Coordinated ARP2/3 and glycolytic activities regulate the morphological and functional fitness of human CD8+ T cells.

Actin cytoskeleton remodeling sustains the ability of cytotoxic T cells to search for target cells and eliminate them. We here investigated the relationship between energetic status, actin remodeling, and functional fitness in human CD8+ effector T cells. Cell spreading during migration or immunological synapse assembly mirrored cytotoxic activity. Morphological and functional fitness were boosted by interleukin-2 (IL-2), which also stimulated the transcription of glycolytic enzymes, actin isoforms, and actin-related protein (ARP)2/3 complex subunits. This molecular program scaled with F-actin content and cell spreading. Inhibiting glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility, and adhesion, while mitochondrial oxidative phosphorylation blockade impacted cell elongation during confined migration. The severe morphological and functional defects of ARPC1B-deficient T cells were only partially corrected by IL-2, emphasizing ARP2/3-mediated actin polymerization as a crucial energy state integrator. The study therefore underscores the tight coordination between metabolic and actin remodeling programs to sustain the cytotoxic activity of CD8+ T cells.

LFA-1 nanoclusters integrate TCR stimulation strength to tune T-cell cytotoxic activity.

T-cell cytotoxic function relies on the cooperation between the highly specific but poorly adhesive T-cell receptor (TCR) and the integrin LFA-1. How LFA-1-mediated adhesion may scale with TCR stimulation strength is ill-defined. Here, we show that LFA-1 conformation activation scales with TCR stimulation to calibrate human T-cell cytotoxicity. Super-resolution microscopy analysis reveals that >1000 LFA-1 nanoclusters provide a discretized platform at the immunological synapse to translate TCR engagement and density of the LFA-1 ligand ICAM-1 into graded adhesion. Indeed, the number of high-affinity conformation LFA-1 nanoclusters increases as a function of TCR triggering strength. Blockade of LFA-1 conformational activation impairs adhesion to target cells and killing. However, it occurs at a lower TCR stimulation threshold than lytic granule exocytosis implying that it licenses, rather than directly controls, the killing decision. We conclude that the organization of LFA-1 into nanoclusters provides a calibrated system to adjust T-cell killing to the antigen stimulation strength.

Lymphocyte migration and retention properties affected by ibrutinib in chronic lymphocytic leukemia.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is widely used for treatment of patients with relapsed/refractory or treatment-naïve chronic lymphocytic leukemia (CLL). A prominent effect of ibrutinib is to disrupt the retention of CLL cells from supportive lymphoid tissues, by altering BTK-dependent adhesion and migration. To further explore the mechanism of action of ibrutinib and its potential impact on non-leukemic cells, we quantified multiple motility and adhesion parameters of human primary CLL cells and non-leukemic lymphoid cells. In vitro, ibrutinib affected CCL19-, CXCL12- and CXCL13-evoked migration behavior of CLL cells and non-neoplastic lymphocytes, by reducing both motility speed and directionality. De-phosphorylation of BTK induced by ibrutinib in CLL cells was associated with defective polarization over fibronectin and inability to assemble the immunological synapse upon B-cell receptor engagement. In patients' samples collected during a 6-month monitoring of therapy, chemokine-evoked migration was repressed in CLL cells and marginally reduced in T cells. This was accompanied by profound modulation of the expression of chemokine receptors and adhesion molecules. Remarkably, the relative expression of the receptors governing lymph node entry (CCR7) versus exit (S1PR1) stood out as a reliable predictive marker of the clinically relevant treatment-induced lymphocytosis. Together, our data reveal a multifaceted modulation of motility and adhesive properties of ibrutinib on both CLL leukemic cell and T-cell populations and point to intrinsic differences in CLL recirculation properties as an underlying cause for variability in treatment response.

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.

Kinetic measurements of human CD8+ T cell cytotoxic activity in a 384-well plate format.

The elimination of infected or cancerous cells by CD8+ cytotoxic T lymphocytes (CTL) is a crucial effector mechanism of the immune system. Upon antigen recognition, CTL stop migrating, establish a tight contact with target cells and deliver cytotoxic molecules such as perforin and granzymes that lead to target cell apoptosis. The ability of CTL to control a population of infected cells or a tumor depends on multiple parameters, such as the relative numbers of CTL and target cells, the intrinsic cytotoxic activity of CTL, the intrinsic resistance of target cells and the repertoire of immune checkpoints tuning cytotoxic activity at the CTL:target cell interface. In this context, in vitro assays to precisely measure CTL:target cell interactions and cytotoxic activity over time are required to monitor natural or therapeutic responses. We here present an image-based method that allows recording of positions and survival of CTL and target cells over time in a high-throughput format. The protocol relies on the staining of CTL and target cells with fluorescent dyes and the automated imaging of cells deposited in wells of a 384-well plate with an automated cell imaging device. We discuss potential applications offered by the kinetic assessment of CTL cytotoxic activity in a high-throughput format.

Biallelic NFATC1 mutations cause an inborn error of immunity with impaired CD8+ T-cell function and perturbed glycolysis.

The nuclear factor of activated T cells (NFAT) family of transcription factors plays central roles in adaptive immunity in murine models; however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified 3 patients who carry germ line biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglobulinemia, and decreased antibody responses. The compound heterozygous NFATC1 variants identified in these patients caused decreased stability and reduced the binding of DNA and interacting proteins. We observed defects in early activation and proliferation of T and B cells from these patients, amenable to rescue upon genetic reconstitution. Stimulation induced early T-cell activation and proliferation responses were delayed but not lost, reaching that of healthy controls at day 7, indicative of an adaptive capacity of the cells. Assessment of the metabolic capacity of patient T cells revealed that NFATc1 dysfunction rendered T cells unable to engage in glycolysis after stimulation, although oxidative metabolic processes were intact. We hypothesized that NFATc1-mutant T cells could compensate for the energy deficit due to defective glycolysis by using enhanced lipid metabolism as an adaptation, leading to a delayed, but not lost, activation responses. Indeed, we observed increased 13C-labeled palmitate incorporation into citrate, indicating higher fatty acid oxidation, and we demonstrated that metformin and rosiglitazone improved patient T-cell effector functions. Collectively, enabled by our molecular dissection of the consequences of loss-of-function NFATC1 mutations and extending the role of NFATc1 in human immunity beyond receptor signaling, we provide evidence of metabolic plasticity in the context of impaired glycolysis observed in patient T cells, alleviating delayed effector responses.

Deciphering actin remodelling in immune cells through the prism of actin-related inborn errors of immunity.

Actin cytoskeleton remodelling drives cell motility, cell to cell contacts, as well as membrane and organelle dynamics. Those cellular activities operate at a particularly high pace in immune cells since these cells migrate through various tissues, interact with multiple cellular partners, ingest microorganisms and secrete effector molecules. The central and multifaceted role of actin cytoskeleton remodelling in sustaining immune cell tasks in humans is highlighted by rare inborn errors of immunity due to mutations in genes encoding proximal and distal actin regulators. In line with the specificity of some of the actin-based processes at work in immune cells, the expression of some of the affected genes, such as WAS, ARPC1B and HEM1 is restricted to the hematopoietic compartment. Exploration of these natural deficiencies highlights the fact that the molecular control of actin remodelling is tuned distinctly in the various subsets of myeloid and lymphoid immune cells and sustains different networks associated with a vast array of specialized tasks. Furthermore, defects in individual actin remodelling proteins are usually associated with partial cellular impairments highlighting the plasticity of actin cytoskeleton remodelling. This review covers the roles of disease-associated actin regulators in promoting the actin-based processes of immune cells. It focuses on the specific molecular function of those regulators across various immune cell subsets and in response to different stimuli. Given the fact that numerous immune-related actin defects have only been characterized recently, we further discuss the challenges lying ahead to decipher the underlying patho-mechanisms.

Computational modelling of cell motility modes emerging from cell-matrix adhesion dynamics.

Lymphocytes have been described to perform different motility patterns such as Brownian random walks, persistent random walks, and Lévy walks. Depending on the conditions, such as confinement or the distribution of target cells, either Brownian or Lévy walks lead to more efficient interaction with the targets. The diversity of these motility patterns may be explained by an adaptive response to the surrounding extracellular matrix (ECM). Indeed, depending on the ECM composition, lymphocytes either display a floating motility without attaching to the ECM, or sliding and stepping motility with respectively continuous or discontinuous attachment to the ECM, or pivoting behaviour with sustained attachment to the ECM. Moreover, on the long term, lymphocytes either perform a persistent random walk or a Brownian-like movement depending on the ECM composition. How the ECM affects cell motility is still incompletely understood. Here, we integrate essential mechanistic details of the lymphocyte-matrix adhesions and lymphocyte intrinsic cytoskeletal induced cell propulsion into a Cellular Potts model (CPM). We show that the combination of de novo cell-matrix adhesion formation, adhesion growth and shrinkage, adhesion rupture, and feedback of adhesions onto cell propulsion recapitulates multiple lymphocyte behaviours, for different lymphocyte subsets and various substrates. With an increasing attachment area and increased adhesion strength, the cells' speed and persistence decreases. Additionally, the model predicts random walks with short-term persistent but long-term subdiffusive properties resulting in a pivoting type of motility. For small adhesion areas, the spatial distribution of adhesions emerges as a key factor influencing cell motility. Small adhesions at the front allow for more persistent motility than larger clusters at the back, despite a similar total adhesion area. In conclusion, we present an integrated framework to simulate the effects of ECM proteins on cell-matrix adhesion dynamics. The model reveals a sufficient set of principles explaining the plasticity of lymphocyte motility.

Altered Basal Lipid Metabolism Underlies the Functional Impairment of Naive CD8+ T Cells in Elderly Humans.

Aging is associated with functional deficits in the naive T cell compartment, which compromise the generation of de novo immune responses against previously unencountered Ags. The mechanisms that underlie this phenomenon have nonetheless remained unclear. We found that naive CD8+ T cells in elderly humans were prone to apoptosis and proliferated suboptimally in response to stimulation via the TCR. These abnormalities were associated with dysregulated lipid metabolism under homeostatic conditions and enhanced levels of basal activation. Importantly, reversal of the bioenergetic anomalies with lipid-altering drugs, such as rosiglitazone, almost completely restored the Ag responsiveness of naive CD8+ T cells. Interventions that favor lipid catabolism may therefore find utility as adjunctive therapies in the elderly to promote vaccine-induced immunity against targetable cancers and emerging pathogens, such as seasonal influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

BioProfiling.jl: profiling biological perturbations with high-content imaging in single cells and heterogeneous populations.

MOTIVATION: High-content imaging screens provide a cost-effective and scalable way to assess cell states across diverse experimental conditions. The analysis of the acquired microscopy images involves assembling and curating raw cellular measurements into morphological profiles suitable for testing biological hypotheses. Despite being a critical step, general-purpose and adaptable tools for morphological profiling are lacking and no solution is available for the high-performance Julia programming language. RESULTS: Here, we introduce BioProfiling.jl, an efficient end-to-end solution for compiling and filtering informative morphological profiles in Julia. The package contains all the necessary data structures to curate morphological measurements and helper functions to transform, normalize and visualize profiles. Robust statistical distances and permutation tests enable quantification of the significance of the observed changes despite the high fraction of outliers inherent to high-content screens. This package also simplifies visual artifact diagnostics, thus streamlining a bottleneck of morphological analyses. We showcase the features of the package by analyzing a chemical imaging screen, in which the morphological profiles prove to be informative about the compounds' mechanisms of action and can be conveniently integrated with the network localization of molecular targets. AVAILABILITY AND IMPLEMENTATION: The Julia package is available on GitHub: https://github.com/menchelab/BioProfiling.jl. We also provide Jupyter notebooks reproducing our analyses: https://github.com/menchelab/BioProfilingNotebooks. The data underlying this article are available from FigShare, at https://doi.org/10.6084/m9.figshare.14784678.v2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular Tuning of Actin Dynamics in Leukocyte Migration as Revealed by Immune-Related Actinopathies.

Motility is a crucial activity of immune cells allowing them to patrol tissues as they differentiate, sample or exchange information, and execute their effector functions. Although all immune cells are highly migratory, each subset is endowed with very distinct motility patterns in accordance with functional specification. Furthermore individual immune cell subsets adapt their motility behaviour to the surrounding tissue environment. This review focuses on how the generation and adaptation of diversified motility patterns in immune cells is sustained by actin cytoskeleton dynamics. In particular, we review the knowledge gained through the study of inborn errors of immunity (IEI) related to actin defects. Such pathologies are unique models that help us to uncover the contribution of individual actin regulators to the migration of immune cells in the context of their development and function.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

Actin Dynamics at the T Cell Synapse as Revealed by Immune-Related Actinopathies.

The actin cytoskeleton is composed of dynamic filament networks that build adaptable local architectures to sustain nearly all cellular activities in response to a myriad of stimuli. Although the function of numerous players that tune actin remodeling is known, the coordinated molecular orchestration of the actin cytoskeleton to guide cellular decisions is still ill defined. T lymphocytes provide a prototypical example of how a complex program of actin cytoskeleton remodeling sustains the spatio-temporal control of key cellular activities, namely antigen scanning and sensing, as well as polarized delivery of effector molecules, via the immunological synapse. We here review the unique knowledge on actin dynamics at the T lymphocyte synapse gained through the study of primary immunodeficiences caused by mutations in genes encoding actin regulatory proteins. Beyond the specific roles of individual actin remodelers, we further develop the view that these operate in a coordinated manner and are an integral part of multiple signaling pathways in T lymphocytes.

Histone deacetylase 1 controls CD4+ T cell trafficking in autoinflammatory diseases.

CD4+ T cell trafficking is a fundamental property of adaptive immunity. In this study, we uncover a novel role for histone deacetylase 1 (HDAC1) in controlling effector CD4+ T cell migration, thereby providing mechanistic insight into why a T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis (EAE). HDAC1-deficient CD4+ T cells downregulated genes associated with leukocyte extravasation. In vitro, HDAC1-deficient CD4+ T cells displayed aberrant morphology and migration on surfaces coated with integrin LFA-1 ligand ICAM-1 and showed an impaired ability to arrest on and to migrate across a monolayer of primary mouse brain microvascular endothelial cells under physiological flow. Moreover, HDAC1 deficiency reduced homing of CD4+ T cells into the intestinal epithelium and lamina propria preventing weight-loss, crypt damage and intestinal inflammation in adoptive CD4+ T cell transfer colitis. This correlated with reduced expression levels of LFA-1 integrin chains CD11a and CD18 as well as of selectin ligands CD43, CD44 and CD162 on transferred circulating HDAC1-deficient CD4+ T cells. Our data reveal that HDAC1 controls T cell-mediated autoimmunity via the regulation of CD4+ T cell trafficking into the CNS and intestinal tissues.

RhoG deficiency abrogates cytotoxicity of human lymphocytes and causes hemophagocytic lymphohistiocytosis.

Exocytosis of cytotoxic granules (CG) by lymphocytes is required for the elimination of infected and malignant cells. Impairments in this process underly a group of diseases with dramatic hyperferritinemic inflammation termed hemophagocytic lymphohistiocytosis (HLH). Although genetic and functional studies of HLH have identified proteins controlling distinct steps of CG exocytosis, the molecular mechanisms that spatiotemporally coordinate CG release remain partially elusive. We studied a patient exhibiting characteristic clinical features of HLH associated with markedly impaired cytotoxic T lymphocyte (CTL) and natural killer (NK) cell exocytosis functions, who beared biallelic deleterious mutations in the gene encoding the small GTPase RhoG. Experimental ablation of RHOG in a model cell line and primary CTLs from healthy individuals uncovered a hitherto unappreciated role of RhoG in retaining CGs in the vicinity of the plasma membrane (PM), a fundamental prerequisite for CG exocytotic release. We discovered that RhoG engages in a protein-protein interaction with Munc13-4, an exocytosis protein essential for CG fusion with the PM. We show that this interaction is critical for docking of Munc13-4+ CGs to the PM and subsequent membrane fusion and release of CG content. Thus, our study illuminates RhoG as a novel essential regulator of human lymphocyte cytotoxicity and provides the molecular pathomechanism behind the identified here and previously unreported genetically determined form of HLH.

Molecular Tuning of Filamin A Activities in the Context of Adhesion and Migration.

The dynamic organization of actin cytoskeleton meshworks relies on multiple actin-binding proteins endowed with distinct actin-remodeling activities. Filamin A is a large multi-domain scaffolding protein that cross-links actin filaments with orthogonal orientation in response to various stimuli. As such it plays key roles in the modulation of cell shape, cell motility, and differentiation throughout development and adult life. The essentiality and complexity of Filamin A is highlighted by mutations that lead to a variety of severe human disorders affecting multiple organs. One of the most conserved activity of Filamin A is to bridge the actin cytoskeleton to integrins, thereby maintaining the later in an inactive state. We here review the numerous mechanisms cells have developed to adjust Filamin A content and activity and focus on the function of Filamin A as a gatekeeper to integrin activation and associated adhesion and motility.

Eomes-Dependent Loss of the Co-activating Receptor CD226 Restrains CD8+ T Cell Anti-tumor Functions and Limits the Efficacy of Cancer Immunotherapy.

CD8+ T cells within the tumor microenvironment (TME) are exposed to various signals that ultimately determine functional outcomes. Here, we examined the role of the co-activating receptor CD226 (DNAM-1) in CD8+ T cell function. The absence of CD226 expression identified a subset of dysfunctional CD8+ T cells present in peripheral blood of healthy individuals. These cells exhibited reduced LFA-1 activation, altered TCR signaling, and a distinct transcriptomic program upon stimulation. CD226neg CD8+ T cells accumulated in human and mouse tumors of diverse origin through an antigen-specific mechanism involving the transcriptional regulator Eomesodermin (Eomes). Despite similar expression of co-inhibitory receptors, CD8+ tumor-infiltrating lymphocyte failed to respond to anti-PD-1 in the absence of CD226. Immune checkpoint blockade efficacy was hampered in Cd226-/- mice. Anti-CD137 (4-1BB) agonists also stimulated Eomes-dependent CD226 loss that limited the anti-tumor efficacy of this treatment. Thus, CD226 loss restrains CD8+ T cell function and limits the efficacy of cancer immunotherapy.

The cytoskeletal regulator HEM1 governs B cell development and prevents autoimmunity.

The WAVE regulatory complex (WRC) is crucial for assembly of the peripheral branched actin network constituting one of the main drivers of eukaryotic cell migration. Here, we uncover an essential role of the hematopoietic-specific WRC component HEM1 for immune cell development. Germline-encoded HEM1 deficiency underlies an inborn error of immunity with systemic autoimmunity, at cellular level marked by WRC destabilization, reduced filamentous actin, and failure to assemble lamellipodia. Hem1-/- mice display systemic autoimmunity, phenocopying the human disease. In the absence of Hem1, B cells become deprived of extracellular stimuli necessary to maintain the strength of B cell receptor signaling at a level permissive for survival of non-autoreactive B cells. This shifts the balance of B cell fate choices toward autoreactive B cells and thus autoimmunity.

Surmounting the obstacles that impede effective CAR T cell trafficking to solid tumors.

Innovative immunotherapies based on immune checkpoint targeting antibodies and engineered T cells are transforming the way we approach cancer treatment. However, although these T cell centered strategies result in marked and durable responses in patients across many different tumor types, they provide therapeutic efficacy only in a proportion of patients. A major challenge of immuno-oncology is thereby to identify mechanisms responsible for resistance to cancer immunotherapy in order to overcome them via adapted strategies that will ultimately improve intrinsic efficacy and response rates. Here, we focus on the barriers that restrain the trafficking of chimeric antigen receptor (CAR)-expressing T cells to solid tumors. Upon infusion, CAR T cells need to home into malignant sites, navigate within complex tumor environments, form productive interactions with cancer cells, deliver their cytotoxic activities, and finally persist. We review the accumulating evidence that the microenvironment of solid tumors contains multiple obstacles that hinder CAR T cells in the dynamic steps underlying their trafficking. We focus on how these hurdles may in part account for the failure of CAR T cell clinical trials in human carcinomas. Given the engineered nature of CAR T cells and possibilities to modify the tumor environment, there are ample opportunities to augment CAR T cell ability to efficiently find and combat tumors. We present some of these strategies, which represent a dynamic field of research with high potential for clinical applicability.