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.

Clonal Deletion Prunes but Does Not Eliminate Self-Specific αß CD8(+) T Lymphocytes.

It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. We found that self-peptide MHC-specific CD8(+) T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome-encoded SMCY antigen, self-specific T cells exhibited only a 3-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors.

A single peptide-major histocompatibility complex ligand triggers digital cytokine secretion in CD4(+) T cells.

We have developed a single-molecule imaging technique that uses quantum-dot-labeled peptide-major histocompatibility complex (pMHC) ligands to study CD4(+) T cell functional sensitivity. We found that naive T cells, T cell blasts, and memory T cells could all be triggered by a single pMHC to secrete tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) cytokines with a rate of ∼1,000, ∼10,000, and ∼10,000 molecules/min, respectively, and that additional pMHCs did not augment secretion, indicating a digital response pattern. We also found that a single pMHC localized to the immunological synapse induced the slow formation of a long-lasting T cell receptor (TCR) cluster, consistent with a serial engagement mechanism. These data show that scaling up CD4(+) T cell cytokine responses involves increasingly efficient T cell recruitment rather than greater cytokine production per cell.

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.

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.

IL-33 fine tunes mast cell degranulation and chemokine production at the single-cell level.

BACKGROUND: Mast cells are versatile key components of allergy and inflammation known to respond to both innate and adaptive immunologic stimuli. However, the response of individual mast cells to cumulative stimuli remains poorly understood. OBJECTIVES: We sought to dissect mast cell responses at the single-cell level and their potentiation by IL-33. METHODS: We monitored mast cell degranulation in real time by exploiting the capacity of fluorochrome-labeled avidin to stain degranulating cells. During the degranulation process, the granule matrix is externalized and immediately bound by fluorochrome-labeled avidin present in the culture medium. The degranulation process is monitored by using either time-lapse microscopy or fluorescence-activated cell sorting analysis. RESULTS: Single-cell analysis revealed a strong heterogeneity of individual mast cell degranulation responses. We observed that the number of degranulating mast cells was graded according to the FcεRI stimulation strength, whereas the magnitude of individual mast cell degranulation remained unchanged, suggesting an all-or-none response of mast cells after FcεRI triggering. IL-33 pretreatment increased not only the number of degranulating and chemokine-producing mast cells but also the magnitude of individual mast cell degranulation and chemokine production. CONCLUSION: We illustrate the effect of IL-33 on mast cell biology at the single-cell level by showing that IL-33 potentiates IgE-mediated mast cell responses by both increasing the number of responding cells and enhancing the responses of individual mast cells.

Immunological synapse: center of attention again.

The functional role of molecular clustering in the center of the immunological synapse is controversial. In this issue of Immunity, Cemerski et al. (2008) report that the synapse center can serve as a major site of sustained signal transduction.

An initial and rapid step of lytic granule secretion precedes microtubule organizing center polarization at the cytotoxic T lymphocyte/target cell synapse.

It is presently assumed that lethal hit delivery by cytotoxic T lymphocytes (CTLs) is mechanistically linked to centrosome polarization toward target cells, leading to dedicated release of lytic granules within a confined secretory domain. Here we provide three lines of evidence showing that this mechanism might not apply as a general paradigm for lethal hit delivery. First, in CTLs stimulated with immobilized peptide-MHC complexes, lytic granules and microtubule organizing center localization into synaptic areas are spatio-temporally dissociated, as detected by total internal reflection fluorescence microscopy. Second, in many CTL/target cell conjugates, lytic granule secretion precedes microtubule polarization and can be detected during the first minute after cell-cell contact. Third, inhibition of microtubule organizing center and centrosome polarization impairs neither lytic granule release at the CTL synapse nor killing efficiency. Our results broaden current views of CTL biology by revealing an extremely rapid step of lytic granule secretion and by showing that microtubule organizing center polarization is dispensable for efficient lethal hit delivery.

Human mast cells drive memory CD4+ T cells toward an inflammatory IL-22+ phenotype.

BACKGROUND: Mast cells are key components of the skin microenvironment in psoriasis, yet their functional role in this T-cell-mediated inflammatory disorder remains to be elucidated. OBJECTIVE: To define the impact of T-cell/mast-cell cognate interactions on the cytokines produced by TH cells. METHODS: We used human primary mast cells and effector/memory CD4(+) T cells for in vitro coculture experiments, and we analyzed TH cells responses by using cytometry. CD4(+) T-cell/mast-cell conjugates in skin lesions from patients with psoriasis were analyzed by using 3-color immunohistochemistry and confocal microscopy. RESULTS: We show that IFN-γ-primed human mast cells formed productive immunologic synapses with antigen-experienced CD4(+) T cells. These interactions promoted the generation of TH22 and IL-22/IFN-γ-producing TH cells from the circulating memory CD4(+) T-cell pool via a TNF-α/IL-6-dependent mechanism. An analysis of human psoriatic skin biopsies showed a rich infiltrate of IL-22(+)CD4(+) T cells frequently found in contact with mast cells. Moreover, most of these mast-cell-conjugated lymphocytes coexpressed IFN-γ, suggesting that IL-22(+)IFN-γ(+) CD4(+) T cells are generated in vivo on interaction with mast cells. CONCLUSIONS: Our findings identify human mast cells as functional partners of TH cells, shaping their responses toward IL-22 production.

Alternative activation of mast cells by CD4+ T helper cells.

Effector CD4+ T lymphocytes (Teff) infiltrate sites of inflammation and orchestrate the immune response by instructing local leukocytes. Mast cells (MCs) are tissue sentinel cells strategically located near blood vessels and T cell rich areas. MC/Teff cells interactions shape Teff cell responses but in turn, Teff cell action on MC is still poorly understood. Here, we analyzed the human MC/Teff cells interplay through both the application of RNAseq and functional assays. We showed that activated Teff cells induce a specific transcriptomic program in MCs including production of both inflammatory cytokines and chemokines, prostaglandin, and a FcεRI-dependent degranulation facilitation thereby driving them toward an inflammatory phenotype. Moreover, Teff cells induce in MCs the capacity to interact with CD4+ T cell through a wide-range of dedicated soluble and membrane ligands and to play the role of antigen presenting cells (APCs).

Distribution, function, and prognostic value of cytotoxic T lymphocytes in follicular lymphoma: a 3-D tissue-imaging study.

CD8+ CTLs are thought to play a role in the control of follicular lymphoma (FL). Yet, the link between CTL tissue distribution, activation status, ability to kill FL cells in vivo, and disease progression is still elusive. Pretreatment lymph nodes from FL patients were analyzed by IHC (n = 80) or by 3-color confocal microscopy (n = 10). IHC revealed a rich infiltrate of CD8+ granzyme B+ (GrzB) cells in FL interfollicular spaces. Accordingly, confocal microscopy showed an increased number of CD3+CD8+GrzB+ CTLs and a brighter GrzB staining in individual CTL in FL samples compared with reactive lymph nodes. CTLs did not penetrate tumor nodules. In 3-dimensional (3-D) image reconstructions, CTLs were detected at the FL follicle border where they formed lytic synapse-like structures with FL B cells and with apoptotic cells, suggesting an in situ cytotoxic function. Finally, although GrzB expression in CTLs did not correlate with risk factors, high GrzB content correlated with prolonged progression free-survival (PFS) after rituximab-combined chemotherapy. Our results show the recruitment of armed CTLs with a tumor-controlling potential into FL lymph nodes and suggest that CTL-associated GrzB expression could influence PFS in FL patients having received rituximab-combined chemotherapy.

Reciprocal polarization of T and B cells at the immunological synapse.

Cognate interactions between T and B lymphocytes lead to the formation of the immunological synapse (IS) where bidirectional activation signals are exchanged. Although the molecular architecture and the function of the IS have been studied extensively on the T cell side, little is known about events occurring during synapse formation in Ag-presenting B cells. We investigated the impact of BCR and TLR signaling on human B cell activation and on the T and B cell side of the IS. On the T cell side, we observed that T cells polarized toward both naive and previously activated B cells. Nevertheless, when T cells interacted with different B cells simultaneously, T cells selectively polarized their secretory machinery toward preactivated B cells. Furthermore, both naive and preactivated B cells reoriented their microtubule-organizing center toward the synaptic T cell during cognate interactions. This phenomenon was rapid and not dependent on T cell secretory activity. Interestingly, not only the microtubule-organizing center but also the Golgi apparatus and Lamp-3(+) and MHC class II(+) vesicles all repositioned beneath the IS, suggesting that the entire endocytic/exocytic B cell compartment was reoriented toward the T cell. Taken together, our results show that the B cell activation status fine-tunes T cell polarization responses and reveal the capacity of naive and activated B cells to polarize toward T cells during cognate interactions.

T-cell antagonism by short half-life pMHC ligands can be mediated by an efficient trapping of T-cell polarization toward the APC.

T-cell activation results from productive T-cell receptor (TCR) engagement by a cognate peptide-MHC (pMHC) complex on the antigen presenting cell (APC) surface, a process leading to the polarization of the T-cell secretory machinery toward the APC interface. We have previously shown that the half-life of the TCR/pMHC interaction and the density of pMHC on the APC are two parameters determining T-cell activation. However, whether the half-life of the TCR/pMHC interaction can modulate the efficiency of T-cell secretory machinery polarization toward an APC still remains unclear. Here, by using altered peptide ligands conferring different half-lives to the TCR/pMHC interaction, we have tested how this parameter can control T-cell polarization. We observed that only TCR/pMHC interactions with intermediate half-lives can promote the assembly of synapses that lead to T-cell activation. Strikingly, intermediate half-life interactions can be competed out by short half-life interactions, which can efficiently promote T-cell polarization and antagonize T-cell activation that was induced by activating intermediate half-life interactions. However, short TCR/pMHC interactions fail at promoting phosphorylation of signaling molecules at the T-cell-APC contact interface, which are needed for T-cell activation. Our data suggest that although intermediate half-life pMHC ligands promote assembly of activating synapses, this process can be inhibited by short half-life antagonistic pMHC ligands, which promote the assembly of non activating synapses.

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.

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.

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.

Wiskott-Aldrich syndrome protein controls antigen-presenting cell-driven CD4+ T-cell motility by regulating adhesion to intercellular adhesion molecule-1.

T-cell scanning for antigen-presenting cells (APC) is a finely tuned process. Whereas non-cognate APC trigger T-cell motility via chemokines and intercellular adhesion molecule-1 (ICAM-1), cognate APC deliver a stop signal resulting from antigen recognition. We tested in vitro the contribution of the actin cytoskeleton regulator Wiskott-Aldrich syndrome protein (WASP) to the scanning activity of primary human CD4(+)  T cells. WASP knock-down resulted in increased T-cell motility upon encounter with non-cognate dendritic cells or B cells and reduced capacity to stop following antigen recognition. The high motility of WASP-deficient T cells was accompanied by a diminished ability to round up and to stabilize pauses. WASP-deficient T cells migrated in a normal proportion towards CXCL12, CCL19 and CCL21, but displayed an increased adhesion and elongation on ICAM-1. The elongated morphology of WASP-deficient T cells was related to a reduced confinement of high-affinity lymphocyte function-associated antigen 1 to the mid-cell zone. Our data therefore indicate that WASP controls CD4(+) T-cell motility upon APC encounter by regulating lymphocyte function-associated antigen 1 spatial distribution.

Activation of the ancestral polarity regulator protein kinase C zeta at the immunological synapse drives polarization of Th cell secretory machinery toward APCs.

A key feature in T lymphocyte biology is that Th cells rapidly polarize their secretory machinery toward cognate APCs. The molecular mechanisms of these dynamic Th cell responses and their impact on APC biology remain to be elucidated. In this study, we demonstrate that protein kinase Czeta (PKCzeta) is rapidly activated at the immunological synapse (IS) in human Th cells interacting with cognate dendritic cells (DCs) and that a functional PKCzeta is required for the polarization of Th cell secretory machinery toward DCs. We also show that PKCzeta-dependent Th cell polarization allows dedicated delivery of IFN-gamma and CD40L at the IS and is required for the activation of cognate DCs to IL-12 production. PKCzeta synaptic activation is a low-threshold phenomenon and, in Th cells interacting with multiple DCs, selectively occurs at the IS formed with the DCs offering the strongest stimulus leading to dedicated Th cell polarization. Our results identify the PKCzeta signaling pathway as a key component of the Th cell polarization machinery and provide a molecular basis for T cell-dedicated activation of cognate DCs.

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.