Cell shape sensing licenses dendritic cells for homeostatic migration to lymph nodes.

Immune cells experience large cell shape changes during environmental patrolling because of the physical constraints that they encounter while migrating through tissues. These cells can adapt to such deformation events using dedicated shape-sensing pathways. However, how shape sensing affects immune cell function is mostly unknown. Here, we identify a shape-sensing mechanism that increases the expression of the chemokine receptor CCR7 and guides dendritic cell migration from peripheral tissues to lymph nodes at steady state. This mechanism relies on the lipid metabolism enzyme cPLA2, requires nuclear envelope tensioning and is finely tuned by the ARP2/3 actin nucleation complex. We also show that this shape-sensing axis reprograms dendritic cell transcription by activating an IKKß-NF-κB-dependent pathway known to control their tolerogenic potential. These results indicate that cell shape changes experienced by immune cells can define their migratory behavior and immunoregulatory properties and reveal a contribution of the physical properties of tissues to adaptive immunity.

Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

"If you please draw me a cell". Insights from immune cells.

Studies in recent years have shed light on the particular features of cytoskeleton dynamics in immune cells, challenging the classical picture drawn from typical adherent cell lines. New mechanisms linking the dynamics of the membrane-cytoskeleton interface to the mechanical properties of immune cells have been uncovered and shown to be essential for immune surveillance functions. In this Essay, we discuss these features, and propose immune cells as a new playground for cell biologists who try to understand how cells adapt to different microenvironments to fulfil their functions efficiently.

Integrating Physical and Molecular Insights on Immune Cell Migration.

The function of most immune cells depends on their ability to migrate through complex microenvironments, either randomly to patrol for the presence of antigens or directionally to reach their next site of action. The actin cytoskeleton and its partners are key conductors of immune cell migration as they control the intrinsic migratory properties of leukocytes as well as their capacity to respond to cues present in their environment. In this review we focus on the latest discoveries regarding the role of the actomyosin cytoskeleton in optimizing immune cell migration in complex environments, with a special focus on recent insights provided by physical modeling.

Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo.

Upon antigen recognition, T cells form either static (synapses) or migratory (kinapses) contacts with antigen-presenting cells. Addressing whether synapses and kinapses result in distinct T cell receptor (TCR) signals has been hampered by the inability to simultaneously assess T cell phenotype and behavior. Here, we introduced dynamic in situ cytometry (DISC), a combination of intravital multiphoton imaging and flow cytometry-like phenotypic analysis. Taking advantage of CD62L shedding as a marker of early TCR signaling, we examined how T cells sense TCR ligands of varying affinities in vivo. We uncovered three modes of antigen recognition: synapses with the strongest TCR signals, kinapses with robust signaling, and kinapses with weak signaling. As illustrated here, the DISC approach should provide unique opportunities to link immune cell behavior to phenotype and function in vivo.

Dynamics of the membrane-cytoskeleton interface in MHC class II-restricted antigen presentation.

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC-T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.

Visualizing the functional diversification of CD8+ T cell responses in lymph nodes.

CD8(+) T cell responses generate effector cells endowed with distinct functional potentials but the contribution of early events in this process is unclear. Here, we have imaged T cells expressing a fluorescent reporter for the activation of the interferon-γ (IFN-γ) locus during priming in lymph nodes. We have demonstrated marked differences in the efficiency of gene activation during stable T cell-dentritic cell (DC) contacts, influenced in part by signal strength. Imaging the first cell division, we have demonstrated that heterogeneity in T cell functional potential was largely apparent as T cells initiated clonal expansion. Moreover, by analyzing the fate of single activated T cells ex vivo, we have provided evidence that these early differences resulted in clonal progenies with distinct functional properties. Thus, the early set of T cell-DC interactions in lymph nodes largely contribute to the heterogeneity of T cell responses through the generation of functionally divergent clonal progenies.

Signal strength regulates antigen-mediated T-cell deceleration by distinct mechanisms to promote local exploration or arrest.

T lymphocytes are highly motile cells that decelerate upon antigen recognition. These cells can either completely stop or maintain a low level of motility, forming contacts referred to as synapses or kinapses, respectively. Whether similar or distinct molecular mechanisms regulate T-cell deceleration during synapses or kinapses is unclear. Here, we used microfabricated channels and intravital imaging to observe and manipulate T-cell kinapses and synapses. We report that high-affinity antigen induced a pronounced deceleration selectively dependent on Ca(2+) signals and actin-related protein 2/3 complex (Arp2/3) activity. In contrast, low-affinity antigens induced a switch of migration mode that promotes T-cell exploratory behavior, characterized by partial deceleration and frequent direction changes. This switch depended on T-cell receptor binding but was largely independent of downstream signaling. We propose that distinct mechanisms of T-cell deceleration can be triggered during antigenic recognition to favor local exploration and signal integration upon suboptimal stimulus and complete arrest on the best antigen-presenting cells.

A virtual lymph node model to dissect the requirements for T-cell activation by synapses and kinapses.

The initiation of T-cell responses in lymph nodes requires T cells to integrate signals delivered by dendritic cells (DCs) during long-lasting contacts (synapses) or more transient interactions (kinapses). However, it remains extremely challenging to understand how a specific sequence of contacts established by T cells ultimately dictates T-cell fate. Here, we have coupled a computational model of T-cell migration and interactions with DCs with a real-time, flow cytometry-like representation of T-cell activation. In this model, low-affinity peptides trigger T-cell proliferation through kinapses but we show that this process is only effective under conditions of high DC densities and prolonged antigen availability. By contrast, high-affinity peptides favor synapse formation and a vigorous proliferation under a wide range of antigen presentation conditions. In line with the predictions, decreasing the DC density in vivo selectively abolished proliferation induced by the low-affinity peptide. Finally, our results suggest that T cells possess a biochemical memory of previous stimulations of at least 1-2 days. We propose that the stability of T-cell-DC interactions, apart from their signaling potency, profoundly influences the robustness of T-cell activation. By offering the ability to control parameters that are difficult to manipulate experimentally, the virtual lymph node model provides new possibilities to tackle the fundamental mechanisms that regulate T-cell responses elicited by infections or vaccines.

Phenotypic CD8+ T cell diversification occurs before, during, and after the first T cell division.

Effector T cell responses rely on a phenotypically and functionally heterogeneous population of cells. Whether this diversity is programmed before clonal expansion or in later phases as a result of stochastic events or asymmetric cell division is not fully understood. In this study, we first took advantage of a sensitive in vitro assay to analyze the composition of single CD8(+) T cell progenies. Heterogeneity was predominantly observed between progenies of distinct clones, but could also be detected within individual progenies. Furthermore, by physically isolating daughter cells of the first T cell division, we showed that differences in paired daughter cell progenies contributed to intraclonal diversification. Finally, we developed an in vivo limiting dilution assay to compare individual T cell progenies following immunization. We provided evidence for simultaneous intraclonal and interclonal diversification in vivo. Our results support the idea that T cell diversification is a continuous process, initiated before clonal expansion and amplified during the first and subsequent cell divisions.

CD74 supports accumulation and function of regulatory T cells in tumors.

Regulatory T cells (Tregs) are plastic cells playing a pivotal role in the maintenance of immune homeostasis. Tregs actively adapt to the microenvironment where they reside; as a consequence, their molecular and functional profiles differ among tissues and pathologies. In tumors, the features acquired by Tregs remains poorly characterized. Here, we observe that human tumor-infiltrating Tregs selectively overexpress CD74, the MHC class II invariant chain. CD74 has been previously described as a regulator of antigen-presenting cell biology, however its function in Tregs remains unknown. CD74 genetic deletion in human primary Tregs reveals that CD74KO Tregs exhibit major defects in the organization of their actin cytoskeleton and intracellular organelles. Additionally, intratumoral CD74KO Tregs show a decreased activation, a drop in Foxp3 expression, a low accumulation in the tumor, and consistently, they are associated with accelerated tumor rejection in preclinical models in female mice. These observations are unique to tumor conditions as, at steady state, CD74KO-Treg phenotype, survival, and suppressive capacity are unaffected in vitro and in vivo. CD74 therefore emerges as a specific regulator of tumor-infiltrating Tregs and as a target to interfere with Treg anti-tumor activity.

Medium-throughput image-based phenotypic siRNA screen to unveil the molecular basis of B cell polarization.

Cell polarity is an essential and highly conserved process governing cell function. Cell polarization is generally triggered by an external signal that induces the relocation of the centrosome, thus defining the polarity axis of the cell. Here, we took advantage of B cells as a model to study cell polarity and perform a medium-throughput siRNA-based imaging screen to identify new molecular regulators of polarization. We first identified candidates based on a quantitative proteomic analysis of proteins differentially associated with the centrosome of resting non-polarized and stimulated polarized B cells. We then targeted 233 candidates in a siRNA screen and identified hits regulating the polarization of the centrosome and/or lysosomes in B cells upon stimulation. Our dataset of proteomics, images, and polarity indexes provides a valuable source of information for a broad community of scientists interested in the molecular mechanisms regulating cell polarity.

The WASp L272P gain-of-function mutation alters dendritic cell coordination of actin dynamics for migration and adhesion.

Dendritic cells (DCs) devoid of the actin regulator Wiskott-Aldrich syndrome protein (WASp) show reduced directed migration and decreased formation of podosome adhesion structures. We examined DCs expressing a gain-of-function mutation in WASp, WASp L272P, identified in X-linked neutropenia patients. Analysis of WASp L272P DCs was compared to WASp-deficient DCs to examine how WASp activity influences DC migratory responses. In confined space, WASp-deficient DCs had increased migration speed whereas WASp L272P DCs had similar average speed but increased speed fluctuations, reduced displacement, and atypical rounded morphology, compared to wild-type (WT) DCs. Using an ear inflammation model and flow cytometry analysis, WT, WASp-deficient, and WASp L272P DCs were found to migrate in comparable numbers to the draining lymph nodes (LNs). However, histology analysis revealed that migratory DCs of WASp deficient and WASp L272P mice were mainly located in the collagenous capsule of the LN whereas WT DCs were located inside the LN. Analysis of ultrastructural features revealed that WASp L272P DCs had reduced cell area but formed larger podosome structures when compared to WT DCs. Together, our data suggest that WASp activity regulates DC migration and that loss-of-function and gain-of-function in WASp activity lead to different and phenotype-specific DC migratory behavior.

Barotaxis: How cells live and move under pressure.

Cell migration is an essential process that controls many physiological functions ranging from development to immunity. In vivo, cells are guided by a combination of physical and chemical cues. Chemokines have been the center of attention for years, but the role of physical properties of tissues has been under-investigated, despite the fact that these properties can be drastically modified in pathology. Here, we discuss the role of one important tissue physical property, hydraulic resistance, in cell guidance, a phenomenon referred to as barotaxis, and describe the underlying physical principles and molecular mechanisms. Finally, we speculate on the putative role of barotaxis in physiological processes involving immune and cancer cells.

Macropinocytosis Overcomes Directional Bias in Dendritic Cells Due to Hydraulic Resistance and Facilitates Space Exploration.

The migration of immune cells can be guided by physical cues imposed by the environment, such as geometry, rigidity, or hydraulic resistance (HR). Neutrophils preferentially follow paths of least HR in vitro, a phenomenon known as barotaxis. The mechanisms and physiological relevance of barotaxis remain unclear. We show that barotaxis results from the amplification of a small force imbalance by the actomyosin cytoskeleton, resulting in biased directional choices. In immature dendritic cells (DCs), actomyosin is recruited to the cell front to build macropinosomes. These cells are therefore insensitive to HR, as macropinocytosis allows fluid transport across these cells. This may enhance their space exploration capacity in vivo. Conversely, mature DCs down-regulate macropinocytosis and are thus barotactic. Modeling suggests that HR may help guide these cells to lymph nodes where they initiate immune responses. Hence, DCs can either overcome or capitalize on the physical obstacles they encounter, helping their immune-surveillance function.

In Vivo Imaging of T Cell Immunological Synapses and Kinapses in Lymph Nodes.

T cells can become activated in lymph nodes following a diverse set of interactions with antigen-presenting cells. These cellular contacts range from short and dynamic to stable and long-lasting interactions, termed kinapses and synapses, respectively. Here, we describe a methodology to generate naïve T cells expressing a fluorescent probe of interest through the generation of bone marrow chimeras and to image T cell dynamics using intravital two-photon microscopy. In these settings, the formation of kinapses and synapses can be triggered by the administration of low and high affinity peptides, respectively. Finally, 3D cell tracking can help classify distinct T cell behaviors. These approaches should offer new possibilities for dissecting the process of T cell activation in vivo.

Microchannels for the Study of T Cell Immunological Synapses and Kinapses.

T Cells can form very stable (synapses) or very transient and migratory (kinapses) contacts with antigen-presenting cells. Here, we describe how microchannels can be used to conveniently study the distinct dynamics of T cells during antigen recognition. Microchannels provide a controlled confined environment that promotes T cell migration and recapitulates kinapse and synapse behaviors when coated with appropriate pMHC molecules. We also depict the advantages of this in vitro approach for addressing mechanistic issues and for analysis.

Caveolin-1 Expression Increases upon Maturation in Dendritic Cells and Promotes Their Migration to Lymph Nodes Thereby Favoring the Induction of CD8+ T Cell Responses.

Dendritic cell (DC) trafficking from peripheral tissues to lymph nodes (LNs) is a key step required to initiate T cell responses against pathogens as well as tumors. In this context, cellular membrane protrusions and the actin cytoskeleton are essential to guide DC migration towards chemotactic signals. Caveolin-1 (CAV1) is a scaffolding protein that modulates signaling pathways leading to remodeling of the actin cytoskeleton and enhanced migration of cancer cells. However, whether CAV1 is relevant for DC function and specifically for DC migration to LNs is unknown. Here, we show that CAV1 expression is upregulated in DCs upon LPS- and TNF-α-induced maturation. CAV1 deficiency did not affect differentiation, maturation, or the ability of DCs to activate CD8+ T cells in vitro. However, CAV1-deficient (CAV1-/-) DCs displayed reduced in vivo trafficking to draining LNs in control and inflammatory conditions. In vitro, CAV1-/- DCs showed reduced directional migration in CCL21 gradients in transwell assays without affecting migration velocity in confined microchannels or three-dimensional collagen matrices. In addition, CAV1-/- DCs displayed reduced activation of the small GTPase Rac1, a regulator of actin cytoskeletal remodeling, and lower numbers of F-actin-forming protrusions. Furthermore, mice adoptively transferred with peptide-pulsed CAV1-/- DCs showed reduced CD8+ T cell responses and antitumor protection. Our results suggest that CAV1 promotes the activation of Rac1 and the formation of membrane protrusions that favor DC chemotactic trafficking toward LNs where they can initiate cytotoxic T cell responses.

TLR3-Induced Maturation of Murine Dendritic Cells Regulates CTL Responses by Modulating PD-L1 Trafficking.

Targeting TLR3 through formulations of polyI:C is widely studied as an adjuvant in cancer immunotherapy. The efficacy of such targeting has been shown to increase in combination with anti-PD-L1 treatment. Nevertheless, the mechanistic details of the effect of polyI:C on DC maturation and the impact on T-DC interactions upon PD-L1 blockade is largely unknown. Here we found that although DC treatment with polyI:C induced a potent inflammatory response including the production of type I interferon, polyI:C treatment of DCs impaired activation of peptide specific CD8+ T cells mainly due to PD-L1. Interestingly, we found that PD-L1 trafficking to the cell surface is regulated in two waves in polyI:C-treated DCs. One induced upon overnight treatment and a second more rapid one, specific to polyI:C treatment, was induced upon CD40 signaling leading to a further increase in surface PD-L1 in DCs. The polyI:C-induced cell surface PD-L1 reduced the times of contact between DCs and T cells, potentially accounting for limited T cell activation. Our results reveal a novel CD40-dependent regulation of PD-L1 trafficking induced upon TLR3 signaling that dictates its inhibitory activity. These results provide a mechanistic framework to understand the efficacy of anti-PD-L1 cancer immunotherapy combined with TLR agonists.