Numerous cell types relate to their immediate environment by exerting a three-dimensional pressure field on their environment, with components both longitudinal and transverse to the cell membrane. This pressure field can in principle be measured by traction force microscopy experiments. Compared to other approaches, the technique of Protrusion Force Microscopy gives access with high spatial resolution to the pressure field by measuring the deformation of a thin elastic membrane using atomic force microscopy (AFM). However, while the pressure field under interest is three-dimensional, the height profile measured by AFM is only one-dimensional. We propose a solution to this inverse problem and we explore its regime of applicability in the experimental context.

Multiple sclerosis (MS) is a complex inflammatory disease of the CNS resulting from an intricate interplay between genetic predisposition and environmental factors. Vitamin D (VD) deficiency is one of the established risk factors for MS. CD46 costimulation of CD4+ T cells induces a switch from Th1 to type I regulatory cells (Tr1), characterized by increased IL-10 production. This switch is impaired in MS T cells but can be restored by VD, which also strongly promotes expression of CD226 on CD46-activated T cells. The rs763361 polymorphism in the CD226 gene, resulting in a non-synonymous Gly307Ser variant, is associated with increased risk for MS. Herein, we show that expression of this CD226 risk allele disrupts the ability of CD46-activated T cells to operate the IFNγ/IL-10 switch upon VD exposure. Mechanistically, the risk variant impairs activation of the integrin LFA-1, which promotes the Tr1 phenotype. LFA-1-mediated Tr1 differentiation is also impaired in MS T cells expressing the CD226 risk allele upon CD46 and VD stimulation. Our study unveils how, in the context of MS susceptibility, a genetic polymorphism and an environmental factor act in concert to control the differentiation of Tr1 cells.

Cytotoxic T Lymphocytes (CTLs) mediate tumor clearance and anti-tumoral responses through the elimination of target cancerous cells upon antigen recognition and TCR engagement. The killing of cancerous cells mediated by CTLs can be modulated by the strength of the TCR engagement, notably through the antigen affinity to the TCR, the quality of the immune synapse, the time of contact between CTLs and targets, among other parameters. Unveiling the mechanisms, kinetics and factors influencing cancer cell elimination by CTLs is essential for the amelioration of lymphocyte-based immunotherapies, such as CAR-T cells, and the understanding of tumoral clearance in vivo. Here, we provide a method to precisely monitor the cytotoxic capacity of murine CTLs in an antigen-specific manner, using ovalbumin (OVA) specific CTLs expressing the modified OT1 TCR and four different tumoral B16-F10 cell lines expressing OVA peptides with variable affinity for the OT1 TCR. We present step-by-step the implementation of a high throughput image-based pipeline allowing, on one hand, the time-lapse monitoring of CTL-mediated cytotoxicity and, on the other hand, the measurement of morphological parameters of CTLs and target cells based on fluorescent labeling. This method is suitable for ex-vivo exploration of lymphocyte cytotoxicity and related parameters, and can be adapted for mechanistic studies or screening approaches.

Actin cytoskeleton remodelling drives the migration of immune cells and their engagement in dynamic cell–cell contacts. The importance of actin cytoskeleton dynamics in immune cell function is highlighted by the discovery of inborn errors of immunity (IEIs) that are caused by defects in individual actin-regulatory proteins, resulting in immune-related actinopathies. In addition to susceptibility to infection, these often present with a vast array of autoimmune and autoinflammatory manifestations. Here, we review the role of actin subnetworks in the activation and function of lymphoid and myeloid cells. We focus on the mechanisms by which actin defects result in aberrant lymphocyte function, including dysregulation of T cell- and B cell-mediated tolerance and biased cytokine production, which can result in autoimmunity. We also highlight the relationship between actin defects and inflammasome activation and other pathomechanisms in myeloid cells as the underlying cause of autoinflammation. Finally, we discuss future avenues for research and therapeutic intervention based on a molecular understanding of immune-related actinopathies. The actin cytoskeleton is essential for immune cell shape, signalling and function. In this Review, the authors examine how germ-line mutations affecting actin-regulatory proteins, called immune-related actinopathies, lead to inborn errors of immunity. These are characterized by susceptibility to infection as well as autoimmune and autoinflammatory disease manifestations. Focusing on WASP, HEM1 and DOCK11 deficiencies, the authors examine the diverse mechanisms that link disturbed actin homeostasis in lymphoid and myeloid cells to autoimmunity and autoinflammation and outline emerging mechanistic insights and therapeutic directions.

Regulated exocytosis controls key cellular functions ranging from neurotransmitter release to the secretion of immune mediators, and its disruption is associated with numerous pathologies. The cytotoxic activity of lymphocytes is particularly dependent on regulated and polarized lytic granule delivery toward infected or malignant cells. Although genetic and mechanistic studies have identified factors regulating exocytosis in cytotoxic lymphocytes, a systematic mapping of the relevant factors and their relationships is lacking. Through a genome-scale CRISPR knockout screen in a human natural killer cell line, we characterized a complex genetic network regulating cytotoxic granule exocytosis, with lipid metabolism and protein lipidation among the most prominent pathways. By combining global protein palmitoylation and lipidomic studies, we found that ZDHHC17 drives palmitoylation of the core SNARE complex protein SNAP23 to target cytotoxic granules to GM1-rich lipid rafts whose assembly is controlled by serine palmitoyltransferase. In summary, our study identifies previously unrecognized factors essential for cytotoxic function in human lymphocytes and uncovers how lipid metabolism and protein palmitoylation are involved in the process of regulated exocytosis.

BackgroundThe majority of monogenic inborn errors of immunity presenting as actinopathies were reported originally from the Middle East and North Africa (MENA) countries indicating a high prevalence of these entities in the region. However, their prognosis is unclear due to rarity and lack of comprehensive treatment outcomes.MethodsWe evaluated clinical, immunological, and genetic abnormalities associated with 15 genetic entities of actinopathies. Based on the function of mutant genes in actin-regulatory pathways, patients were classified into CDC42- and RAC2-related subcategories.ResultsA total of 503 individuals (29.5% females) from 17 countries were considered with a median age of 120 months. Although most patients presented initially with allergic phenotypes (37.7%), the most prevalent manifestations throughout the lifespan were infection in respiratory tracts (72.2%). Primary clinical diagnosis was mainly combined immunodeficiencies (48.3%) and the majority of cases were molecularly assigned to the CDC42 pathway (64.8%). The most common genetic defects were reported within the DOCK8 (n = 209) followed by the WAS (n = 94) and the CARMIL2 (n = 15) genes. Hematopoietic stem cell transplantation (HSCT) was conducted on 24.0% of patients, which significantly improved survival in patients with defects in WAS, DOCK8 and DOCK2. Overall mortality was 23.0%, mainly due to sepsis and malignancy.ConclusionPatients with defects in RAC2-associated regulators of actin usually present with late-onset symptoms due to normal immune profiles, but a higher rate of EBV and HPV infections, autoimmune cytopenia, asthma, and lymphoproliferation compared to defects in the CDC42 pathway. The severity of mutations in patients of the CDC42 group helps to estimate the prognosis of the disease and prioritization of HSCT.

ABSTRACT:Immune cell functionality is highly dependent on the actin cytoskeleton. The actin cytoskeleton is regulated by a complex molecular machinery that involves multiple genes. Mutations in these genes can cause inborn errors of immunity, also termed immunoactinopathies, of which Wiskott-Aldrich syndrome is the best-characterized entity. Currently, mutations in 23 genes can be considered causative of immunoactinopathies. Immunoactinopathies are rare disease entities with complex combinations of clinical manifestations, including immunodeficiency, immune dysregulation, malignancies, atopy, thrombocytopenia and bleeding, skin involvement, or congenital defects. Prompt diagnosis is crucial, because hematopoietic stem cell transplantation in an early phase can offer cure and prevent further complications. This review provides a detailed summary of the clinical experience with immunoactinopathies so far, elaborates on the most distinguishing features of immunoactinopathies by providing a clinical categorization, and links this information to the underlying biological pathways. This information may be of help to clinicians in the diagnosis of patients and to eventually improve patient care.

Multiple sclerosis (MS) is a complex inflammatory disease of the central nervous system (CNS), resulting from an intricate interplay between genetic predisposition and environmental factors. Vitamin D (VD) deficiency is one of the established risk factors for MS. CD46 costimulation of CD4+ T cells induces a switch from Th1 to type I regulatory cells (Tr1), characterized by increased IL-10 production. This switch is impaired in MS T cells but can be restored by VD, which also strongly promotes expression of CD226 on CD46-activated T cells. The rs763361 polymorphism in the CD226 gene, resulting in a non-synonymous Gly307Ser variant, is associated with increased risk for MS. Herein, we show that expression of this CD226 risk allele disrupts the ability of CD46-activated T cells to operate the IFNγ/IL-10 switch upon VD exposure. Mechanistically, the risk variant impairs activation of the integrin LFA-1, which sustains homotypic adhesion associated with acquisition of the Tr1 phenotype. LFA-1-mediated Tr1 differentiation is also impaired in MS T cells expressing the CD226 risk allele upon CD46 and VD stimulation. Our study unveils how, in the context of MS susceptibility, a genetic polymorphism and an environmental factor act in concert to control the differentiation of Tr1 cells.

Despite the efficacy of natalizumab, which targets the integrin VLA-4, in treating multiple sclerosis (MS), approximately 35% patients with MS present evidence of disease activity two years after treatment initiation. Individual heterogeneity of leukocyte response to VLA-4 on natalizumab-mediated blockade may underlie disparities in treatment efficacy. Here we use a high-content cell imaging (HCI) pipeline to profile the in vitro effects of natalizumab on VLA-4-stimulated PBMCs from MS patients prior to natalizumab treatment. Unsupervised clustering of image data partially discriminates non-responder MS patients based on morphology, F-actin organization and signaling-related features in CD8+ T cells. Furthermore, through a random forest approach, treatment response can be predicted with a performance of 92% for a discovery cohort and 88% for a validation cohort. Unfavorable treatment response is associated with a distinct actin remodeling response of natalizumab-exposed CD8+ T cells and a residual ability of these cells to spread on VCAM-1. Our study thus unveils that CD8+ T cells from individual MS patients display heterogeneous susceptibility to natalizumab in vitro and highlights the potential of HCI-based pretreatment monitoring to assist individualized treatment prescription.

T-cell antigen receptors (TCRs) exhibit inherent cross-reactivity which broadens the spectrum of epitopes that are recognizable by a finite TCR-repertoire but also carries the risk of autoimmunity. However, TCRs support also a high level of antigen specificity as they allow T-cells to discriminate single antigenic peptide/MHC complexes (pMHCs) against millions of structurally related self-pMHCs, in some cases based on the absence or presence of a single methyl-group. How TCRs manage to convey such seemingly contrary properties and why some T-cells become over time autoreactive despite negative thymic selection, has remained elusive. Here, we devised a non-invasive molecular live cell imaging platform to investigate the biophysical parameters governing stimulatory TCR:pMHC interactions in settings of autoreactivity and anti-viral responses - two extremes in T-cell antigen recognition. We show that CMV-specific CD8+ RA14-T-cells respond effectively to even a single HLA-A2/CMV (A2/CMV) antigen, with synaptic TCR:pMHC lifetimes lasting seconds. In contrast, cross-reactivity of type 1 diabetes (T1D)-associated CD8+ 1E6 T-cells towards HLA-A2/preproinsulin (A2/PPI) self-epitopes involved ten-fold less stable synaptic TCR interactions resulting in severely attenuated ZAP70 recruitment and downstream signaling. Compared to A2/CMV-engaged RA14 T-cells, 1E6-T-cells required for activation 4000 or more A2/PPI and at least 100-times as many simultaneously pMHC-engaged TCRs. In support of antigen discrimination, CD8 co-engagement of MHC class I (MHCI) strengthened both settings of TCR:pMHC interactions equally but was essential only for sensitized virus detection but not autorecognition (1000- versus 5-fold enhancement). We conclude that the binding dynamics of TCRs and CD8 with pMHC shape the boundaries of central tolerance in the physiological context of the phenomenal yet also differential T-cell antigen detection capacity, TCR-cross-reactivity and self-antigen abundance. Gained insights are integral to a molecular and quantitative understanding of CD8+ T-cell mediated autoimmunity and protective immunity against infections and cancer. ### Competing Interest Statement The authors have declared no competing interest.

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.

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.

Background One of the first clinical observations of ibrutinib activity in the treatment of chronic lymphocytic leukemia (CLL) is a rapid decline in lymph nodes size. This phenomenon is accompanied by an hyperlymphocytosis, either transient or prolonged, which is associated with distinct clinical responses and thus has an impact on long-term outcomes. Understanding which factors determine distinct disease courses upon ibrutinib treatment remains a scientific challenge. Methods and findings From 2016 to 2021, we conducted a longitudinal and observational study in 2 cohorts of patients with chronic lymphocytic leukemia (CLL) (cohort 1, n = 41; cohort 2, n = 81). These cohorts reflect the well-known clinical features of CLL patients, such as Male/Female sex ratio of 2/1, a median age of 70 years at diagnosis, and include patients in first-line therapy (27%) or relapsed/refractory patients (73%). Blood cell counts were followed for each patient during 2 years of ibrutinib treatment. In addition, immunophenotyping and whole-body magnetic resonance imaging (MRI) were assessed in patients from cohort 1. These data were integrated in a newly built mathematical model, inspired by previous mathematical works on CLL treatment and combining dynamical and statistical models, leading to the identification of biological mechanisms associated with the 2 types of clinical responses. This multidisciplinary approach allowed to identify baseline parameters that dictated lymphocytes kinetics upon ibrutinib treatment. Indeed, ibrutinib-induced lymphocytosis defined 2 CLL patient subgroups, transient hyperlymphocytosis (tHL) or prolonged hyperlymphocytosis (pHL), that can be discriminated, before the treatment, by absolute counts of CD4+ T lymphocytes (p = 0.026) and regulatory CD4 T cells (p = 0.007), programmed cell death protein 1 PD1 (p = 0.022) and CD69 (p = 0.03) expression on B leukemic cells, CD19/CD5high/CXCR4low level (p = 0.04), and lymph node cellularity. We also pinpointed that the group of patients identified by the transient hyperlymphocytosis has lower duration response and a poor clinical outcome. The mathematical approach led to the reproduction of patient-specific dynamics and the estimation of associated patient-specific biological parameters, and highlighted that the differences between the 2 groups were mainly due to the production of leukemic B cells in lymph node compartments, and to a lesser extent to T lymphocytes and leukemic B cell egress into bloodstream. Access to additional data, especially longitudinal MRI data, could strengthen the conclusions regarding leukemic B cell dynamics in lymph nodes and the relevance of 2 distinct groups of patients. Conclusions Altogether, our multidisciplinary study provides a better understanding of ibrutinib response and highlights new pharmacodynamic parameters before and along ibrutinib treatment. Since our results highlight a reduced duration response and outcome in patients with transient hyperlymphocytosis, our approach provides support for managing ibrutinib therapy after 3 months of treatment. Trial registration ClinicalTrials.gov NCT02824159.

Understanding human T-cell antigen recognition in health and disease is becoming increasingly instrumental for monitoring T-cell responses to pathogen challenge and for the rational design of T-cell-based therapies targeting cancer, autoimmunity and organ transplant rejection. Here we showcase a quantitative imaging platform which is based on the use of planar glass-supported lipid bilayers (SLBs). The latter are functionalized with antigen (peptide-loaded HLA) as adhesion and costimulatory molecules (ICAM-1, B7-1) to serve as surrogate antigen presenting cell for antigen recognition by T-cells, which are equipped with T-cell antigen receptors (TCRs) sequenced from antigen-specific patient T-cells. We outline in detail, how the experimental use of SLBs supports recoding and analysis of synaptic antigen engagement and calcium signaling at the single cell level in response to user-defined antigen densities for quantitative comparison.

Despite the efficacy of natalizumab in Multiple Sclerosis (MS) treatment, approximately 30% of patients do not respond favorably. Individual heterogeneity of T-cell response to VLA-4 natalizumab-mediated blockade may underlie disparities in treatment efficacy. Here, a high-content cell imaging (HCI) pipeline was implemented to profile the in vitro effects of natalizumab on VLA-4-stimulated leukocytes from MS patients prior to treatment. Unsupervised clustering of image data partially discriminated non-responder MS patients based on morphology, F-actin organization, and signaling-related features in CD8+ T cells. Treatment response was assessed through a Random Forest approach with predictive performance of 91% for a discovery cohort and 70% for a validation cohort. Unfavorable treatment response was associated with the inefficacy of natalizumab to impair the ability of pretreated CD8+ T cells to spread over VCAM-1. Our study unveils that CD8+ T cells from individual MS patients display heterogeneous susceptibility to natalizumab in vitro and highlights the potential of HCI-based pretreatment monitoring to assist individualized treatment prescription.

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.

PDF file - 93K, CIP4 is dispensable for CCR7 internalization (S1), CIP4 knock-down impairs the polarized morphology of JVM3 cells (S2).

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.

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.