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Modulating cholesterol metabolism can improve CD8 + T-cell-mediated immunity against tumours; genetic or pharmacological inhibition of the cholesterol esterification enzyme ACAT1 led to higher plasma membrane cholesterol levels, better T-cell receptor clustering and signalling, improved immunological synapse maturation, and enhanced antitumour activity in mice. T cell antitumour activity boosted This study reports a new approach to cancer immunotherapy through the modulation of T cell cholesterol metabolism. Chenqi Xu and colleagues demonstrate that inhibition of the cellular cholesterol esterification pathway in mice, either by genetic ablation or by pharmacological inhibition of acetyl-CoA acetyltransferase 1 (ACAT1) and ACAT2, increases plasma membrane cholesterol levels, T-cell receptor clustering and signalling, and significantly potentiates the antitumour response of CD8 + T cells in mice. To test the potential of ACAT1 as a drug target for cancer immunotherapy, the authors treated melanoma-bearing mice with avasimibe, an ACAT inhibitor that has been used to treat atherosclerosis in clinical trials. An antitumour effect was observed and a combination of avasimibe and anti-PD-1 antibody was more effective than either alone. CD8 + T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment 1 , 2 , 3 , 4 . Reactivating the cytotoxicity of CD8 + T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8 + T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme 5 , led to potentiated effector function and enhanced proliferation of CD8 + but not CD4 + T cells. This is due to the increase in the plasma membrane cholesterol level of CD8 + T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8 + T cells were better than wild-type CD8 + T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile 6 , 7 , to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

Interactions between receptors and ligands on immune cells are visualized in vivo and in vitro using an enzyme-tagged ligand that, when cells interact, leaves behind a detectable label on the receptor-expressing cell. LIPSTIC for immune cell interactions Gabriel Victora and colleagues describe a new approach to studying cell–cell interactions in the immune system. The method, which they called Labelling Immune Partnerships by SorTagging Intercellular Contacts (LIPSTIC), relies on chemical labelling (tagging) of genetically modified receptor–ligand pairs, a process mediated by the bacterial enzyme sortase. The history of ligand–receptor interactions is revealed by the presence of reporter tags that can be detected by flow cytometry or microscopy. Using this technique, the authors observe unanticipated CD40–CD40L interactions between T cells and dendritic cells at the non-cognate stage, when the T cell receptor is no longer engaged in the interaction. The method can potentially be applied to other in vitro and in vivo systems. Interactions between different cell types are essential for multiple biological processes, including immunity, embryonic development and neuronal signalling. Although the dynamics of cell–cell interactions can be monitored in vivo by intravital microscopy 1 , this approach does not provide any information on the receptors and ligands involved or enable the isolation of interacting cells for downstream analysis. Here we describe a complementary approach that uses bacterial sortase A-mediated cell labelling across synapses of immune cells to identify receptor–ligand interactions between cells in living mice, by generating a signal that can subsequently be detected ex vivo by flow cytometry. We call this approach for the labelling of ‘kiss-and-run’ interactions between immune cells ‘Labelling Immune Partnerships by SorTagging Intercellular Contacts’ (LIPSTIC). Using LIPSTIC, we show that interactions between dendritic cells and CD4 + T cells during T-cell priming in vivo occur in two distinct modalities: an early, cognate stage, during which CD40–CD40L interactions occur specifically between T cells and antigen-loaded dendritic cells; and a later, non-cognate stage during which these interactions no longer require prior engagement of the T-cell receptor. Therefore, LIPSTIC enables the direct measurement of dynamic cell–cell interactions both in vitro and in vivo . Given its flexibility for use with different receptor–ligand pairs and a range of detectable labels, we expect that this approach will be of use to any field of biology requiring quantification of intercellular communication.

Cancer-associated fibroblasts (CAFs) have emerged as a dominant non-hematopoietic cell population in the tumour microenvironment, serving diverse functions in tumour progression. However, the mechanisms via which CAFs influence the anti-tumour immunity remain poorly understood. Here, using multiple tumour models and biopsies from cancer patients, we report that α-SMA + CAFs can form immunological synapses with Foxp3 + regulatory T cells (Tregs) in tumours. Notably, α-SMA + CAFs can phagocytose and process tumour antigens and exhibit a tolerogenic phenotype which instructs movement arrest, activation and proliferation in Tregs in an antigen-specific manner. Moreover, α-SMA + CAFs display double-membrane structures resembling autophagosomes in their cytoplasm. Single-cell transcriptomic data showed an enrichment in autophagy and antigen processing/presentation pathways in α-SMA-expressing CAF clusters. Conditional knockout of Atg5 in α-SMA + CAFs promoted inflammatory re-programming in CAFs, reduced Treg cell infiltration and attenuated tumour development. Overall, our findings reveal an immunosuppressive mechanism entailing the formation of synapses between α-SMA + CAFs and Tregs in an autophagy-dependent manner. Cancer-associated fibroblasts (CAFs) are a predominant stromal cell population in the tumour microenvironment. Here, the authors demonstrate that αSMA + CAFs can form an immunological synapse with regulatory T cells (Tregs) in tumours, which results in Treg activation and expansion in a process that is antigen- and autophagy-dependent.

Activated Vγ9Vδ2 (γδ2) T lymphocytes that sense parasite-produced phosphoantigens are expanded in Plasmodium falciparum– infected patients. Although previous studies suggested that γδ2 T cells help control erythrocytic malaria, whether γδ2 T cells recognize infected red blood cells (iRBCs) was uncertain. Here we show that iRBCs stained for the phosphoantigen sensor butyrophilin 3A1 (BTN3A1). γδ2 T cells formed immune synapses and lysed iRBCs in a contact, phosphoantigen, BTN3A1 and degranulation-dependent manner, killing intracellular parasites. Granulysin released into the synapse lysed iRBCs and delivered death-inducing granzymes to the parasite. All intra-erythrocytic parasites were susceptible, but schizonts were most sensitive. A second protective γδ2 T cell mechanism was identified. In the presence of patient serum, γδ2 T cells phagocytosed and degraded opsonized iRBCs in a CD16-dependent manner, decreasing parasite multiplication. Thus, γδ2 T cells have two ways to control blood-stage malaria–γδ T cell antigen receptor (TCR)-mediated degranulation and phagocytosis of antibody-coated iRBCs. Junqueira et al. show that human γδ T cells control erythrocytic Plasmodium falciparum infection by multiple mechanisms: antibody-dependent phagocytosis, cytotoxic-granule-mediated erythrocyte lysis and direct parasite killing.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Clarification of the cytotoxic function of T cells is crucial for understanding human immune responses and immunotherapy procedures. Here, we report a high-throughput Bessel oblique plane microscopy (HBOPM) platform capable of 3D live imaging and phenotyping of chimeric antigen receptor (CAR)-modified T-cell cytotoxicity against cancer cells. The HBOPM platform has the following characteristics: an isotropic subcellular resolution of 320 nm, large-scale scouting over 400 interacting cell pairs, long-term observation across 5 hours, and quantitative analysis of the Terabyte-scale 3D, multichannel, time-lapse image datasets. Using this advanced microscopy platform, several key subcellular events in CAR-T cells are captured and comprehensively analyzed; these events include the instantaneous formation of immune synapses and the sustained changes in the microtubing morphology. Furthermore, we identify the actin retrograde flow speed, the actin depletion coefficient, the microtubule polarization and the contact area of the CAR-T/target cell conjugates as essential parameters strongly correlated with CAR-T-cell cytotoxic function. Our approach will be useful for establishing criteria for quantifying T-cell function in individual patients for all T-cell-based immunotherapies. Fluorescence microscopy can be exploited to visualize and study T cell mediated cytotoxicity. Here the authors describe a high-throughput Bessel oblique plane microscopy platform for 3D imaging and phenotyping of cytotoxic CAR-T cells in co-culture with cancer cells.

The advent of immunotherapy, especially checkpoint inhibitor-based immunotherapy, has provided novel and powerful weapons against cancer. Because only a subset of cancer patients exhibit durable responses, further exploration of the mechanisms underlying the resistance to immunotherapy in the bulk of cancer patients is merited. Such efforts may help to identify which patients could benefit from immune checkpoint blockade. Given the existence of a great number of pathways by which cancer can escape immune surveillance, and the complexity of tumor-immune system interaction, development of various combination therapies, including those that combine with conventional therapies, would be necessary. In this review, we summarize the current understanding of the mechanisms by which resistance to checkpoint blockade immunotherapy occurs, and outline how actionable combination strategies may be derived to improve clinical outcomes for patients.

Mechanical forces acting on ligand-engaged T-cell receptors (TCR) have previously been implicated in T-cell antigen recognition and ligand discrimination, yet their magnitude, frequency, and impact remain unclear. Here, we quantitatively assess forces across various TCR:pMHC pairs with different bond lifetimes at single-molecule resolution, both before and during T-cell activation, on platforms that either include or exclude tangential force registration. For this purpose, we use glass-supported lipid bilayers presenting pMHC conjugated to a molecular force sensor unit at its base, adhesion factors and costimulatory molecules to the approaching T-cells. Our results imply that CD4 + T-cell TCRs experience significantly lower forces than previously estimated, with only a small fraction of ligand-engaged TCRs being subjected to these forces during antigen scanning. These rare and minute mechanical forces do not impact the global lifetime distribution of the TCR:ligand bond. We propose that the immunological synapse is created as biophysically stable environment to prevent pulling forces from disturbing antigen recognition. Mechanical forces at the immunological synapse are believed to influence antigen recognition by the T cell receptor (TCR). Here the authors analyse these forces at single-molecule resolution to show that the ligand-engaged TCR of CD4+ T-cells create a stable environment with only a small fraction of TCR:pMHC complexes experiencing mechanistic forces at any given time during antigen surveillance and upon T-cell activation.

The lytic immunological synapse (IS) is a discrete structural entity formed after the ligation of specific activating receptors that leads to the destruction of a cancerous cell. The formation of an effector cell IS in cytotoxic T lymphocytes or natural killer cells is a hierarchical and stepwise rearrangement of structural and signaling components and targeted release of the contents of lytic granules. While recent advances in the generation and testing of cytotoxic lymphocytes expressing chimeric antigen receptors (CARs) has demonstrated their efficacy in the targeted lysis of tumor targets, the contribution and dynamics of IS components have not yet been extensively investigated in the context of engineered CAR cells. Understanding the biology of the CAR IS will be a powerful approach to efficiently guide the engineering of new CARs and help identify mechanistic problems in existing CARs. Here, we review the formation of the lytic IS and describe quantitative imaging-based measurements using multiple microscopy techniques at a single cell level that can be used in conjunction with established population-based assays to provide insight into the important cytotoxic function of CAR cells. The inclusion of this approach in the pipeline of CAR product design could be a novel and valuable innovation for the field. [Display omitted] Evaluation of the CAR immune synapse provides avenues for rational CAR design so as to offset failure of CAR products. Mukherjee et al. review quantitative imaging approaches to study the CAR immunological synapse.

From the thymus to the peripheral lymph nodes, integrin-mediated interactions with neighbor cells and the extracellular matrix tune T cell behavior by organizing cytoskeletal remodeling and modulating receptor signaling. LFA-1 (αLβ2 integrin) and VLA-4 (α4β1 integrin) play a key role throughout the T cell lifecycle from thymocyte differentiation to lymphocyte extravasation and finally play a fundamental role in organizing immune synapse, providing an essential costimulatory signal for the T cell receptor. Apart from tuning T cell signaling, integrins also contribute to homing to specific target organs as exemplified by the importance of α4β7 in maintaining the gut immune system. However, apart from those well-characterized examples, the physiological significance of the other integrin dimers expressed by T cells is far less understood. Thus, integrin-mediated cell-to-cell and cell-to-matrix interactions during the T cell lifespan still represent an open field of research.