Explainable machine learning for profiling the immunological synapse and functional characterization of therapeutic antibodies.

Therapeutic antibodies are widely used to treat severe diseases. Most of them alter immune cells and act within the immunological synapse; an essential cell-to-cell interaction to direct the humoral immune response. Although many antibody designs are generated and evaluated, a high-throughput tool for systematic antibody characterization and prediction of function is lacking. Here, we introduce the first comprehensive open-source framework, scifAI (single-cell imaging flow cytometry AI), for preprocessing, feature engineering, and explainable, predictive machine learning on imaging flow cytometry (IFC) data. Additionally, we generate the largest publicly available IFC dataset of the human immunological synapse containing over 2.8 million images. Using scifAI, we analyze class frequency and morphological changes under different immune stimulation. T cell cytokine production across multiple donors and therapeutic antibodies is quantitatively predicted in vitro, linking morphological features with function and demonstrating the potential to significantly impact antibody design. scifAI is universally applicable to IFC data. Given its modular architecture, it is straightforward to incorporate into existing workflows and analysis pipelines, e.g., for rapid antibody screening and functional characterization.

Actin' off: PD-1 suppresses F-actin rearrangement and degranulation at the immunological synapse.

Programmed cell death molecule 1 (PD-1) is a negative regulator of T cell activation; however, the mechanisms by which it acts are unclear. In this issue of Science Signaling, Paillon et al. show that PD-1 inhibits actin cytoskeletal rearrangements and associated effector responses in cytotoxic T cells.

PD-1 inhibits T cell actin remodeling at the immunological synapse independently of its signaling motifs.

Engagement of the receptor programmed cell death molecule 1 (PD-1) by its ligands PD-L1 and PD-L2 inhibits T cell-mediated immune responses. Blocking such signaling provides the clinical effects of PD-1-targeted immunotherapy. Here, we investigated the mechanisms underlying PD-1-mediated inhibition. Because dynamic actin remodeling is crucial for T cell functions, we characterized the effects of PD-1 engagement on actin remodeling at the immunological synapse, the interface between a T cell and an antigen-presenting cell (APC) or target cell. We used microscopy to analyze the formation of immunological synapses between PD-1+ Jurkat cells or primary human CD8+ cytotoxic T cells and APCs that presented T cell-activating antibodies and were either positive or negative for PD-L1. PD-1 binding to PD-L1 inhibited T cell spreading induced by antibody-mediated activation, which was characterized by the absence of the F-actin-dense distal lamellipodial network at the immunological synapse and the Arp2/3 complex, which mediates branched actin formation. PD-1-induced inhibition of actin remodeling also prevented the characteristic deformation of T cells that contact APCs and the release of cytotoxic granules. We showed that the effects of PD-1 on actin remodeling did not require its tyrosine-based signaling motifs, which are thought to mediate the co-inhibitory effects of PD-1. Our study highlights a previously unappreciated mechanism of PD-1-mediated suppression of T cell activity, which depends on the regulation of actin cytoskeleton dynamics in a signaling motif-independent manner.

Actin cytoskeleton remodeling at the cancer cell side of the immunological synapse: good, bad, or both?

Cytotoxic lymphocytes (CLs), specifically cytotoxic T lymphocytes and natural killer cells, are indispensable guardians of the immune system and orchestrate the recognition and elimination of cancer cells. Upon encountering a cancer cell, CLs establish a specialized cellular junction, known as the immunological synapse that stands as a pivotal determinant for effective cell killing. Extensive research has focused on the presynaptic side of the immunological synapse and elucidated the multiple functions of the CL actin cytoskeleton in synapse formation, organization, regulatory signaling, and lytic activity. In contrast, the postsynaptic (cancer cell) counterpart has remained relatively unexplored. Nevertheless, both indirect and direct evidence has begun to illuminate the significant and profound consequences of cytoskeletal changes within cancer cells on the outcome of the lytic immunological synapse. Here, we explore the understudied role of the cancer cell actin cytoskeleton in modulating the immune response within the immunological synapse. We shed light on the intricate interplay between actin dynamics and the evasion mechanisms employed by cancer cells, thus providing potential routes for future research and envisioning therapeutic interventions targeting the postsynaptic side of the immunological synapse in the realm of cancer immunotherapy. This review article highlights the importance of actin dynamics within the immunological synapse between cytotoxic lymphocytes and cancer cells focusing on the less-explored postsynaptic side of the synapse. It presents emerging evidence that actin dynamics in cancer cells can critically influence the outcome of cytotoxic lymphocyte interactions with cancer cells.

Impact of Mycobacterium tuberculosis Glycolipids on the CD4+ T Cell-Macrophage Immunological Synapse.

Mycobacterium tuberculosis cell-wall glycolipids such as mannosylated lipoarabinomannan (ManLAM) can inhibit murine CD4+ T cells by blocking TCR signaling. This results in suppression of IL-2 production, reduced T cell proliferation, and induction of CD4+ T cell anergy. This study extended these findings to the interaction between primary human CD4+ T cells and macrophages infected by mycobacteria. Exposure of human CD4+ T cells to ManLAM before activation resulted in loss of polyfunctionality, as measured by IL-2, IFN-γ, and TNF-α expression, and reduced CD25 expression. This was not associated with upregulation of inhibitory receptors CTLA-4, PD-1, TIM-3, and Lag-3. By confocal microscopy and imaging flow cytometry, ManLAM exposure reduced conjugate formation between macrophages and CD4+ T cells. ManLAM colocalized to the immunological synapse (IS) and reduced translocation of lymphocyte-specific protein tyrosine kinase (LCK) to the IS. When CD4+ T cells and Mycobacterium bovis BCG-infected monocytes were cocultured, ManLAM colocalized to CD4+ T cells, which formed fewer conjugates with infected monocytes. These results demonstrate that mycobacterial cell-wall glycolipids such as ManLAM can traffic from infected macrophages to disrupt productive IS formation and inhibit CD4+ T cell activation, contributing to immune evasion by M. tuberculosis.

Reprogramming dendritic cells through the immunological synapse: A two-way street.

Dendritic cells (DCs) bridge innate and adaptive immunity. Their main function is to present antigens to prime T cells and initiate and shape adaptive responses. Antigen presentation takes place through intimate contacts between the two cells, termed immune synapses (IS). During the formation of IS, information travels towards the T-cell side to induce and tune its activation; but it also travels in reverse via engagement of membrane receptors and within extracellular vesicles transferred to the DC. Such reverse information transfer and its consequences on DC fate have been largely neglected. Here, we review the events and effects of IS-mediated antigen presentation on DCs. In addition, we discuss novel technological advancements that enable monitoring DCs interactions with T lymphocytes, the main effects of DCs undergoing productive IS (postsynaptic DCs, or psDCs), and how reverse information transfer could be harnessed to modulate immune responses for therapeutic intervention.

End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network.

The organization of the mitochondrial network is relevant for the metabolic fate of T cells and their ability to respond to TCR stimulation. This arrangement depends on cytoskeleton dynamics in response to TCR and CD28 activation, which allows the polarization of the mitochondria through their change in shape, and their movement along the microtubules towards the immune synapse. This work focus on the role of End-binding protein 1 (EB1), a protein that regulates tubulin polymerization and has been previously identified as a regulator of intracellular transport of CD3-enriched vesicles. EB1-interferred cells showed defective intracellular organization and metabolic strength in activated T cells, pointing to a relevant connection of the cytoskeleton and metabolism in response to TCR stimulation, which leads to increased AICD. By unifying the organization of the tubulin cytoskeleton and mitochondria during CD4+ T cell activation, this work highlights the importance of this connection for critical cell asymmetry together with metabolic functions such as glycolysis, mitochondria respiration, and cell viability.

Live-cell imaging for analysis of the NK cell immunological synapse.

The immunological synapse (IS) between NK cells and cancer cells is instrumental for the initiation of tumor-specific cytotoxicity. Improper function of processes at the IS can lead to NK cell unresponsiveness, contributing to tumor immune escape. Critical steps at the IS include target cell recognition, conjugation of NK cell and cancer cell, cytotoxic granule convergence to the microtubule-organizing center (MTOC), granule polarization to the IS, and degranulation. Here, we describe confocal live-cell imaging methods for the analysis of these processes at the immunological synapse, with a focus on mechanisms of cancer cell resistance facilitating escape from NK cell cytotoxicity.

Metrics of 2D immunological synapses in human T cells via high-content confocal cell imaging.

Immunological synapses (IS) are the privileged site of complex information transfer between T cells and antigen presenting cells. IS are highly structured in terms of actin and tubulin cytoskeleton organization, receptor and proximal signal patterning, and intracellular organelle polarization. The magnitude and quality of T cell responses upon antigen recognition is dependent on IS molecular organization. For that reason, methods to precisely assess IS parameters are crucial to monitor T cell activation and function in health and disease, but also for T cell centered therapeutic intervention. Confocal and super-resolution microscopy approaches have allowed to characterize the complex structure of the T cell IS. However, those approaches suffer from a low-throughput and low-content format precluding multi-parametric classification of IS across large numbers of samples or stimulatory conditions. Here, we present a protocol of high-content confocal cell imaging in a 384-well plate format adapted to the unbiased analysis of primary T cells forming IS over pre-coated stimulatory molecules. The protocol focuses on the staining of F-actin, pericentrin and granzyme B in CD8+ T cells, but is transposable to other IS molecular markers and lymphocyte subsets. We discuss potential applications offered by the multi-parametric characterization of T cell IS in a high-throughput format.

Imaging PIP2 and BCR microclusters in B cell immunological synapse.

The humoral immune response is dependent on B cell activation and differentiation, which is typically triggered by the formation of immunological synapses at the interface between B cells and the antigen presenting surfaces. However, due to the highly dynamic and transient feature of immunological synapses, it has been difficult to capture and investigate the molecular events that occur within them. The planar lipids bilayer (PLB) supported antigen presenting surface combined with high-resolution high-speed total internal reflection fluorescence microscope (TIRFM) live cell imaging system has been proved to be a powerful tool that allows us to visualize the dynamic events in immunological synapse. In addition, the phospholipid phosphatidylinositol-(4,5)-biphosphate (PIP2) plays a unique role in B cell activation, and it is difficult to investigate the synaptic dynamics of PIP2 molecules. Hence, we describe here the general procedures for the utilization of a PLB based antigen presenting system combining TIRFM based imaging methods to visualize the spatial-temporal co-distribution of PIP2 and BCR microcluster within the B cell immunological synapse.

Activation kinetics of regulatory molecules during immunological synapse in T cells.

T cell activation through TCR stimulation leads to the formation of the immunological synapse (IS), a specialized adhesion organized between T lymphocytes and antigen presenting cells (APCs) in which a dynamic interaction among signaling molecules, the cytoskeleton and intracellular organelles achieves proper antigen-mediated stimulation and effector function. The kinetics of molecular reactions at the IS is essential to determine the quality of the response to the antigen stimulation. Herein, we describe methods based on biochemistry, flow cytometry and imaging in live and fixed cells to study the activation state and dynamics of regulatory molecules at the IS in the Jurkat T cell line CH7C17 and primary human and mouse CD4+ T lymphocytes stimulated by antigen presented by Raji and HOM2 B cell lines and human and mouse dendritic cells.

Immunological synapse-driven transfer of extracellular vesicle microRNAs in primary lymphocytes.

Cell-to-cell communication is necessary to orchestrate effective immune responses against disease-causing agents and in homeostasis. During immune synapsis, transfer of small extracellular vesicles that contain bioactive molecules, including microRNAs, occurs from the T lymphocyte to the antigen-presenting cell. In this chapter, we describe the methodology to identify and validate specific microRNAs shuttled from T lymphocytes to B cells upon immune synapse formation, and to analyze their functional impact on post-synaptic antigen-presenting cells.

Correlative light and electron microscopy to explore the lytic immunological synapse between natural killer cells and cancer cells.

Cytotoxic lymphocytes, such as natural killer (NK) cells and cytotoxic T cells, can recognize and kill tumor cells by establishing a highly specialized cell-cell contact called the immunological synapse. The formation and lytic activity of the immunological synapse are accompanied by local changes in the organization, dynamics and molecular composition of the cell membrane, as well as the polarization of various cellular components, such as the cytoskeleton, vesicles and organelles. Characterization and understanding of the molecular and cellular processes underlying immunological synapse formation and activity requires the combination of complementary types of information provided by different imaging modalities, the correlation of which can be difficult. Correlative light and electron microscopy (CLEM) allows for the accurate correlation of functional information provided by fluorescent light microscopy with ultrastructural features provided by high-resolution electron microscopy. In this chapter, we present a detailed protocol describing each step to generate cell-cell conjugates between NK cells and cancer cells, and to analyze these conjugates by CLEM using separate confocal laser-scanning and transmission electron microscopes.

Population dynamics of immunological synapse formation induced by bispecific T cell engagers predict clinical pharmacodynamics and treatment resistance.

Effector T cells need to form immunological synapses (IS) with recognized target cells to elicit cytolytic effects. Facilitating IS formation is the principal pharmacological action of most T cell-based cancer immunotherapies. However, the dynamics of IS formation at the cell population level, the primary driver of the pharmacodynamics of many cancer immunotherapies, remains poorly defined. Using classic immunotherapy CD3/CD19 bispecific T cell engager (BiTE) as our model system, we integrate experimental and theoretical approaches to investigate the population dynamics of IS formation and their relevance to clinical pharmacodynamics and treatment resistance. Our models produce experimentally consistent predictions when defining IS formation as a series of spatiotemporally coordinated events driven by molecular and cellular interactions. The models predict tumor-killing pharmacodynamics in patients and reveal trajectories of tumor evolution across anatomical sites under BiTE immunotherapy. Our models highlight the bone marrow as a potential sanctuary site permitting tumor evolution and antigen escape. The models also suggest that optimal dosing regimens are a function of tumor growth, CD19 expression, and patient T cell abundance, which confer adequate tumor control with reduced disease evolution. This work has implications for developing more effective T cell-based cancer immunotherapies.

Building the synapse engine to drive B lymphocyte function.

B cell receptor (BCR)-mediated antigen-specific recognition activates B lymphocytes and drives the humoral immune response. This enables the generation of antibody-producing plasma cells, the effector arm of the B cell immune response, and of memory B cells, which confer protection against additional encounters with antigen. B cells search for cognate antigen in the complex cellular microarchitecture of secondary lymphoid organs, where antigens are captured and exposed on the surface of different immune cells. While scanning the cell network, the BCR can be stimulated by a specific antigen and elicit the establishment of the immune synapse with the antigen-presenting cell. At the immune synapse, an integrin-enriched supramolecular domain is assembled at the periphery of the B cell contact with the antigen-presenting cell, ensuring a stable and long-lasting interaction. The coordinated action of the actomyosin cytoskeleton and the microtubule network in the inner B cell space provides a structural framework that integrates signaling events and antigen uptake through the generation of traction forces and organelle polarization. Accordingly, the B cell immune synapse can be envisioned as a temporal engine that drives the molecular mechanisms needed for successful B cell activation. Here, I review different aspects of the B cell synapse engine and provide insights into other aspects poorly known or virtually unexplored.

Curving out a new path: CD28/B7 cis interactions.

Important signaling events at the immunological synapse have increasingly been linked to cis interactions between receptors on T cells. In this issue of Immunity, Zhao et al.1 implicate cis CD28/B7 interactions facilitated by curved membrane invaginations in boosting tumor immunity.

Endothelial mechanical stretch regulates the immunological synapse interface of renal endothelial cells in a sex-dependent manner.

Increased mechanical endothelial cell stretch contributes to the development of numerous cardiovascular and renal pathologies. Recent studies have shone a light on the importance of sex-dependent inflammation in the pathogenesis of renal disease states. The endothelium plays an intimate and critical role in the orchestration of immune cell activation through upregulation of adhesion molecules and secretion of cytokines and chemokines. While endothelial cells are not recognized as professional antigen-presenting cells, in response to cytokine stimulation, endothelial cells can express both major histocompatibility complex (MHC) I and MHC II. MHCs are essential to forming a part of the immunological synapse interface during antigen presentation to adaptive immune cells. Whether MHC I and II are increased under increased mechanical stretch is unknown. Due to hypertension being multifactorial, we hypothesized that increased mechanical endothelial stretch promotes the regulation of MHCs and key costimulatory proteins on mouse renal endothelial cells (MRECs) in a stretch-dependent manner. MRECs derived from both sexes underwent 5%, 10%, or 15% uniaxial cyclical stretch, and immunological synapse interface proteins were determined by immunofluorescence microscopy, immunoblot analysis, and RNA sequencing. We found that increased endothelial mechanical stretch conditions promoted downregulation of MHC I in male MRECs but upregulation in female MRECs. Moreover, MHC II was upregulated by mechanical stretch in both male and female MRECs, whereas CD86 and CD70 were regulated in a sex-dependent manner. By bulk RNA sequencing, we found that increased mechanical endothelial cell stretch promoted differential gene expression of key antigen processing and presentation genes in female MRECs, demonstrating that females have upregulation of key antigen presentation pathways. Taken together, our data demonstrate that mechanical endothelial stretch regulates endothelial activation and immunological synapse interface formation in renal endothelial cells in a sex-dependent manner.NEW & NOTEWORTHY Endothelial cells contribute to the development of renal inflammation and have the unique ability to express antigen presentation proteins. Whether increased endothelial mechanical stretch regulates immunological synapse interface proteins remains unknown. We found that antigen presentation proteins and costimulatory proteins on renal endothelial cells are modulated by mechanical stretch in a sex-dependent manner. Our data provide novel insights into the sex-dependent ability of renal endothelial cells to present antigens in response to endothelial mechanical stimuli.

LFA-1 Activation in T-Cell Migration and Immunological Synapse Formation.

Integrin LFA-1 plays a critical role in T-cell migration and in the formation of immunological synapses. LFA-1 functions through interacting with its ligands with differing affinities: low, intermediate, and high. Most prior research has studied how LFA-1 in the high-affinity state regulates the trafficking and functions of T cells. LFA-1 is also presented in the intermediate-affinity state on T cells, however, the signaling to activate LFA-1 to the intermediate-affinity state and the role of LFA-1 in this affinity state both remain largely elusive. This review briefly summarizes the activation and roles of LFA-1 with varied ligand-binding affinities in the regulation of T-cell migration and immunological synapse formation.

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.