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.

Thoracic Proton Minibeam Radiation Therapy: Tissue Preservation and Survival Advantage Over Conventional Proton Therapy.

PURPOSE: Proton minibeam radiation therapy (pMBRT) is an innovative radiation therapy approach that highly modulates the spatial dimension of the dose delivery using narrow, parallel, and submillimetric proton beamlets. pMBRT has proven its remarkable healthy tissue preservation in the brain and skin. This study assesses the potential advantages of pMBRT for thoracic irradiations compared with conventional radiation therapy in terms of normal tissue toxicity. The challenge here was the influence of respiratory motion on the typical peak and valley dose patterns of pMBRT and its potential biologic effect. METHODS AND MATERIALS: The whole thorax of naïve C57BL/6 mice received one fraction of high dose (18 Gy) pMBRT or conventional proton therapy (CPT) without any respiratory control. The development of radiation-induced pulmonary fibrosis was longitudinally monitored using cone beam computed tomography. Anatomopathologic analysis was carried out at 9 months postirradiation and focused on the reaction of the lungs' parenchyma and the response of cell types involved in the development of radiation-induced fibrosis and lung regeneration as alveolar type II epithelial cells, club cells, and macrophages. RESULTS: pMBRT has milder effects on survival, skin reactions, and lung fibrosis compared with CPT. The pMBRT-induced lung changes were more regional and less severe, with evidence of potential reactive proliferation of alveolar type II epithelial cells and less extensive depletion of club cells and macrophage invasion than the more damaging effects observed in CPT. CONCLUSIONS: pMBRT appears suitable to treat moving targets, holding a significant ability to preserve healthy lung tissue, even without respiratory control or precise targeting.

Plasma Membrane Blebbing Is Controlled by Subcellular Distribution of Vimentin Intermediate Filaments.

The formation of specific cellular protrusions, plasma membrane blebs, underlies the amoeboid mode of cell motility, which is characteristic for free-living amoebae and leukocytes, and can also be adopted by stem and tumor cells to bypass unfavorable migration conditions and thus facilitate their long-distance migration. Not all cells are equally prone to bleb formation. We have previously shown that membrane blebbing can be experimentally induced in a subset of HT1080 fibrosarcoma cells, whereas other cells in the same culture under the same conditions retain non-blebbing mesenchymal morphology. Here we show that this heterogeneity is associated with the distribution of vimentin intermediate filaments (VIFs). Using different approaches to alter the VIF organization, we show that blebbing activity is biased toward cell edges lacking abundant VIFs, whereas the VIF-rich regions of the cell periphery exhibit low blebbing activity. This pattern is observed both in interphase fibroblasts, with and without experimentally induced blebbing, and during mitosis-associated blebbing. Moreover, the downregulation of vimentin expression or displacement of VIFs away from the cell periphery promotes blebbing even in cells resistant to bleb-inducing treatments. Thus, we reveal a new important function of VIFs in cell physiology that involves the regulation of non-apoptotic blebbing essential for amoeboid cell migration and mitosis.

PD-1 inhibits T cell actin remodeling at the immunological synapse independently of its signaling motifs.

Engagement of the receptor programmed cell death molecule 1 (PD-1) by its ligands PD-L1 and PD-L2 inhibits T cell-mediated immune responses. Blocking such signaling provides the clinical effects of PD-1-targeted immunotherapy. Here, we investigated the mechanisms underlying PD-1-mediated inhibition. Because dynamic actin remodeling is crucial for T cell functions, we characterized the effects of PD-1 engagement on actin remodeling at the immunological synapse, the interface between a T cell and an antigen-presenting cell (APC) or target cell. We used microscopy to analyze the formation of immunological synapses between PD-1+ Jurkat cells or primary human CD8+ cytotoxic T cells and APCs that presented T cell-activating antibodies and were either positive or negative for PD-L1. PD-1 binding to PD-L1 inhibited T cell spreading induced by antibody-mediated activation, which was characterized by the absence of the F-actin-dense distal lamellipodial network at the immunological synapse and the Arp2/3 complex, which mediates branched actin formation. PD-1-induced inhibition of actin remodeling also prevented the characteristic deformation of T cells that contact APCs and the release of cytotoxic granules. We showed that the effects of PD-1 on actin remodeling did not require its tyrosine-based signaling motifs, which are thought to mediate the co-inhibitory effects of PD-1. Our study highlights a previously unappreciated mechanism of PD-1-mediated suppression of T cell activity, which depends on the regulation of actin cytoskeleton dynamics in a signaling motif-independent manner.

Nuclear envelope disruption triggers hallmarks of aging in lung alveolar macrophages.

Aging is characterized by gradual immune dysfunction and increased disease risk. Genomic instability is considered central to the aging process, but the underlying mechanisms of DNA damage are insufficiently defined. Cells in confined environments experience forces applied to their nucleus, leading to transient nuclear envelope rupture (NER) and DNA damage. Here, we show that Lamin A/C protects lung alveolar macrophages (AMs) from NER and hallmarks of aging. AMs move within constricted spaces in the lung. Immune-specific ablation of lamin A/C results in selective depletion of AMs and heightened susceptibility to influenza virus-induced pathogenesis and lung cancer growth. Lamin A/C-deficient AMs that persist display constitutive NER marks, DNA damage and p53-dependent senescence. AMs from aged wild-type and from lamin A/C-deficient mice share a lysosomal signature comprising CD63. CD63 is required to limit damaged DNA in macrophages. We propose that NER-induced genomic instability represents a mechanism of aging in AMs.

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.

Homeostatic activation of aryl hydrocarbon receptor by dietary ligands dampens cutaneous allergic responses by controlling Langerhans cells migration.

Dietary compounds can affect the development of inflammatory responses at distant sites. However, the mechanisms involved remain incompletely understood. Here, we addressed the influence on allergic responses of dietary agonists of aryl hydrocarbon receptor (AhR). In cutaneous papain-induced allergy, we found that lack of dietary AhR ligands exacerbates allergic responses. This phenomenon was tissue-specific as airway allergy was unaffected by the diet. In addition, lack of dietary AhR ligands worsened asthma-like allergy in a model of 'atopic march.' Mice deprived of dietary AhR ligands displayed impaired Langerhans cell migration, leading to exaggerated T cell responses. Mechanistically, dietary AhR ligands regulated the inflammatory profile of epidermal cells, without affecting barrier function. In particular, we evidenced TGF-ß hyperproduction in the skin of mice deprived of dietary AhR ligands, explaining Langerhans cell retention. Our work identifies an essential role for homeostatic activation of AhR by dietary ligands in the dampening of cutaneous allergic responses and uncovers the importance of the gut-skin axis in the development of allergic diseases.

RELA tunes innate-like interferon I/III responses in human T cells.

In innate immune cells, intracellular sensors such as cGAS-STING stimulate type I/III interferon (IFN) expression, which promotes antiviral defense and immune activation. However, how IFN-I/III expression is controlled in adaptive cells is poorly understood. Here, we identify a transcriptional rheostat orchestrated by RELA that confers human T cells with innate-like abilities to produce IFN-I/III. Despite intact cGAS-STING signaling, IFN-I/III responses are stunted in CD4+ T cells compared with dendritic cells or macrophages. We find that lysine residues in RELA tune the IFN-I/III response at baseline and in response to STING stimulation in CD4+ T cells. This response requires positive feedback driven by cGAS and IRF7 expression. By combining RELA with IRF3 and DNA demethylation, IFN-I/III production in CD4+ T cells reaches levels observed in dendritic cells. IFN-I/III production provides self-protection of CD4+ T cells against HIV infection and enhances the elimination of tumor cells by CAR T cells. Therefore, innate-like functions can be tuned and leveraged in human T cells.

Release of HIV-1 particles from macrophages is promoted by an anchored cytoskeleton and driven by mechanical constraints.

A feature of HIV-1 replication in macrophages is that viral assembly occurs at the limiting membrane of a compartment often named the virus-containing compartment (VCC). Assembled virions accumulate in the lumen of the VCC, from where they can be released into the extracellular medium via mechanisms that remain poorly described. Here, we show that the actin cytoskeleton contributes to this process by performing experiments combining pharmacological and mechanical perturbations with imaging and biochemical analysis. We found that jasplakinolide inhibited HIV-1 release from macrophages and led to scattering of the compartment. Concomitantly, both the integrin CD18 (ß2-integrin) and the phosphorylated form of PYK2 (also known as PTK2B) were displaced away from the VCC. Inhibition of PYK2 activity promoted retention of viral particles in VCCs that lost their connections to the surface. Finally, in infected macrophages undergoing frustrated phagocytosis, VCCs rapidly trafficked to the basal membrane and released their viral content, in a manner dependent on their association with the actin cytoskeleton. These results highlight that the trafficking of VCCs and virus release are intimately linked to a reorganization of the macrophage actin cytoskeleton that can be modulated by external physical cues.

Spatial Positioning and Matrix Programs of Cancer-Associated Fibroblasts Promote T-cell Exclusion in Human Lung Tumors.

It is currently accepted that cancer-associated fibroblasts (CAF) participate in T-cell exclusion from tumor nests. To unbiasedly test this, we used single-cell RNA sequencing coupled with multiplex imaging on a large cohort of lung tumors. We identified four main CAF populations, two of which are associated with T-cell exclusion: (i) MYH11+αSMA+ CAF, which are present in early-stage tumors and form a single cell layer lining cancer aggregates, and (ii) FAP+αSMA+ CAF, which appear in more advanced tumors and organize in patches within the stroma or in multiple layers around tumor nests. Both populations orchestrate a particular structural tissue organization through dense and aligned fiber deposition compared with T cell-permissive CAF. Yet they produce distinct matrix molecules, including collagen IV (MYH11+αSMA+ CAF) and collagen XI/XII (FAP+αSMA+ CAF). Hereby, we uncovered unique molecular programs of CAF driving T-cell marginalization, whose targeting should increase immunotherapy efficacy in patients bearing T cell-excluded tumors. SIGNIFICANCE: The cellular and molecular programs driving T-cell marginalization in solid tumors remain unclear. Here, we describe two CAF populations associated with T-cell exclusion in human lung tumors. We demonstrate the importance of pairing molecular and spatial analysis of the tumor microenvironment, a prerequisite to developing new strategies targeting T cell-excluding CAF. See related commentary by Sherman, p. 2501. This article is highlighted in the In This Issue feature, p. 2483.

Endocytic membrane repair by ESCRT-III controls antigen export to the cytosol during antigen cross-presentation.

Despite its crucial role in initiation of cytotoxic immune responses, the molecular pathways underlying antigen cross-presentation remain incompletely understood. The mechanism of antigen exit from endocytic compartments into the cytosol is a long-standing matter of controversy, confronting two main models: transfer through specific channels/transporters or rupture of endocytic membranes and leakage of luminal content. By monitoring the occurrence of intracellular damage in conventional dendritic cells (cDCs), we show that cross-presenting cDC1s display more frequent endomembrane injuries and increased recruitment of endosomal sorting complex required for transport (ESCRT)-III, the main repair system for intracellular membranes, relative to cDC2s. Silencing of CHMP2a or CHMP4b, two effector subunits of ESCRT-III, enhances cytosolic antigen export and cross-presentation. This phenotype is partially reversed by chemical inhibition of RIPK3, suggesting that endocytic damage is related to basal activation of the necroptosis pathway. Membrane repair therefore proves crucial in containing antigen export to the cytosol and cross-presentation in cDCs.

HLA-independent T cell receptors for targeting tumors with low antigen density.

Chimeric antigen receptors (CARs) are receptors for antigen that direct potent immune responses. Tumor escape associated with low target antigen expression is emerging as one potential limitation of their efficacy. Here we edit the TRAC locus in human peripheral blood T cells to engage cell-surface targets through their T cell receptor-CD3 complex reconfigured to utilize the same immunoglobulin heavy and light chains as a matched CAR. We demonstrate that these HLA-independent T cell receptors (HIT receptors) consistently afford high antigen sensitivity and mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. We demonstrate that the functional persistence of HIT T cells can be augmented by constitutive coexpression of CD80 and 4-1BBL. Finally, we validate the increased antigen sensitivity afforded by HIT receptors in xenograft mouse models of B cell leukemia and acute myeloid leukemia, targeting CD19 and CD70, respectively. Overall, HIT receptors are well suited for targeting cell surface antigens of low abundance.

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.

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.

Compromised nuclear envelope integrity drives TREX1-dependent DNA damage and tumor cell invasion.

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.

Inhibition of HIV infection by structural proteins of the inner nuclear membrane is associated with reduced chromatin dynamics.

The human immunodeficiency virus (HIV) enters the nucleus to establish infection, but the role of nuclear envelope proteins in this process is incompletely understood. Inner nuclear transmembrane proteins SUN1 and SUN2 connect nuclear lamins to the cytoskeleton and participate in the DNA damage response (DDR). Increased levels of SUN1 or SUN2 potently restrict HIV infection through an unresolved mechanism. Here, we find that the antiviral activities of SUN1 and SUN2 are distinct. HIV-1 and HIV-2 are preferentially inhibited by SUN1 and SUN2, respectively. We identify DNA damage inducers that stimulate HIV-1 infection and show that SUN1, but not SUN2, neutralizes this effect. Finally, we show that chromatin movements and nuclear rotations are associated with the effects of SUN proteins and Lamin A/C on infection. These results reveal an emerging role of chromatin dynamics and the DDR in the control of HIV infection by structural components of the nuclear envelope.

Specificities of exosome versus small ectosome secretion revealed by live intracellular tracking of CD63 and CD9.

Despite their roles in intercellular communications, the different populations of extracellular vesicles (EVs) and their secretion mechanisms are not fully characterized: how and to what extent EVs form as intraluminal vesicles of endocytic compartments (exosomes), or at the plasma membrane (PM) (ectosomes) remains unclear. Here we follow intracellular trafficking of the EV markers CD9 and CD63 from the endoplasmic reticulum to their residency compartment, respectively PM and late endosomes. We observe transient co-localization at both places, before they finally segregate. CD9 and a mutant CD63 stabilized at the PM are more abundantly released in EVs than CD63. Thus, in HeLa cells, ectosomes are more prominent than exosomes. By comparative proteomic analysis and differential response to neutralization of endosomal pH, we identify a few surface proteins likely specific of either exosomes (LAMP1) or ectosomes (BSG, SLC3A2). Our work sets the path for molecular and functional discrimination of exosomes and small ectosomes in any cell type.

Macrophages Maintain Epithelium Integrity by Limiting Fungal Product Absorption.

The colon is primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be toxic to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows rapid quality check of absorbed fluids to avoid intoxication of colonocytes. This mechanism relies on a population of distal colon macrophages that are equipped with "balloon-like" protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids containing fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis. VIDEO ABSTRACT.

HIV-2-Infected Macrophages Produce and Accumulate Poorly Infectious Viral Particles.

A significant proportion of HIV-2-infected patients exhibit natural virological control that is generally absent from HIV-1-infected patients. Along with CD4+ T cells, HIV-1 targets macrophages which may contribute to viral spreading and the latent reservoir. We have studied the relationship between macrophages and HIV-2, focusing on post-entry steps. HIV-2-infected monocyte-derived macrophages (MDMs) produced substantial amounts of viral particles that were largely harbored intracellularly. New viruses assembled at the limiting membrane of internal compartments similar to virus-containing compartments (VCCs) described for HIV-1. VCCs from MDMs infected with either virus shared protein composition and morphology. Strikingly, HIV-2 Gag was mostly absent from the cytosol and almost exclusively localized to the VCCs, whereas HIV-1 Gag was distributed in both locations. Ultrastructural analyses of HIV-2-infected MDMs revealed the presence of numerous VCCs containing both immature and mature particles in the lumen. HIV-2 particles produced de novo by MDMs were poorly infectious in reporter cells and in transmission to activated T cells through a process that appeared independent of BST2 restriction. Rather than being involved in viral spreading, HIV-2-infected macrophages may represent a cell-associated source of viral antigens that can participate in the immune control of HIV-2 infection.

Actomyosin-driven force patterning controls endocytosis at the immune synapse.

An important channel of cell-to-cell communication is direct contact. The immune synapse is a paradigmatic example of such type of interaction: it forms upon engagement of antigen receptors in lymphocytes by antigen-presenting cells and allows the local exchange of molecules and information. Although mechanics has been shown to play an important role in this process, how forces organize and impact on synapse function is unknown. We find that mechanical forces are spatio-temporally patterned at the immune synapse: global pulsatile myosin II-driven tangential forces are observed at the synapse periphery while localised forces generated by invadosome-like F-actin protrusions are detected at its centre. Noticeably, we observe that these force-producing actin protrusions constitute the main site of antigen extraction and endocytosis and require myosin II contractility to form. The interplay between global and local forces dictated by the organization of the actomyosin cytoskeleton therefore controls endocytosis at the immune synapse.