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.

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.

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.

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.

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.

Human regulatory T cells inhibit polarization of T helper cells toward antigen-presenting cells via a TGF-beta-dependent mechanism.

The molecular mechanisms used by regulatory T cells (Treg) to inhibit the effector phase of adaptive immune responses are still elusive. In the present work, we investigated the possibility that Treg may interfere with a basic biological function of T helper cells (T(H)): polarization of secretory machinery for dedicated help delivery. To address this question, we visualized by confocal microscopy different parameters of activation in T(H) and Treg cells interacting simultaneously with individual antigen-presenting cells (APC). Our results show that, although productive TCR engagement in T(H)/APC conjugates was unaffected by the presence of adjacent Treg, the reorientation of T(H) secretory machinery toward APC was strongly inhibited. Blocking TGF-beta completely reverted Treg induced inhibition of T(H) polarization. Our results identify a previously undescribed mechanism by which Treg inhibit effector T cells. TGF-beta produced by adjacent Treg interferes with polarization of T(H) secretory machinery toward APC, thus affecting a crucial step of T(H)-mediated amplification of the immune response.

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.

Visualizing CTL/melanoma cell interactions: multiple hits must be delivered for tumour cell annihilation.

It is well established that cytotoxic T lymphocytes (CTL) can kill target cells offering a very small number of specific peptide/MHC complexes (pMHC). It is also known that lethal hit delivery is a very rapid response that occurs within a few minutes after cell-cell contact. Whether cytotoxicity is efficient and rapid in the context of CTL interaction with target cells derived from solid tumours is still elusive. We addressed this question by visualizing the dynamics of human CTL interaction with melanoma cells and their efficiency in eliciting cytotoxicity. Our results show that in spite of CTL activation to lethal hit delivery, killing of melanoma cells is not efficient. Time-lapse microscopy experiments demonstrate that individual CTL rapidly polarize their lytic machinery towards target cells, yet the apoptotic process in melanoma cells is defective or 'delayed' as compared to conventional targets. These results indicate that although CTL activation to lethal hit delivery can be viewed as a 'digital' phenomenon rapidly triggered by a few ligands, melanoma cell annihilation is an 'analogue' response requiring multiple hits and prolonged contact time.

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.

Analysis of peptide/MHC-induced TCR downregulation: deciphering the triggering kinetics.

The interaction of T-lymphocytes with antigen-presenting cells displaying a small number of specific peptide/major histocompatibility complexes results in the downregulation of a large number of T-cell receptors (TCR), suggesting serial TCR triggering. However, the details of TCR downregulation are controversial. In particular, the level of comodulation of nonengaged TCR reported by different authors ranges from essentially none to considerable levels. Here, we address this controversy using complementary experimental and mathematical techniques. We find that TCR downregulation is very rapid during the first 2-4 min after T-cell antigen-presenting cells contact formation. After this phase, TCR downregulation proceeds at a relatively slow rate. Statistical and computational analyses show that this pronounced change in downregulation kinetics is compatible with the notion of initial serial triggering of clustered TCR followed by serial triggering of individual TCR. We further propose a compatible mechanism for concurrent triggering of multiple TCR by a single peptide/major histocompatibility complex. We provide a unified picture of productive TCR engagement and downregulation in which TCR triggering characteristics evolve from an initial cooperative phase to a sustained phase of signal accumulation.

Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse.

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients.

The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells.

Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells.

T-cell activation is accompanied by an ubiquitination process occurring at the immunological synapse.

The immunological synapse (IS) is a specialized signaling area formed at the contact site between T-cells and antigen-presenting cells (APC), where sustained engagement and signaling of TCR and accessory molecules occur. A key feature of T-cell antigen recognition is that the process of TCR/peptide-MHC interaction is self-limited by the internalization and degradation of triggered TCR and recruited signaling components. The mechanism of signaling component degradation involves their ubiquitination and targeting for degradation. Yet, the relationship between the ubiquitination process and TCR signaling as well as the cellular localization of TCR-induced ubiquitination are still elusive. In the present work, we visualize for the first time ubiquitination at the TCR signaling area. We show an enrichment of ubiquitin staining in TCR/CD3 caps in T-lymphocytes stimulated by anti-CD3 antibodies. Remarkably, we also show the recruitment of the ubiquitin ligase Cbl-b and a significant ubiquitination at the immunological synapse in antigen-stimulated T-cells. Our results identify the immunological synapse as the cellular area where TCR-induced protein ubiquitination occurs. They imply that the synapse is a specialized site where the activation process is not only triggered, but also controlled via ubiquitination of signaling actors.

Individual Human Cytotoxic T Lymphocytes Exhibit Intraclonal Heterogeneity during Sustained Killing.

The killing of antigen-bearing cells by clonal populations of cytotoxic T lymphocytes (CTLs) is thought to be a rapid phenomenon executed uniformly by individual CTLs. We combined bulk and single-CTL killing assays over a prolonged time period to provide the killing statistics of clonal human CTLs against an excess of target cells. Our data reveal efficiency in sustained killing at the population level, which relied on a highly heterogeneous multiple killing performance at the individual level. Although intraclonal functional heterogeneity was a stable trait in clonal populations, it was reset in the progeny of individual CTLs. In-depth mathematical analysis of individual CTL killing data revealed a substantial proportion of high-rate killer CTLs with burst killing activity. Importantly, such activity was delayed and required activation with strong antigenic stimulation. Our study implies that functional heterogeneity allows CTL populations to calibrate prolonged cytotoxic activity to the size of target cell populations.

A biased competition theory of cytotoxic T lymphocyte interaction with tumor nodules.

The dynamics of the interaction between Cytotoxic T Lymphocytes (CTL) and tumor cells has been addressed in depth, in particular using numerical simulations. However, stochastic mathematical models that take into account the competitive interaction between CTL and tumors undergoing immunoediting, a process of tumor cell escape from immunesurveillance, are presently missing. Here, we introduce a stochastic dynamical particle interaction model based on experimentally measured parameters that allows to describe CTL function during immunoediting. The model describes the competitive interaction between CTL and melanoma cell nodules and allows temporal and two-dimensional spatial progression. The model is designed to provide probabilistic estimates of tumor eradication through numerical simulations in which tunable parameters influencing CTL efficacy against a tumor nodule undergoing immunoediting are tested. Our model shows that the rate of CTL/tumor nodule productive collisions during the initial time of interaction determines the success of CTL in tumor eradication. It allows efficient cytotoxic function before the tumor cells acquire a substantial resistance to CTL attack, due to mutations stochastically occurring during cell division. Interestingly, a bias in CTL motility inducing a progressive attraction towards a few scout CTL, which have detected the nodule enhances early productive collisions and tumor eradication. Taken together, our results are compatible with a biased competition theory of CTL function in which CTL efficacy against a tumor nodule undergoing immunoediting is strongly dependent on guidance of CTL trajectories by scout siblings. They highlight unprecedented aspects of immune cell behavior that might inspire new CTL-based therapeutic strategies against tumors.

UV-C light induces raft-associated acid sphingomyelinase and JNK activation and translocation independently on a nuclear signal.

The initiation of UV light-induced signaling in mammalian cells is largely considered to be subsequent to DNA damage. Several studies have also described ceramide (CER), a lipid second messenger, as a major contributor in mediating UV light-induced c-Jun N-terminal kinase (JNK) activation and cell death. It is demonstrated here that UV-C light irradiation of U937 cells results in the activation and translocation of a Zn2+-independent acid sphingomyelinase, leading to CER accumulation in raft microdomains. These CER-enriched rafts aggregate and play a functional role in JNK activation. The observation that UV-C light also induced CER generation and the externalization of acid sphingomyelinase and JNK in human platelets conclusively rules out the involvement of a nuclear signal generated by DNA damage in the initiation of a UV light response, which is generated at the plasma membrane.

Immunological synapses are versatile structures enabling selective T cell polarization.

Helper T cells discriminate among different antigen-presenting cells to provide their help in a selective fashion. The molecular mechanisms leading to this exquisite selectivity are still elusive. Here, we demonstrate that immunological synapses are dynamic and adaptable structures allowing T cells to communicate with multiple cells. We show that T cells can form simultaneous immunological synapses with cells presenting different levels of antigenic ligands but eventually polarize toward the strongest stimulus. Remarkably, living T cells form discrete foci of signal transduction of different intensities during the interaction with different antigen-presenting cells and rapidly relocate TCR and Golgi apparatus toward the cell providing the strongest stimulus. Our results illustrate that, although T cell activation requires sustained signaling, T cells are capable of rapid synapse remodeling and swift polarization responses. The combination of sustained signaling with preferential and rapid polarization provides a mechanism for the high sensitivity and selectivity of T cell responses.

CD4+ T cells downregulate Bcl-2 in germinal centers.

Germinal centers (GCs) are the main site of T cell-dependent antibody responses. Upon antigen challenge, GCs comprise mostly B cells undergoing proliferation, somatic hypermutation and antigen-affinity selection. GC B cells down-modulate the expression of Bcl-2 protein and are highly sensitive to apoptosis to eliminate autoreactive or low-affinity cells. Bcl-2 is still expressed in a few GC cells, whose identity remains unclear. To address this issue, we examined by confocal microscopy the expression of Bcl-2 by different GC lymphocyte subsets in hyperplastic tonsils. We found that the vast majority of Bcl-2(+) GC cells are T lymphocytes. Conversely, while in the mantle zone and in the interfollicular areas T cells are almost exclusively Bcl-2(+), in the GC, most T lymphocytes are Bcl-2(-). In addition, most of the CD4(+) GC T cells are Bcl-2(-), while nearly 100% of the CD8(+) GC T cells are Bcl-2(+). The Bcl-2 downregulation by both B and CD4(+) T GC cells supports the concept that these two subsets may undergo a selection process in this microenvironment.