometry and confocal microscopy. Our results showed that tumor-associated MCs (TAMCs) harbor a distinct phenotype as compared with MCs present in non-lesional counterpart of the lung. Moreover, we found two TAMCs subsets based on the expression of CD103 (also named alphaE integrin). CD103+ TAMCs appeared more mature, more prone to interact with CD4+ T cells, and located closer to cancer cells than their CD103- counterpart. In spite of these characteristics, we did not observe a prognosis advantage of a high frequency of CD103+ TAMCs, while a high frequency of total TAMC correlated with better overall survival and progression free survival. Together, this study reveals that TAMCs constitute a heterogeneous population and indicates that MC subsets should be considered for patients' stratification and management in future research.

Identification of distinct cytotoxic granules as the origin of supramolecular attack particles in T lymphocytes.

Cytotoxic T lymphocytes (CTL) kill malignant and infected cells through the directed release of cytotoxic proteins into the immunological synapse (IS). The cytotoxic protein granzyme B (GzmB) is released in its soluble form or in supramolecular attack particles (SMAP). We utilize synaptobrevin2-mRFP knock-in mice to isolate fusogenic cytotoxic granules in an unbiased manner and visualize them alone or in degranulating CTLs. We identified two classes of fusion-competent granules, single core granules (SCG) and multi core granules (MCG), with different diameter, morphology and protein composition. Functional analyses demonstrate that both classes of granules fuse with the plasma membrane at the IS. SCG fusion releases soluble GzmB. MCGs can be labelled with the SMAP marker thrombospondin-1 and their fusion releases intact SMAPs. We propose that CTLs use SCG fusion to fill the synaptic cleft with active cytotoxic proteins instantly and parallel MCG fusion to deliver latent SMAPs for delayed killing of refractory targets.

Ultrarapid lytic granule release from CTLs activates Ca2+-dependent synaptic resistance pathways in melanoma cells.

Human cytotoxic T lymphocytes (CTLs) exhibit ultrarapid lytic granule secretion, but whether melanoma cells mobilize defense mechanisms with commensurate rapidity remains unknown. We used single-cell time-lapse microscopy to offer high spatiotemporal resolution analyses of subcellular events in melanoma cells upon CTL attack. Target cell perforation initiated an intracellular Ca2+ wave that propagated outward from the synapse within milliseconds and triggered lysosomal mobilization to the synapse, facilitating membrane repair and conferring resistance to CTL induced cytotoxicity. Inhibition of Ca2+ flux and silencing of synaptotagmin VII limited synaptic lysosomal exposure and enhanced cytotoxicity. Multiplexed immunohistochemistry of patient melanoma nodules combined with automated image analysis showed that melanoma cells facing CD8+ CTLs in the tumor periphery or peritumoral area exhibited significant lysosomal enrichment. Our results identified synaptic Ca2+ entry as the definitive trigger for lysosomal deployment to the synapse upon CTL attack and highlighted an unpredicted defensive topology of lysosome distribution in melanoma nodules.

Self-activation of Vγ9Vδ2 T cells by exogenous phosphoantigens involves TCR and butyrophilins.

The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.

Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior.

Cytotoxic immune cells are endowed with a high degree of heterogeneity in their lytic function, but how this heterogeneity is generated is still an open question. We therefore investigated if human CD8+ T cells could segregate their lytic components during telophase, using imaging flow cytometry, confocal microscopy, and live-cell imaging. We show that CD107a+-intracellular vesicles, perforin, and granzyme B unevenly segregate in a constant fraction of telophasic cells during each division round. Mathematical modeling posits that unequal lytic molecule inheritance by daughter cells results from the random distribution of lytic granules on the two sides of the cleavage furrow. Finally, we establish that the level of lytic compartment in individual cytotoxic T lymphocyte (CTL) dictates CTL killing capacity.

Sequential adjustment of cytotoxic T lymphocyte densities improves efficacy in controlling tumor growth.

Understanding the human cytotoxic T lymphocyte (CTL) biology is crucial to develop novel strategies aiming at maximizing their lytic capacity against cancer cells. Here we introduce an agent-based model, calibrated on population-scale experimental data that allows quantifying human CTL per capita killing. Our model highlights higher individual CTL killing capacity at lower CTL densities and fits experimental data of human melanoma cell killing. The model allows extending the analysis over prolonged time frames, difficult to investigate experimentally, and reveals that initial high CTL densities hamper efficacy to control melanoma growth. Computational analysis forecasts that sequential addition of fresh CTL cohorts improves tumor growth control. In vivo experimental data, obtained in a mouse melanoma model, confirm this prediction. Taken together, our results unveil the impact that sequential adjustment of cellular densities has on enhancing CTL efficacy over long-term confrontation with tumor cells. In perspective, they can be instrumental to refine CTL-based therapeutic strategies aiming at controlling tumor growth.

Theophylline induces differentiation and modulates cytoskeleton dynamics and cytokines secretion in human melanoma-initiating cells.

AIMS: Cutaneous melanoma is the most aggressive skin cancer, derived from neoplastic transformation of melanocytes. Since several evidences highlighted the importance of a hierarchical model of differentiation among cancer cells, closely related to resistance mechanisms and tumor relapse, we investigated the effects of theophylline (Theo), a methylxanthine commonly used in treatment of respiratory diseases, on melanoma cells with different degree of differentiation, including patient-derived melanoma-initiating cells. MATERIALS AND METHODS: The antiproliferative and antimetastatic effects of Theo was demonstrated by cell counting, adhesion and migration assays on A375 and SK-MEL-30 cells. Further, Theo ability to reduce cell growth was highly significant in A375-derived spheroids and in two patient-derived melanoma-initiating cells (MICs). In order to identify pathways potentially involved in the antineoplastic properties of Theo, a comparative mass spectrometry proteomic analysis was used. Then, melanin content, tyrosinase and tissue transglutaminase activities as differentiation markers and actin re-organization through confocal microscopy were evaluated. Furthermore, a secretome profile of MICs after Theo treatments was performed by multiplex immunoassay. KEY FINDINGS: Obtained results demonstrate inhibitory effects of Theo on melanoma cell proliferation and migration, mainly in MICs, together with the induction of differentiation parameters. Moreover, our data indicate that the known anti-melanoma effect of Theo is due also to its ability to interfere with cytoskeleton dynamics and to induce the secretion of inflammatory molecules involved in recruitment of immunosuppressive cells in tumor microenvironment. SIGNIFICANCE: Data strongly suggest that Theo supplement, either as drug or as dietary supply, may represent a potent additional weapon against melanoma.

Multiparametric analysis of CD8+ T cell compartment phenotype in chronic lymphocytic leukemia reveals a signature associated with progression toward therapy.

CD8+ T cells are frontline defenders against cancer and primary targets of current immunotherapies. In CLL, specific functional alterations have been described in circulating CD8+ T cells, yet a global view of the CD8+ T cell compartment phenotype and of its real impact on disease progression is presently elusive. We developed a multidimensional statistical analysis of CD8+ T cell phenotypic marker expression based on whole blood multi-color flow-cytometry. The analysis comprises both unsupervised statistics (hClust and PCA) and supervised classification methods (Random forest, Adaboost algorithm, Decision tree learning and logistic regression) and allows to cluster patients by comparing multiple phenotypic markers expressed by CD8+ T cells. Our results reveal a global CD8+ T cell phenotypic signature in CLL patients that is significantly modified when compared to healthy donors. We also uncover a CD8+ T cell signature characteristic of patients evolving toward therapy within 6 months after phenotyping. The unbiased, not predetermined and multimodal approach highlights a prominent role of the memory compartment in the prognostic signature. The analysis also reveals that imbalance of the central/effector memory compartment in CD8+ T cells can occur irrespectively of the elapsed time after diagnosis. Taken together our results indicate that, in CLL patients, CD8+ T cell phenotype is imprinted by disease clinical progression and reveal that CD8+ T cell memory compartment alteration is not only a hallmark of CLL disease but also a signature of disease evolution toward the need for therapy.

Hydroxychloroquine as a novel therapeutic approach in mast cell activation diseases.

There is no therapeutic agent approved in cutaneous mastocytosis and mast cell activation syndrome. We report the efficacy of hydroxychloroquine in four patients with cutaneous mastocytosis (n = 2) and mast cell activation syndrome (n = 2). We show that this molecule reduces the long-term survival of primary human mast cells, interferes with lysosome function and leads to the accumulation of non-functional tryptase in the mast cell granules. Furthermore, hydroxychloroquine decreases the production of pro-inflammatory mediators.

The Wiskott-Aldrich Syndrome Protein Contributes to the Assembly of the LFA-1 Nanocluster Belt at the Lytic Synapse.

T lymphocyte cytotoxicity relies on a synaptic ring of lymphocyte function-associated antigen 1 (LFA-1), which permits polarized delivery of lytic granules. How LFA-1 organization is controlled by underlying actin cytoskeleton dynamics is poorly understood. Here, we explored the contribution of the actin cytoskeleton regulator WASP to the topography of LFA-1 using a combination of microscopy modalities. We uncover that the reduced cytotoxicity of Wiskott-Aldrich syndrome patient-derived CD8+ T lymphocytes lacking WASP is associated with reduced LFA-1 activation, unstable synapse, and delayed lethal hit. At the nanometric scale, WASP constrains high-affinity LFA-1 into dense nanoclusters located in actin meshwork interstices. At the cellular scale, WASP is required for the assembly of a radial belt composed of hundreds of LFA-1 nanoclusters and for lytic granule docking within this belt. Our study unravels the nanoscale topography of LFA-1 at the lytic synapse and identifies WASP as a molecule controlling individual LFA-1 cluster density and LFA-1 nanocluster belt integrity.

New roles and controls of mast cells.

Mast cells are innate immune cells implicated in immune surveillance and defense. They are filled with secretory granules where a vast array of molecules endowed with multiple biological activities are stored. The process of granule secretion, named degranulation, is a tightly controlled biological phenomenon that allows mast cells to rapidly and efficiently release bioactive mediators in response to extracellular stimuli. MC degranulation allows fighting pathogens, limiting envenomation and contributes to tissue homeostasis. However, it is also a potentially harmful response that plays a key role in the development of allergy and inflammatory diseases. Recent findings revealed that MC degranulation is a complex modular process that can be controlled at multiple levels. First, mast cells can decode different activation stimuli into two main patterns of degranulation that differently impact inflammatory responses. Second, mast cells in contact with antibody-opsonized cells or parasites form antibody-dependent degranulatory synapse for dedicated secretion and defense. Third, IL-33 fine-tunes FcR-mediated degranulation at the single cell level. Together these recent findings show how mast cells adapt their degranulation responses to environmental cues and highlight the remarkable functional plasticity of these cells.