Natural killer (NK) cells engage target cells via the immunological synapse (IS), where inhibitory and activating signals determine whether NK cell cytotoxicity is suppressed or activated. We previously reported that cancer cells can rapidly remodel their actin cytoskeleton upon NK cell engagement, leading to F-actin accumulation at the synapse. Here, we show that this process inhibits NK cell activation as indicated by impaired MTOC and lytic granule polarization. Exploring the underlying mechanism, we demonstrate that actin remodeling drives the recruitment of inhibitory ligands, such as HLA-A, -B, and -C, to the synapse. Disrupting HLA interaction with their cognate inhibitory receptors KIRs restores NK cell activation. Using NK cells expressing inhibitory KIR receptors, matched or unmatched to HLA molecules on cancer cells, we show that synaptic F-actin accumulation and matching KIR–HLA interactions jointly suppress NK cell cytotoxicity. Our findings reveal an immune evasion strategy in which cancer cells impair NK cell activation by altering synaptic signaling through actin cytoskeleton–driven recruitment of inhibitory signals to the IS.

Natural killer (NK) cells engage target cells via the immunological synapse, where inhibitory and activating signals determine whether NK cell cytotoxicity is suppressed or activated. We report that cancer cells can rapidly remodel their actin cytoskeleton upon NK cell engagement, leading to F-actin accumulation at the synapse. This process inhibits NK cell activation as indicated by impaired MTOC and lytic granule polarization. Exploring the underlying mechanism, we found that actin remodelling drives the recruitment of inhibitory ligands, such as HLA-A, -B, and -C, to the synapse. Disrupting HLA interaction with their cognate inhibitory receptors KIRs restored NK cell activation. Using NK cells expressing inhibitory KIR receptors, matched or unmatched to HLA molecules on cancer cells, we show that synaptic F-actin accumulation and matching KIR-HLA interactions jointly suppress NK cell cytotoxicity. Our findings reveal a novel immune evasion strategy in which cancer cells impair NK cell activation by altering synaptic signalling through actin cytoskeleton-driven recruitment of inhibitory signals to the immunological synapse. ### Competing Interest Statement The authors have declared no competing interest.

The formation of a lytic immunological synapse (IS) is crucial for cytotoxic lymphocytes to accurately target and effectively eliminate malignant cells. While significant attention has been focused on the lymphocyte side of the IS, particularly its role as a secretory domain for lytic granules, the cancer cell side of the IS has remained relatively underexplored. Recent findings have revealed that cancer cells can rapidly polarize their actin cytoskeleton toward the IS upon interaction with natural killer (NK) cells, thereby evading NK cell-mediated cytotoxicity. In this Brief Research Report, we present preliminary findings suggesting that actin cytoskeleton remodeling at the cancer cell side of the IS is associated with the targeted secretion of small extracellular vesicles towards the interacting NK cell. We observed that multivesicular bodies (MVBs) preferentially accumulate in the synaptic region in cancer cells exhibiting synaptic accumulation of F-actin, compared to those lacking actin cytoskeleton remodeling. Extracellular immunofluorescence staining revealed increased surface exposure of CD63 at the cancer cell side of the IS, suggestive of the fusion of MVBs with the plasma membrane. This hypothesis was supported by a pH-sensitive probe demonstrating dynamic trafficking of CD63 to the extracellular region of the IS. Collectively, our data support the notion that cancer cells can engage in targeted secretion of extracellular vesicles in response to NK cell attack, underscoring the need for further research into the potential role of this process in facilitating cancer cell immune evasion.

Cytotoxic lymphocytes, such as cytotoxic T cells and natural killer (NK) cells, are instrumental in the recognition and eradication of pathogenic cells, notably those undergoing malignant transformation. Cytotoxic lymphocytes establish direct contact with cancer cells via the formation of a specialized cell-cell junction known as the lytic immunological synapse. This structure serves as a critical platform for lymphocytes to integrate surface signals from potential cancer cells and to direct their cytolytic apparatus toward the confirmed targets. Conversely, cancer cells evolve synaptic defense strategies to evade lymphocyte cytotoxicity. This chapter delineates protocols using imaging flow cytometry to examine and quantify important subcellular processes occurring within cytotoxic lymphocytes and cancer cells engaged into an immunological synapse. These processes encompass the spatial redistribution of cytoskeletal components, vesicles, organelles and cell surface molecules. We specifically describe methods to generate and select conjugates between MDA-MB-231 breast cancer cells or K-562 leukemic cells and either the NK-92MI cell line or primary human NK cells. In addition, we detail procedures to evaluate the synaptic polarization of the actin cytoskeleton, CD63-positive vesicular compartments, MHC class I molecules, as well as the microtubule-organizing center in effector cells.

Typical actin cytoskeleton organization observed in most NK cell-conjugated MCF-7 cells. The cell conjugate was acquired on a LSM880 laser scanning confocal microscope (Carl Zeiss) with Airyscan in the high-resolution mode, and the 3D projection was assembled and animated using Zeiss Zen Software. Then NK cell was stained in red (PKH26) whereas the LifeAct-mEGFP-expressing tumour cell appears in green.

The actin cytoskeleton plays a critical role in cancer cell invasion and metastasis; however, the coordination of its multiple functions remains unclear. Actin dynamics in the cytoplasm control the formation of invadopodia, which are membrane protrusions that facilitate cancer cell invasion by focusing the secretion of extracellular matrix-degrading enzymes, including matrix metalloproteinases (MMPs). In this study, we investigated the nuclear role of cysteine-rich protein 2 (CRP2), a two LIM domain-containing F-actin-binding protein that we previously identified as a cytoskeletal component of invadopodia, in breast cancer cells. We found that F-actin depolymerization stimulates the translocation of CRP2 into the nucleus, resulting in an increase in the transcript levels of pro-invasive and pro-metastatic genes, including several members of the MMP gene family. We demonstrate that in the nucleus, CRP2 interacts with the transcription factor serum response factor (SRF), which is crucial for the expression of MMP-9 and MMP-13 . Our data suggest that CRP2 and SRF cooperate to modulate of MMP expression levels. Furthermore, Kaplan-Meier analysis revealed a significant association between high-level expression of SRF and shorter overall survival and distant metastasis-free survival in breast cancer patients with a high CRP2 expression profile. Our findings suggest a model in which CRP2 mediates the coordination of cytoplasmic and nuclear processes driven by actin dynamics, ultimately resulting in the induction of invasive and metastatic behavior in breast cancer cells.

<p>Time lapse imaging movie showing typical actin cytoskeleton configurations and dynamics in MCF-7 cells upon NK cell attack. Individual Z stacks were taken every minute over a period of 77 minutes. Time lapse movies were assembled from the Z stacks in NIH ImageJ and played at a fast frame rate (5 frames per second). NK cells are stained in red (PKH26) whereas the LifeAct-mEGFP-expressing tumour cell appears in green.</p>

Here, we present a protocol to identify and quantify phosphopeptides during the dynamic formation of an immunological synapse. We describe steps for mixing isotope-labeled immune and target cells, the stabilization of cell-to-cell conjugates by cross-linking, and their isolation by fluorescence-activated cell sorting. We detail the isolation of phosphopeptides by phosphopeptide enrichment and their subsequent measurement by mass spectrometry. Finally, we describe the analysis of the resulting data to separate cell-specific phosphopeptides using the isotope label and label-free quantification.

Cancer immune evasion is a major obstacle to effective anticancer immunotherapies. Recently, we unveiled a mechanism crucial in cancer escape from natural killer (NK)-mediated killing. The identified process is characterized by a rapid and massive accumulation of actin filaments at the cancer cell side of the immunological synapse (IS). We termed this process “actin response” (AR) and discovered that its inhibition is sufficient to restore cancer cell susceptibility to NK-mediated killing in vitro. Importantly, the AR is conserved across a wide range of malignancies, highlighting the broad translational potential of targeting this pathway. In this study, we investigated the molecular mechanisms underlying AR-mediated cancer cell immune evasion. Cancer-NK cell conjugates were analyzed by imaging flow cytometry (IFC) to investigate the polarization of inhibitory and activating ligands to the cancer cell side of the IS relative to the AR. We found that the AR is associated with and necessary for polarization of several inhibitory ligands. For instance, HLA-A,-B,-C molecules, which act as potent inhibitors of NK cells, were 2-fold increased at the IS of cancer cells with an AR when compared to cancer cells without an AR. Using confocal microscopy, we found that such polarization of inhibitory molecules is associated with the loss of MTOC and lytic granule polarization in conjugated human primary NK cells. In addition, correlative light and electron microscopy analysis revealed the presence of small vesicles (100 nm in size) and multivesicular bodies (MVBs) in the synaptic region of cancer cells with an AR. Quantification of vesicle markers at the IFC revealed a significant enrichment of CD63+/CD9+/CD81+ vesicles in the synaptic region of cancer cells with an AR. Interestingly, ultrastructure analysis showed that the AR is largely made of long actin-rich protrusions projecting into and altering the morphology of the synaptic cleft. Moreover, these protrusions were decorated with inhibitory molecules, such as PD-L1, and CD63. We identified a novel, highly conserved immune escape mechanism that exploits the fast remodeling of the actin cytoskeleton in cancer cells to induce the polarization of inhibitory ligands along with small tumor vesicles towards the IS. These findings suggest that actin remodeling in tumor cells at the IS actively promotes immune evasion. Further insights on the molecular and cellular mechanisms underpinning this pathway, such as the identification of selective targeting of linker proteins between the actin cytoskeleton and inhibitory ligands or small vesicles, represent a promising therapeutic strategy to improve the efficacy of anticancer immunotherapies. Nevertheless, additional research is needed in order to evaluate the translational potential of targeting the AR in a clinical setting. Citation Format: Andrea Michela Biolato, Liza Filali, Céline Hoffmann, Felix Kleine-Borgmann, Elena Ockfen, Max Krecke, Michel Mittelbronn, Clément Thomas. Actin remodeling, inhibitory ligand polarization and small vesicle recruitment hinder effective anti-tumor immunity by shaping the tumor cell side of the immune synapse [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6439.

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.

<p>Time lapse imaging movie showing typical actin cytoskeleton configurations and dynamics in MDA-MB-231 cells upon NK cell attack. Individual Z stacks were taken every minute over a period of 2 hours. Time lapse movies were assembled from the Z stacks in NIH ImageJ and played at a fast frame rate (5 frames per second). NK cells are stained in red (PKH26) whereas the LifeAct-mEGFP-expressing tumour cell appears in green.</p>

<p>Typical actin cytoskeleton organization observed in most NK cell-conjugated MDA-MB-231 cells. The cell conjugate was acquired on a LSM880 laser scanning confocal microscope (Carl Zeiss) with Airyscan in the high-resolution mode, and the 3D projection was assembled and animated using Zeiss Zen Software. Then NK cell was stained in red (PKH26) whereas the LifeAct-mEGFP-expressing tumour cell appears in green.</p>

<p>Time lapse imaging movie showing typical actin cytoskeleton configurations and dynamics in MCF-7 cells upon NK cell attack. Individual Z stacks were taken every minute over a period of 77 minutes. Time lapse movies were assembled from the Z stacks in NIH ImageJ and played at a fast frame rate (5 frames per second). NK cells are stained in red (PKH26) whereas the LifeAct-mEGFP-expressing tumour cell appears in green.</p>

Supplementary Materials and Methods

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.

Supplementary Figures S1-S6 and their legends

Natural killer (NK) cells are innate effector lymphocytes with strong antitumor effects against hematologic malignancies such as chronic lymphocytic leukemia (CLL). However, NK cells fail to control CLL progression on the long term. For effective lysis of their targets, NK cells use a specific cell-cell interface, known as the immunological synapse (IS), whose assembly and effector function critically rely on dynamic cytoskeletal changes in NK cells. Here we explored the role of CLL cell actin cytoskeleton during NK cell attack. We found that CLL cells can undergo fast actin cytoskeleton remodeling which is characterized by a NK cell contact-induced accumulation of actin filaments at the IS. Such polarization of the actin cytoskeleton was strongly associated with resistance against NK cell-mediated cytotoxicity and reduced amounts of the cell-death inducing molecule granzyme B in target CLL cells. Selective pharmacological targeting of the key actin regulator Cdc42 abrogated the capacity of CLL cells to reorganize their actin cytoskeleton during NK cell attack, increased levels of transferred granzyme B and restored CLL cell susceptibility to NK cell cytotoxicity. This resistance mechanism was confirmed in primary CLL cells from patients. In addition, pharmacological inhibition of actin dynamics in combination with blocking antibodies increased conjugation frequency and improved CLL cell elimination by NK cells. Together our results highlight the critical role of CLL cell actin cytoskeleton in driving resistance against NK cell cytotoxicity and provide new potential therapeutic point of intervention to target CLL immune escape.

How cytotoxic lymphocytes are protected against their own weapons during close combat with diseased target cells is an important and long-standing question in immunology. A study in this issue provides new insights into the mechanisms by which natural killer (NK) cells avoid self-destruction.

Invadopodia are actin-rich membrane protrusions that facilitate cancer cell dissemination by focusing on proteolytic activity and clearing paths for migration through physical barriers, such as basement membranes, dense extracellular matrices, and endothelial cell junctions. Invadopodium formation and activity require spatially and temporally regulated changes in actin filament organization and dynamics. About three decades of research have led to a remarkable understanding of how these changes are orchestrated by sequential recruitment and coordinated activity of different sets of actin-binding proteins. In this chapter, we provide an update on the roles of the actin cytoskeleton during the main stages of invadopodium development with a particular focus on actin polymerization machineries and production of pushing forces driving extracellular matrix remodeling.

Congenital defects of the immune system called primary immunodeficiency disorders (PID) describe a group of diseases characterized by a decrease, an absence, or a malfunction of at least one part of the immune system. As a result, PID patients are more prone to develop life-threatening complications, including cancer. PID currently include over 400 different disorders, however, the variety of PID-related cancers is narrow. We discuss here reasons for this clinical phenotype. Namely, PID can lead to cell intrinsic failure to control cell transformation, failure to activate tumor surveillance by cytotoxic cells or both. As the most frequent tumors seen among PID patients stem from faulty lymphocyte development leading to leukemia and lymphoma, we focus on the extensive genomic alterations needed to create the vast diversity of B and T lymphocytes with potential to recognize any pathogen and why defects in these processes lead to malignancies in the immunodeficient environment of PID patients. In the second part of the review, we discuss PID affecting tumor surveillance and especially membrane trafficking defects caused by altered exocytosis and regulation of the actin cytoskeleton. As an impairment of these membrane trafficking pathways often results in dysfunctional effector immune cells, tumor cell immune evasion is elevated in PID. By considering new anti-cancer treatment concepts, such as transfer of genetically engineered immune cells, restoration of anti-tumor immunity in PID patients could be an approach to complement standard therapies.