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A high diversity of αβ T cell receptors (TCRs), capable of recognizing virtually any pathogen but also self-antigens, is generated during T cell development in the thymus. Nevertheless, a strict developmental program supports the selection of a self-tolerant T cell repertoire capable of responding to foreign antigens. The steps of T cell selection are controlled by cortical and medullary stromal niches, mainly composed of thymic epithelial cells and dendritic cells. The integration of important cues provided by these specialized niches, including ( a) the TCR signal strength induced by the recognition of self-peptide-MHC complexes, ( b) costimulatory signals, and ( c) cytokine signals, critically controls T cell repertoire selection. This review discusses our current understanding of the signals that coordinate positive selection, negative selection, and agonist selection of Foxp3+regulatory T cells. It also highlights recent advances that have unraveled the functional diversity of thymic antigen-presenting cell subsets implicated in T cell selection.

International audience; Lung-resident memory B cells (MBCs) provide localized protection against reinfection in respiratory airways. Currently, the biology of these cells remains largely unexplored. Here, we combined influenza and SARS-CoV-2 infection with fluorescent-reporter mice to identify MBCs regardless of antigen specificity. We found that two main transcriptionally distinct subsets of MBCs colonized the lung peribronchial niche after infection. These subsets arose from different progenitors and were both class switched, somatically mutated, and intrinsically biased in their differentiation fate toward plasma cells. Combined analysis of antigen specificity and B cell receptor repertoire segregated these subsets into “bona fide” virus-specific MBCs and “bystander” MBCs with no apparent specificity for eliciting viruses generated through an alternative permissive process. Thus, diverse transcriptional programs in MBCs are not linked to specific effector fates but rather to divergent strategies of the immune system to simultaneously provide rapid protection from reinfection while diversifying the initial B cell repertoire.

SummaryNatural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein ‘cytokine inducible SH2-containing protein’ (CISH) in NK cells to evaluate the impact on their functions and antitumor properties.MethodsTo further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions.ResultsIn Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions.ConclusionThis study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

International audience; Natural Killer (NK) cells are innate lymphoid cells (ILCs) capable of recognizing and directly killing tumor cells. They also secrete cytokines and chemokines, which participate in the shaping of the adaptive response. NK cells identify tumor cells and are activated through a net positive signal from inhibitory and activating receptors. Several activating NK cell receptors are coupled to adaptor molecules containing an immunoreceptor tyrosine-based activation motif (ITAM). These receptors include CD16 and the natural cytotoxic receptors NKp46, NKp44, NKp30 in humans. The powerful antitumor NK cell response triggered by these activating receptors has made them attractive targets for exploitation in immunotherapy. In this review, we will discuss the different activating receptors associated with ITAM-bearing cell surface receptors expressed on NK cells, their modulations in the tumor context and the various therapeutic tools developed to boost NK cell responses in cancer patients.

International audience; DCs do not just excel in antigen presentation. They orchestrate information transfer from innate to adaptive immunity by sensing and integrating a variety of danger signals, and translating them to naïve T cells, to mount specifically tailored immune responses. This is accomplished by distinct DC types specialized in different functions and because each DC is functionally plastic, assuming different activation states depending on the input signals received. Mouse models hold the key to untangle this complexity and determine which DC types and activation states contribute to which functions. Here, we aim to provide comprehensive information for selecting the most appropriate mutant mouse strains to address specific research questions on DCs, considering three in vivo experimental approaches: (i) interrogating the roles of DC types through their depletion; (ii) determining the underlying mechanisms by specific genetic manipulations; (iii) deciphering the spatiotemporal dynamics of DC responses. We summarize the advantages, caveats, suggested use, and perspectives for a variety of mutant mouse strains, discussing in more detail the most widely used or accurate models. Finally, we discuss innovative strategies to improve targeting specificity and next-generation mutant mouse models, and briefly address how humanized mouse models can accelerate translation into the clinic.

Interactions of developing T cells with Aire+ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEChi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire+ mTEChi. However, it remains unknown whether thymocytes control the precursors of Aire+ mTEChi that are contained in mTEClo cells or other mTEClo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4+ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4+ thymocytes induce key transcriptional regulators in mTEClo and control the composition of mTEClo subsets, including Aire+ mTEChi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEClo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4+ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4+ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.

International audience; Natural Killer (NK) cells are potent anti-leukemic immune effectors. However, they display multiple defects in acute myeloid leukemia (AML) patients leading to reduced anti-tumor potential. Our limited understanding of the mechanisms underlying these defects hampers the development of strategies to restore NK cell potential. Here, we have used a mouse model of AML to gain insight into these mechanisms. We found that leukemia progression resulted in NK cell maturation defects and functional alterations. Next, we assessed NK cell cytokine signaling governing their behavior. We showed that NK cells from leukemic mice exhibit constitutive IL-15/mTOR signaling and type I IFN signaling. However, these cells failed to respond to IL-15 stimulation in vitro as illustrated by reduced activation of the mTOR pathway. Moreover, our data suggest that mTOR-mediated metabolic responses were reduced in NK cells from AML-bearing mice. Noteworthy, the reduction of mTOR-mediated activation of NK cells during AML development partially rescued NK cell metabolic and functional defects. Altogether, our data strongly suggest that NK cells from leukemic mice are metabolically and functionally exhausted as a result of a chronic cytokine activation, at least partially IL-15/mTOR signaling. NK cells from AML patients also displayed reduced IL-2/15Rβ expression and showed cues of reduced metabolic response to IL-15 stimulation in vitro , suggesting that a similar mechanism might occur in AML patients. Our study pinpoints the dysregulation of cytokine stimulation pathways as a new mechanism leading to NK cell defects in AML.

Copyright © 2021 Varghese, Mukherjee, Al-Mohanna, Saleh, Almajhdi, Beirag, Alkahtani, Rajkumari, Nal Rogier, Sim, Idicula-Thomas, Madan, Murugaiah and Kishore. The complement system is designed to recognise and eliminate invading pathogens via activation of classical, alternative and lectin pathways. Human properdin stabilises the alternative pathway C3 convertase, resulting in an amplification loop that leads to the formation of C5 convertase, thereby acting as a positive regulator of the alternative pathway. It has been noted that human properdin on its own can operate as a pattern recognition receptor and exert immune functions outside its involvement in complement activation. Properdin can bind directly to microbial targets via DNA, sulfatides and glycosaminoglycans, apoptotic cells, nanoparticles, and well-known viral virulence factors. This study was aimed at investigating the complement-independent role of properdin against Influenza A virus infection. As one of the first immune cells to arrive at the site of IAV infection, we show here that IAV challenged neutrophils released properdin in a time-dependent manner. Properdin was found to directly interact with haemagglutinin, neuraminidase and matrix 1 protein Influenza A virus proteins in ELISA and western blot. Furthermore, modelling studies revealed that properdin could bind HA and NA of the H1N1 subtype with higher affinity compared to that of H3N2 due to the presence of an HA cleavage site in H1N1. In an infection assay using A549 cells, properdin suppressed viral replication in pH1N1 subtype while promoting replication of H3N2 subtype, as revealed by qPCR analysis of M1 transcripts. Properdin treatment triggered an anti-inflammatory response in H1N1-challenged A549 cells and a pro-inflammatory response in H3N2-infected cells, as evident from differential mRNA expression of TNF-α, NF-κB, IFN-α, IFN-β, IL-6, IL-12 and RANTES. Properdin treatment also reduced luciferase reporter activity in MDCK cells transduced with H1N1 pseudotyped lentiviral particles; however, it was increased in the case of pseudotyped H3N2 particles. Collectively, we conclude that infiltrating neutrophils at the site of IAV infection can release properdin, which then acts as an entry inhibitor for pandemic H1N1 subtype while suppressing viral replication and inducing an anti-inflammatory response. H3N2 subtype can escape this immune restriction due to altered haemagglutinin and neuraminindase, leading to enhanced viral entry, replication and pro-inflammatory response. Thus, depending on the subtype, properdin can either limit or aggravate IAV infection in the host. Department of Biotechnology (DBT), India [BT/PR40165/BTIS/137/12/2021]; Department of Science and Technology [SR/WOS-A/LS-38/2018] research fellowship; Researchers Supporting Project (RSP 2021/26), King Saud University, Riyadh, Saudi Arabia.

Summary Specific antigen recognition by T cell receptor (TCR) activates TCR signaling pathway, leading to T cell proliferation and differentiation into effector and memory cells. Herein, we describe protocols for TCR stimulation assays, including procedures for the isolation and enrichment of mouse splenic T cells for ex vivo TCR stimulation with anti-CD3/CD28 antibodies, and the use of ovalbumin-OT-II mouse model for in vivo TCR stimulation. We applied this protocol to show that MYC protein is essential for T cell proliferation and differentiation. For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021) .

Infection with the novel coronavirus SARS-CoV-2 triggers severe illness with high mortality in a subgroup of patients. Such a critical course of COVID-19 is thought to be associated with the development of cytokine storm, a condition seen in macrophage activation syndrome (MAS) and secondary hemophagocytic lymphohistiocytosis (HLH). However, specific data demonstrating a clear association of cytokine storm with severe COVID-19 are still lacking. The aim of this study was to directly address whether immune activation in COVID-19 does indeed mimic the conditions found in these classic cytokine storm syndromes.Levels of 22 biomarkers were quantified in serum samples from patients with COVID-19 (n = 30 patients, n = 83 longitudinal samples in total), patients with secondary HLH/MAS (n = 50), and healthy controls (n = 9). Measurements were performed using bead array assays and single-marker enzyme-linked immunosorbent assay. Serum biomarker levels were assessed for correlations with disease outcome.In patients with secondary HLH/MAS, we observed pronounced activation of the interleukin-18 (IL-18)-interferon-γ axis, increased serum levels of IL-1 receptor antagonist, intercellular adhesion molecule 1, and IL-8, and strongly reduced levels of soluble Fas ligand in the course of SARS-CoV-2 infection. These observations appeared to discriminate immune dysregulation in critical COVID-19 from the well-recognized characteristics of other cytokine storm syndromes.Serum biomarker profiles clearly separate COVID-19 from MAS or secondary HLH in terms of distinguishing the severe systemic hyperinflammation that occurs following SARS-CoV-2 infection. These findings could be useful in determining the efficacy of drugs targeting key molecules and pathways specifically associated with systemic cytokine storm conditions in the treatment of COVID-19.

International audience; Follicular T helper cells (Tfh) are a specialized subset of CD4 effector T cells that are crucial for germinal center (GC) reactions and for selecting B cells to undergo affinity maturation. Despite this central role for humoral immunity, only few data exist about their clonal distribution when multiple lymphoid organs are exposed to the same antigen (Ag) as it is the case in autoimmunity. Here, we used an autoantibody-mediated disease model of the skin and injected one auto-Ag into the two footpads of the same mouse and analyzed the T cell receptor (TCR)β sequences of Tfh located in GCs of both contralateral draining lymph nodes. We found that over 90% of the dominant GC-Tfh clonotypes were shared in both lymph nodes but only transiently. The initially dominant Tfh clonotypes especially declined after establishment of chronic disease while GC reaction and autoimmune disease continued. Our data demonstrates a dynamic behavior of Tfh clonotypes under autoimmune conditions and emphasizes the importance of the time point for distinguishing auto-Ag-specific Tfh clonotypes from potential bystander activated ones.

Upon antigen exposure, activated B cells in antigen-draining lymphoid organs form microanatomical structures, called germinal centers (GCs), where affinity maturation occurs. Within the GC microenvironment, GC B cells undergo proliferation and B cell receptor (BCR) genes somatic hypermutation in the dark zone (DZ), and affinity-based selection in the light zone (LZ). In the current paradigm of GC dynamics, high-affinity LZ B cells may be selected by cognate T- follicular helper cells to either differentiate into plasma cells or memory B cells, or re-enter the DZ and initiate a new round of proliferation and BCR diversification, before migrating back to the LZ. Given the diversity of cell states and potential cell fates that GC B cells may adopt, the two-state DZ-LZ paradigm has been challenged by studies that explored GC B-cell heterogeneity with a variety of single-cell technologies. Here, we review studies and single-cell technologies which have allowed to refine the working model of GC B-cell cellular and molecular heterogeneity during affinity maturation. This review also covers the use of single-cell quantitative data for mathematical modeling of GC reactions, and the application of single-cell genomics to the study of GC-derived malignancies.

International audience; Lymphomas are cancers deriving from lymphocytes, arising preferentially in secondary lymphoid organs, and represent the 6th cancer worldwide and the most frequent blood cancer. The majority of B cell Non-Hodgkin lymphomas (B-NHL) develop from germinal center (GC) experienced mature B cells. GCs are transient structures that form in lymphoid organs in response to antigen exposure of naive B cells, and where B cell receptor (BCR) affinity maturation occurs to promote B cell differentiation into memory B and plasma cells producing high-affinity antibodies. Genomic instability associated with the somatic hypermutation (SHM) and class-switch recombination (CSR) processes during GC transit enhance susceptibility to malignant transformation. Most B cell differentiation steps in the GC are at the origin of frequent B cell malignant entities, namely Follicular Lymphoma (FL) and GCB diffuse large B cell lymphomas (GCB-DLBCL). Over the past decade, large sequencing efforts have provided a great boost in the identification of candidate oncogenes and tumor suppressors involved in FL and DLBCL oncogenesis. Mouse models have been instrumental to accurately mimic in vivo lymphoma-specific mutations and interrogate their normal function in the GC context and their oncogenic function leading to lymphoma onset. The limited access of biopsies during the initiating steps of the disease, the cellular and (epi)genetic heterogeneity of individual tumors across and within patients linked to perturbed dynamics of GC ecosystems make the development of genetically engineered mouse models crucial to decipher lymphomagenesis and disease progression and eventually to test the effects of novel targeted therapies. In this review, we provide an overview of some of the important genetically engineered mouse models that have been developed to recapitulate lymphoma-associated (epi)genetic alterations of two frequent GC-derived lymphoma entities: FL and GCB-DLCBL and describe how those mouse models have improved our knowledge of the molecular processes supporting GC B cell transformation.

The main avenue for the development of an HIV-1 vaccine remains the induction of protective antibodies. A rationale approach is to target antigen to specific receptors on dendritic cells (DC) via fused monoclonal antibodies (mAb). In mouse and non-human primate models, targeting of skin Langerhans cells (LC) with anti-Langerin mAbs fused with HIV-1 Gag antigen drives antigen-specific humoral responses. The development of these immunization strategies in humans requires a better understanding of early immune events driven by human LC. We therefore produced anti-Langerin mAbs fused with the HIV-1 gp140z Envelope (αLC.Env). First, we show that primary skin human LC and in vitro differentiated LC induce differentiation and expansion of naïve CD4+ T cells into T follicular helper (Tfh) cells. Second, when human LC are pre-treated with αLC.Env, differentiated Tfh cells significantly promote the production of specific IgG by B cells. Strikingly, HIV-Env-specific Ig are secreted by HIV-specific memory B cells. Consistently, we found that receptors and cytokines involved in Tfh differentiation and B cell functions are upregulated by LC during their maturation and after targeting Langerin. Finally, we show that subcutaneous immunization of mice by αLC.Env induces germinal center (GC) reaction in draining lymph nodes with higher numbers of Tfh cells, Env-specific B cells, as well as specific IgG serum levels compared to mice immunized with the non-targeting Env antigen. Altogether, we provide evidence that human LC properly targeted may be licensed to efficiently induce Tfh cell and B cell responses in GC., Author summary In recent years, the place of innovative vaccines based on the induction/regulation and modulation of the immune response with the aim to elicit an integrated T- and B cell immune responses against complex antigens has emerged besides “classical” vaccine vectors. Targeting antigens to dendritic cells is a vaccine technology concept supported by more than a decade of animal models and human pre-clinical experimentation. Recent investigations in animals underscored that Langerhans cells (LC) are an important target to consider for the induction of antibody responses by DC targeting vaccine approaches. Nonetheless, the development of these immunization strategies in humans remains elusive. We therefore developed and produced an HIV vaccine candidate targeting specifically LC through the Langerin receptor. We tested the ability of our vaccine candidate of targeting LC from skin explant and of inducing in vitro the differentiation of T follicular helper (Tfh) cells. Using complementary in vitro models, we demonstrated that Tfh cells induced by human LC are functional and the targeting of LC by our vaccine candidate promotes the secretion of anti-HIV IgG by memory B cells from HIV-infected individuals. In this study human LC exhibit key cellular functions able to drive potent anti-HIV-1 humoral responses providing mechanistic evidence of the Tfh- and B cell stimulating functions of primary skin targeted LC. Finally, we demonstrated in Xcr1DTA mice the significant advantage of LC targeting for inducing Tfh and germinal center (GC)-B cells and anti-HIV-1 antibodies. Therefore, the targeting of the human Langerin receptor appears to be a promising strategy for developing efficient HIV-1 vaccine.

Simple Summary Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative option for high-risk hematologic malignancies. However, disease recurrence after allo-HSCT remains a critical issue, underlining the need to develop maintenance therapy. In this context, NK cell-based immunotherapies could enhance graft-versus-tumor effect without triggering graft-versus-host disease. In this prospective phase I clinical trial, we demonstrated the safety of donor-derived NK cell infusion as a prophylactic treatment after allo-HSCT for patients with hematological malignancies. This opens perspectives for future developments of NK cell based therapeutic strategies after allo-HSCT with low incidence of GVHD, representing an advantage over post-transplant T cell modulations that are commonly used in clinical routine. Abstract Background: NK cell-based immunotherapy to prevent relapse after allogeneic transplantation is an appealing strategy because NK cells can provide strong antitumor effect without inducing graft-versus-host disease (GVHD). Thus, we designed a phase-I clinical trial evaluating the safety of a prophylactic donor-derived ex vivo IL-2 activated NK cell (IL-2 NK) infusion after allo-HSCT for patients with hematologic malignancies. Methods: Donor NK cells were purified and cultured ex vivo with IL-2 before infusion, at three dose levels. To identify the maximum tolerated dose was the main objective. In addition, we performed phenotypical and functional characterization of the NK cell therapy product, and longitudinal immune monitoring of NK cell phenotype in patients. Results: Compared to unstimulated NK cells, IL-2 NK cells expressed higher levels of activating receptors and exhibited increased degranulation and cytokine production in vitro. We treated 16 patients without observing any dose-limiting toxicity. At the last follow up, 11 out of 16 treated patients were alive in complete remission of hematologic malignancies without GVHD features and immunosuppressive treatment. Conclusions: Prophylactic donor-derived IL-2 NK cells after allo-HSCT is safe with low incidence of GVHD. Promising survivals and IL-2 NK cell activated phenotype may support a potential clinical efficacy of this strategy.

Immuno-oncology is revolutionizing the treatment of cancers, by inducing the recognition and elimination of tumor cells by the immune system. Recent advances have focused on generating or unleashing tumor antigen-specific T-cell responses, leading to alternative treatment paradigms for many cancers. Despite these successes, the clinical benefit has been limited to a subset of patients and certain tumor types, highlighting the need for alternative strategies. One innovative approach is to broaden and amplify antitumoral immune responses by targeting innate immunity. Particularly, the aim has been to develop new antibody formats capable of stimulating the antitumor activity of innate immune cells, boosting not only their direct role in tumor elimination, but also their function in eliciting multicellular immune responses ultimately resulting in long-lasting tumor control by adaptive immunity. This review covers the development of a new class of synthetic molecules, natural killer cell engagers (NKCEs), which are built from fragments of monoclonal antibodies (mAbs) and are designed to harness the immune functions of NK cells in cancer. As currently shown in preclinical studies and clinical trials, NKCEs are promising candidates for the next generation of tumor immunotherapies.

International audience; Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Lymphocytes are essential for microbial immunity, tumor surveillance, and tissue homeostasis. However, the in vivo development and function of helper-like innate lymphoid cells (ILCs) in humans remain much less well understood than those of T, B, and NK cells. We monitored hematopoietic stem cell transplantation (HSCT) to determine the kinetics of ILC development in both children and adults. It was found that, unlike NK cells, helper-like ILCs recovered slowly, mirroring the pattern observed for T cells, with normalization achieved at 1 year. The type of graft and the proportion of CD34+ cells in the graft did not significantly affect ILC reconstitution. As HSCT is often complicated by acute or chronic graft-versus-host disease (GVHD), the potential role of ILC subsets in maintaining tissue integrity in these conditions was also analyzed. It was found that GVHD was associated with lower levels of activated and gut-homing NKp44+ ILCP, consistent with a non-redundant role of this ILC subset in preventing this life-threatening disorder in lymphopenic conditions.

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans‐endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase‐activating proteins (GAPs). T and B cells express several RHO‐GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time‐lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T‐cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T‐cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T‐cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells., T and B cells continually recirculate between blood and secondary lymphoid organs. The RHO GTPase activating protein ARHGAP45 regulates naïve T cell deformability, motility, and chemotaxis, and thereby regulates key trafficking steps of T and B cells in vivo.

We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.

Rationale Immunoglobulin (Ig) G4-related disease (IgG4-RD) is a novel clinical disease entity characterized by an elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4-positive plasma cells. Pathological changes are most frequently seen in the pancreas, lacrimal glands, and salivary glands, but pathological changes in the lung also exist. Linker for activation of T cell (LAT)Y136F knock-in mice show Th2-dominant immunoreactions with elevated serum IgG1 levels, corresponding to human IgG4. We have reported that LATY136F knock-in mice display several characteristic features of IgG4-RD and concluded that they constitute an appropriate model of human IgG4-RD in salivary glands, pancreas, and kidney lesions. Objectives The aim of this study is to evaluate whether lung lesions in LATY136F knock-in mice can be a model of IgG4-related lung disease. Methods Lung tissue samples from LATY136F knock-in mice (LAT) and wild-type mice (WT) were immunostained for IgG1 and obtained for pathological evaluation, and cell fractions and cytokine levels in broncho-alveolar lavage fluid (BALF) were analyzed. Results In the LAT group, IgG1-positive inflammatory cells increased starting at 4 weeks of age and peaked at 10 weeks of age. The total cell count and percentage of lymphocytes increased significantly in BALF in the LAT group compared to the WT group. In BALF, Th2-dominant cytokines and transforming growth factor-β were also increased. In the LAT group, marked inflammation around broncho-vascular bundles peaked at 10 weeks of age. After 10 weeks, fibrosis around broncho-vascular bundles and bronchiectasis were observed in LATY136F knock-in mice but not WT mice. Conclusions LATY136F knock-in mice constitute an appropriate model of lung lesions in IgG4-RD.

Contains fulltext : 232076.pdf (Publisher’s version ) (Open Access) Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85-111, 117-143, and 157-165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

International audience; Foxp3 + regulatory T cells (Treg) maintain the integrity of the organism by preventing excessive immune responses. These cells protect against autoimmune diseases but are also important regulators of other immune responses including inflammation, allergy, infection, and tumors. Furthermore, they exert non-immune functions such as tissue repair and regeneration. In the periphery, Foxp3 + Treg have emerged as a highly heterogeneous cell population with distinct molecular and functional properties. Foxp3 + Treg mainly develop within the thymus where they receive instructive signals for their differentiation. Recent studies have revealed that thymic Treg are also heterogeneous with two distinct precursors that give rise to mature Foxp3 + Treg exhibiting non-overlapping regulatory activities characterized by a differential ability to control different types of autoimmune reactions. Furthermore, the thymic Treg cell pool is not only composed of newly developing Treg, but also contain a large fraction of recirculating peripheral cells. Here, we review the two pathways of thymic Treg cell differentiation and their potential impact on Treg activity in the periphery. We also summarize our current knowledge on recirculating peripheral Treg in the thymus.

International audience; Thymic epithelial cells (TECs) provide essential clues for the proliferation, survival, migration, and differentiation of thymocytes. Recent advances in mouse and human have revealed that TECs constitute a highly heterogeneous cell population with distinct functional properties. Importantly, TECs are sensitive to thymic damages engendered by myeloablative conditioning regimen used for bone marrow transplantation. These detrimental effects on TECs delay de novo T-cell production, which can increase the risk of morbidity and mortality in many patients. Alike that TECs guide the development of thymocytes, reciprocally thymocytes control the differentiation and organization of TECs. These bidirectional interactions are referred to as thymic crosstalk. The tumor necrosis factor receptor superfamily (TNFRSF) member, receptor activator of nuclear factor kappa-B (RANK) and its cognate ligand RANKL have emerged as key players of the crosstalk between TECs and thymocytes. RANKL, mainly provided by positively selected CD4 + thymocytes and a subset of group 3 innate lymphoid cells, controls mTEC proliferation/differentiation and TEC regeneration. In this review, I discuss recent advances that have unraveled the high heterogeneity of TECs and the implication of the RANK-RANKL signaling axis in TEC differentiation and regeneration. Targeting this cell-signaling pathway opens novel therapeutic perspectives to recover TEC function and T-cell production.

Zdhhc family genes are composed of 24 members that regulate palmitoylation, a post-translational modification process for proteins. Mutations in genes that alter palmitoylation or de-palmitoylation could result in neurodegenerative diseases and inflammatory disorders. In this study, we found that Zdhhc2 was robustly induced in psoriatic skin and loss of Zdhhc2 in mice by CRISPR/Cas9 dramatically inhibited pathology of the ear skin following imiquimod treatment. As psoriasis is an inflammatory disorder, we analyzed tissue infiltrating immune cells and cytokine production. Strikingly we found that a master psoriatic cytokine interferon-α (IFN-α) in the lesioned skin of wildtype (WT) mice was 23-fold higher than that in Zdhhc2 deficient counterparts. In addition, we found that CD45+ white blood cells (WBC) infiltrating in the skin of Zdhhc2 deficient mice were also significantly reduced. Amelioration in psoriasis and dramatically reduced inflammation of Zdhhc2 deficient mice led us to analyze the cellular components that were affected by loss of Zdhhc2. We found that imiquimod induced plasmacytoid dendritic cell (pDC) accumulation in psoriatic skin, spleen, and draining lymph nodes (DLN) were drastically decreased in Zdhhc2 deficient mice, and the expression of pDC activation marker CD80 also exhibited significantly inhibited in psoriatic skin. In further experiments, we confirmed the cell intrinsic effect of Zdhhc2 on pDCs as we found that loss of zDHHC2 in human CAL-1 pDC dampened both interferon regulatory factor 7 (IRF7) phosphorylation and IFN-α production. Therefore, we identified novel function of Zdhhc2 in controlling inflammatory response in psoriasis in mice and we also confirmed that crucial role of Zdhhc2 in pDCs by regulating IRF7 activity and production of the critical cytokine. Our results finding the dependence of IFN-α production on Zdhhc2 in inflamed murine skin and in human pDCs provide rationale for targeting this new molecule in treatment of inflammation.

Checkpoint blockade therapy is effective against many cancers; however, new targets need to be identified to treat patients who do not respond to current treatment or demonstrate immune escape. Here, we showed that blocking the inhibitory receptor Killer cell lectin-like receptor G1 (KLRG1) enhances anti-tumor immunity mediated by NK cells and CD8+ T cells. We found that loss of KLRG1 signaling alone significantly decreased melanoma and breast cancer tumor growth in the lungs of mice. In addition, we demonstrated that KLRG1 blockade can synergize with PD-1 checkpoint therapy to increase the therapeutic efficacy compared to either treatment alone. This effect was even observed with tumors that do not respond to PD-1 checkpoint therapy. Double blockade therapy led to significantly decreased tumor size, increased frequency and activation of CD8+ T cells, and increased NK cell frequency and maturation in the tumor microenvironment. These findings demonstrate that KLRG1 is a novel checkpoint inhibitor target that affects NK and T cell anti-tumor immunity, both alone and in conjunction with established immunotherapies.

Severe COVID-19 is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members, denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against SARS-CoV-2. In contrast, compared to subjects with other infectious or non-infectious lung pathologies, IFNs are over-represented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites., An in-depth analysis of interferons in COVID-19 reveals differences in their roles based on anatomical location, viral load, age and disease severity. In the upper respiratory tract, high levels of IFN-III are protective and result in mild disease in spite of higher SARS-CoV-2 viral burden while the lower airways of patients with severe COVID-19 demonstrate elevated IFN-I and III, cell death and a reduction in interferon stimulated genes.

Summary Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) driven by viruses or bacteria, as well as in numerous immune-mediated disorders. Histone citrullination by the enzyme peptidylarginine deiminase 4 (PAD4) and the consequent decondensation of chromatin are hallmarks in the induction of NETs. Nevertheless, additional histone modifications that may govern NETosis are largely overlooked. Herein, we show that histone deacetylases (HDACs) play critical roles in driving NET formation in human and mouse neutrophils. HDACs belonging to the zinc-dependent lysine deacetylases family are necessary to deacetylate histone H3, thus allowing the activity of PAD4 and NETosis. Of note, HDAC inhibition in mice protects against microbial-induced pneumonia and septic shock, decreasing NETosis and inflammation. Collectively, our findings illustrate a new fundamental step that governs the release of NETs and points to HDAC inhibitors as therapeutic agents that may be used to protect against ARDS and sepsis., Graphical abstract, Highlights • Zn-dependent lysine deacetylases govern neutrophil extracellular trap (NET) formation • Class I/IIb HDACs deacetylate histone H3 allowing its citrullination by PAD4 • HDAC inhibition with ricolinostat protects against viral and bacterial pneumonia • In a model of endotoxic shock, ricolinostat inhibits NETosis and dampens inflammation, Molecular biology; Immunology; Cell biology; Functional aspects of cell biology

Description, Perivascular macrophages orchestrate the expansion of pericyte-like mesenchymal cells to create a proangiogenic niche in cancer., Tumor-associated macrophages (TAMs) are a highly plastic stromal cell type that support cancer progression. Using single-cell RNA sequencing of TAMs from a spontaneous murine model of mammary adenocarcinoma (MMTV-PyMT), we characterize a subset of these cells expressing lymphatic vessel endothelial hyaluronic acid receptor 1 (Lyve-1) that spatially reside proximal to blood vasculature. We demonstrate that Lyve-1+ TAMs support tumor growth and identify a pivotal role for these cells in maintaining a population of perivascular mesenchymal cells that express α-smooth muscle actin and phenotypically resemble pericytes. Using photolabeling techniques, we show that mesenchymal cells maintain their prevalence in the growing tumor through proliferation and uncover a role for Lyve-1+ TAMs in orchestrating a selective platelet-derived growth factor–CC–dependent expansion of the perivascular mesenchymal population, creating a proangiogenic niche. This study highlights the inter-reliance of the immune and nonimmune stromal network that supports cancer progression and provides therapeutic opportunities for tackling the disease.

T‐cell receptor (TCR) ligation‐mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time‐resolved high‐resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T‐cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast‐track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T‐cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T‐cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas., The study presents LymphoAtlas, a phosphoproteomic dataset enabling the identification and visualization of phosphorylation dynamics during the first 10 min after TCR stimulation of primary mouse T cells.

Macrophages reside in the body cavities where they maintain serosal homeostasis and provide immune surveillance. Peritoneal macrophages are implicated in the etiology of pathologies including peritonitis, endometriosis and metastatic cancer thus understanding the factors that govern their behavior is vital. Using a combination of fate mapping techniques, we have investigated the impact of sex and age on murine peritoneal macrophage differentiation, turnover and function. We demonstrate that the sexually dimorphic replenishment of peritoneal macrophages from the bone marrow, which is high in males and very low in females, is driven by changes in the local microenvironment that arise upon sexual maturation. Population and single cell RNAseq revealed striking dimorphisms in gene expression between male and female peritoneal macrophages that was in part explained by differences in composition of these populations. By estimating the time of residency of different subsets within the cavity and assessing development of dimorphisms with age and in monocytopenic Ccr2–/– mice, we demonstrate that key sex-dependent features of peritoneal macrophages are a function of the differential rate of replenishment from the bone marrow while others are reliant on local microenvironment signals. Importantly, we demonstrate that the dimorphic turnover of peritoneal macrophages contributes to differences in the ability to protect against pneumococcal peritonitis between the sexes. These data highlight the importance of considering both sex and age in susceptibility to inflammatory and infectious diseases.

Etzerodt et al. identify a unique subset of CD163+ Tim4+ tissue-resident macrophages in omentum that are maintained independently of bone marrow–derived monocytes and have a specific role in the metastatic spread of ovarian cancer cells., Experimental and clinical evidence suggests that tumor-associated macrophages (TAMs) play important roles in cancer progression. Here, we have characterized the ontogeny and function of TAM subsets in a mouse model of metastatic ovarian cancer that is representative for visceral peritoneal metastasis. We show that the omentum is a critical premetastatic niche for development of invasive disease in this model and define a unique subset of CD163+ Tim4+ resident omental macrophages responsible for metastatic spread of ovarian cancer cells. Transcriptomic analysis showed that resident CD163+ Tim4+ omental macrophages were phenotypically distinct and maintained their resident identity during tumor growth. Selective depletion of CD163+ Tim4+ macrophages in omentum using genetic and pharmacological tools prevented tumor progression and metastatic spread of disease. These studies describe a specific role for tissue-resident macrophages in the invasive progression of metastatic ovarian cancer. The molecular pathways of cross-talk between tissue-resident macrophages and disseminated cancer cells may represent new targets to prevent metastasis and disease recurrence., Graphical Abstract

Follicular lymphoma B cells undergo continuous somatic hypermutation (SHM) of their immunoglobulin variable region genes, generating a heterogeneous tumor population. SHM introduces DNA sequences encoding N-glycosylation sites asparagine-X-serine/threonine (N-gly sites) within the V-region that are rarely found in normal B-cell counterparts. Unique attached oligomannoses activate B-cell receptor signaling pathways after engagement with calcium-dependent lectins expressed by tissue macrophages. This novel interaction appears critical for tumor growth and survival. To elucidate the significance of N-gly site presence and loss during ongoing SHM, we tracked site behavior during tumor evolution and progression in a diverse group of patients through next-generation sequencing. A hierarchy of subclones was visualized through lineage trees based on SHM semblance between subclones and their discordance from the germline sequence. We observed conservation of N-gly sites in more than 96% of subclone populations within and across diagnostic, progression, and transformation events. Rare N-gly-negative subclones were lost or negligible from successive events, in contrast to N-gly-positive subclones, which could additionally migrate between anatomical sites. Ongoing SHM of the N-gly sites resulted in subclones with different amino acid compositions across disease events, yet the vast majority of resulting DNA sequences still encoded for an N-gly site. The selection and expansion of only N-gly-positive subclones is evidence of the tumor cells’ dependence on sites, despite the changing genomic complexity as the disease progresses. N-gly sites were gained in the earliest identified lymphoma cells, indicating they are an early and stable event of pathogenesis. Targeting the inferred mannose-lectin interaction holds therapeutic promise.

In the present study, Louis et al. identify an inflammatory cascade mediated by IL-18 and GM-CSF–producing NK cells that promotes autoantibody-driven arthritis. GM-CSF signaling in myeloid cells is subsequently restrained by the suppressor of cytokine signaling protein CIS to prevent aberrant GM-CSF–mediated inflammation., Despite increasing recognition of the importance of GM-CSF in autoimmune disease, it remains unclear how GM-CSF is regulated at sites of tissue inflammation. Using GM-CSF fate reporter mice, we show that synovial NK cells produce GM-CSF in autoantibody-mediated inflammatory arthritis. Synovial NK cells promote a neutrophilic inflammatory cell infiltrate, and persistent arthritis, via GM-CSF production, as deletion of NK cells, or specific ablation of GM-CSF production in NK cells, abrogated disease. Synovial NK cell production of GM-CSF is IL-18–dependent. Furthermore, we show that cytokine-inducible SH2-containing protein (CIS) is crucial in limiting GM-CSF signaling not only during inflammatory arthritis but also in experimental allergic encephalomyelitis (EAE), a murine model of multiple sclerosis. Thus, a cellular cascade of synovial macrophages, NK cells, and neutrophils mediates persistent joint inflammation via production of IL-18 and GM-CSF. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor. These findings shed new light on GM-CSF biology in sterile tissue inflammation and identify several potential therapeutic targets., Graphical Abstract

International audience; Glucocorticoids (GCs) are endogenous hormones that are crucial for the homeostasis of the organism and adaptation to the external environment. Because of their anti-inflammatory effects, synthetic GCs are also extensively used in clinical practice. However, almost all cells in the body are sensitive to GC regulation. As a result, these mediators have pleiotropic effects, which may be undesirable or detrimental to human health. Here, we summarize the recent findings that contribute to deciphering the molecular mechanisms downstream of glucocorticoid receptor activation. We also discuss the complex role of GCs in infectious diseases such as sepsis and COVID-19, in which the balance between pathogen elimination and protection against excessive inflammation and immunopathology needs to be tightly regulated. An understanding of the cell type-and context-specific actions of GCs from the molecular to the organismal level would help to optimize their therapeutic use.

Contains fulltext : 229325.pdf (Publisher’s version ) (Open Access) Currently three bona fide dendritic cell (DC) types are distinguished in human blood. Herein we focus on type 2 DCs (DC2s) and compare the three defining markers CD1c, CD172, and CD301. When using CD1c to define DC2s, a CD14(+) and a CD14(-) subset can be detected. The CD14(+) subset shares features with monocytes, and this includes substantially higher expression levels for CD64, CD115, CD163, and S100A8/9. We review the current knowledge of these CD1c(+)CD14(+) cells as compared to the CD1c(+)CD14(-) cells with respect to phenotype, function, transcriptomics, and ontogeny. Here, we discuss informative mutations, which suggest that two populations have different developmental requirements. In addition, we cover subsets of CD11c(+)CD8(-) DC2s in the mouse, where CLEC12A(+)ESAM(low) cells, as compared to the CLEC12A(-)ESAM(high) subset, also express higher levels of monocyte-associated markers CD14, CD3, and CD115. Finally, we summarize, for both man and mouse, the data on lower antigen presentation and higher cytokine production in the monocyte-marker expressing DC2 subset, which demonstrate that the DC2 subsets are also functionally distinct.

Introduction Angioimmunoblastic T-cell lymphoma (AITL) is the most frequent of nodal peripheral T-cell lymphomas. AITL results from the transformation of T follicular helper (T FH) cells and is characterized by chemo-resistance and poor survival (5-year OS around 30%). Recent data from prospective clinical trials suggest that disease outcome may be impacted by factors other than genomic features, such as the tumor microenvironment (TME) and overall intra-tumoral heterogeneity. Our understanding of AITL intra-tumoral genetic, transcriptional and functional heterogeneity is limited because most molecular data generated so far have come from bulk analyses. Single-cell RNA sequencing (scRNA-seq) enables fine characterization of cell types and functional cell states. When focused on T or B cells, 5'-end scRNA-seq also yields the TCR or BCR sequences that allow tracking clonally related cells. Here we studied the intra-tumor heterogeneity of AITL tumors using integrative scRNA-seq. Methods We analyzed lymph node live cell suspensions from AITL patients (n=10) using droplet-based 10x Genomics 5'-end scRNA-seq. Malignant T cells from 4 AITL samples were also analyzed by FACS index sorting and plate-based 5'-end scRNA-seq to link cell surface phenotype and gene expression profile. We identified malignant T cell clones by intersecting the gene expression and TCR sequencing data, and performed separate focused analyses of TME subsets and malignant T cells. We compared subsets of malignant T cells from all patients using marker gene-based metaclustering to identify AITL T cell states conserved across patients. We explored the genetic heterogeneity of malignant T cells by mapping RHOA G17V mutations and inferring copy number variation (CNV) subclones from scRNA-seq data. In select cases, we performed in situ analysis by immunohistochemistry (IHC) or spatial transcriptomics to characterize the spatial distribution of malignant T cell subsets identified by scRNA-seq. Results Based on gene expression, malignant T cells grouped in patient-specific clusters, while non-malignant T, B and myeloid TME cells from all patients clustered by cell type or cell state. Among TME cells, we identified 7 subsets of B cells (including activated B cells, plasma cells, and one patient-specific monoclonal B cell proliferation), 6 subsets of myeloid cells (including macrophages, conventional and plasmacytoid dendritic cells), and 8 subsets of non-malignant T cells (including activated cytotoxic T lymphocytes (CTL) with clonal expansions). Patient-specific malignant T cells were heterogeneous and divided into several gene-expression based clusters. Metaclustering of malignant T cell subsets identified T central memory (T CM)-like and T FH-like states in 10/10 samples. We also identified in 3/10 samples clusters of CTL-like malignant T cells expressing characteristic marker genes (including NKG7, GNLY, GZMK, PRF1). We observed an intra-sample continuum of gene expression states from quiescent T CM-like to proliferating T FH-like states. T FH-like cells were larger in size and expressed higher levels of surface PD1 and ICOS than T CM-like and CTL-like subsets. We detected the RHOA G17V mutation in malignant T cells of 4/4 mutated cases, with no evidence of subclonal heterogeneity for that mutation. We detected clonal and subclonal CNV in most AITL malignant T cells. CTL-like states were enriched in specific CNV subclones, but the T CM-like to T FH-like continuum was observed in all CNV subclones, suggesting that functional plasticity and subclonal genetic evolution may occur independently. In situ staining of markers for T FH-like (PD1, ICOS, CD200) and CTL-like (GZMK, GZMA) cells showed that T FH-like and CTL-like cells occupied distinct tissue niches within the tumor. In spatial transcriptomics analysis, T FH-like cells mapped to follicular dendritic cell (FDC)-rich areas, while T CM-like cells were associated with T-zone reticular cells. Conclusions Our analyses recapitulate known characteristics of AITL TME, and uncover previously unrecognized heterogeneity among malignant T cells across multiple patients. The distinct gene expression programs, phenotypes, genetics, and locations of T FH-like, T CM-like and CTL-like states suggest that AITL malignant T cells undergo significant functional plasticity and genetic divergence, which could influence response to therapy and overall clinical course. Figure 1 Figure 1. Disclosures Lemonnier: Institut Roche: Research Funding; Gilead: Other: travel grant. Gaulard: Gilead: Consultancy; Innate Pharma: Research Funding; Sanofi: Research Funding; Alderaan: Research Funding; Takeda: Consultancy, Honoraria. Milpied: Institut Roche: Research Funding; Innate Pharma: Research Funding; Bristol Myers Squibb: Research Funding.

A cardinal feature of COVID-19 is lung inflammation and respiratory failure. In a prospective multi-country cohort of COVID-19 patients, we found that increased Notch4 expression on circulating regulatory T (Treg) cells was associated with disease severity, predicted mortality, and declined upon recovery. Deletion of Notch4 in Treg cells or therapy with anti-Notch4 antibodies in conventional and humanized mice normalized the dysregulated innate immunity and rescued disease morbidity and mortality induced by a synthetic analog of viral RNA or by influenza H1N1 virus. Mechanistically, Notch4 suppressed the induction by interleukin-18 of amphiregulin, a cytokine necessary for tissue repair. Protection by Notch4 inhibition was recapitulated by therapy with Amphiregulin and, reciprocally, abrogated by its antagonism. Amphiregulin declined in COVID-19 subjects as a function of disease severity and Notch4 expression. Thus, Notch4 expression on Treg cells dynamically restrains amphiregulin-dependent tissue repair to promote severe lung inflammation, with therapeutic implications for COVID-19 and related infections., Graphical abstract, Harb, Benamar, et al. find that interleukin-6 increases Notch4 expression on lung regulatory T cells, which, in turn, restrains production of the tissue repair cytokine amphiregulin and promotes severe lung inflammation. Their findings have implications for treatment of COVID-19 and other respiratory viral infections.

The innate lymphoid cell (ILC) family consists of natural killer (NK) cells, helper-like lymphoid cells (ILC1s, ILC2s, and ILC3s), and lymphoid tissue inducer (LTi) cells. Helper-like ILCs are considered the innate counterpart of T-helper cells because of similarities in their cytokine output and expression of key transcription factors. ILCs provide and regulate innate immune functions before the development of adaptive immunity. They are involved in host defense against pathogens, inflammation, tissue repair, and metabolic homeostasis. However, they can also be involved in inflammatory disorders and carcinogenesis. In this review, we summarize the latest research on ILC development and plasticity in humans and mice, focusing on the pathogenic role of helper-like ILCs in inflammatory disorders, such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA).

International audience; Systemic lupus erythematosus (SLE) patients have increased prevalence of metabolic syndrome but the underlying mechanisms are unknown. Toll-like receptor 7 (TLR7) that detects single stranded-RNA plays a key role in antimicrobial host defense and also contributes to the initiation and progression of SLE both in mice and humans. Here, we report the implication of TLR7 signaling in high fat diet (HFD)-induced metabolic syndrome and exacerbation of lupus autoimmunity in TLR8-deficient (TLR8ko) mice, which develop spontaneous lupus-like disease due to increased TLR7 signaling by dendritic cells (DCs). The aggravated SLE pathogenesis in HFD-fed TLR8ko mice was characterized by increased overall immune activation, anti-DNA autoantibody production, and IgG/IgM glomerular deposition that were coupled with increased kidney histopathology. Moreover, upon HFD TLR8ko mice developed metabolic abnormalities, including liver inflammation. In contrast, upon HFD TLR7/8ko mice did not develop SLE and both TLR7ko and TLR7/8ko mice were fully protected from metabolic abnormalities, including body weight gain, insulin resistance, and liver inflammation. Interestingly, HFD led to an increase of TLR7 expression in WT mice, that was coupled with increased TNF production by DCs, and this phenotype was more profound in TLR8ko mice. Our study uncovers the implication of TLR7 signaling in the interconnection of SLE and metabolic abnormalities, indicating that TLR7 might be a novel approach as a tailored therapy in SLE and metabolic diseases.

NK cells are broadly distributed innate lymphoid cells (ILCs) encompassing distinct populations based on CD11b and CD27 expression in mice or CD56 intensity in humans. Involved in anti-viral and anti-tumor immunity thanks to their cytokines and chemokines secretion as well as their cytotoxic capabilities, NK cells have emerged as a promising therapeutic target in several solid tumors and hematological malignancies. To view this Snapshot, open or download the PDF.

International audience; Follicular helper (TFH) and regulatory (TFR) cells are critical players in managing germinal center (GC) reactions that accomplish effective humoral immune responses. Transcriptome analyses were done comparing gene regulation of TFH and TFR cells isolated from Peyer's Patches (PP) and immunized peripheral lymph nodes (pLNs) revealing many regulatory patterns common to all follicular cells. However, in contrast to TFH cells, the upregulation or downregulation of many genes was attenuated substantially in pLN TFR cells when compared to those of PP. Additionally, PP but not pLN TFR cells were largely unresponsive to IL2 and expressed Il4 as well as Il21. Together with fundamental differences in gene expression that were found between cells of both compartments this emphasizes specific adaptations of follicular T cell functions to their micro-milieu. Moreover, although GL7 expression distinguishes matured follicular T cells, GL7(+) as well as GL7(-) cells are present in the GC.

International audience; T cells are critical components of adaptive immunity. As such, their activation is regulated by the T cell receptor (TCR) that constantly scan peptides associated with major histocompatibility complexes (MHC). TCR engagement initiates a series of molecular events leading to cytokine secretion, proliferation, and differentiation of T cells. As a second coincident event, activation of co-stimulatory molecules, such as CD28, synergize with the TCR in order to prolong and/or amplify intracellular signals. With the recent advances in immunotherapies targeting T cells, co-inhibitory receptors are of growing interest for immunologists due to their potential modulatory properties on T cell functions. However, special attention should be dedicated to avoid unwanted clinical outcomes (1). In particular, Manichean categorization of receptors based on incomplete functional knowledge can lead to an over-simplistic view of complex cellular regulations. Thus, analysis of the functions that characterize these receptors in diverse physiological contexts remains essential for their rational use in therapeutic protocols. Here we focus on CD5, a transmembrane receptor that regulates T cell functions and development but remains poorly characterized at the molecular level. We will review its roles in physiological conditions and suggest potential molecular effectors that could account for CD5-dependent regulation of TCR signaling.

International audience; The lipopolysaccharide (LPS) is a major virulence factor of Brucella, a facultative intracellular pathogenic Gram-negative bacterium. Brucella LPS exhibits a low toxicity and its atypical structure was postulated to delay the host immune response, favouring the establishment of chronic disease. Here we carried out an in-depth in vitro and in vivo characterisation of the immunomodulatory effects of Brucella LPS on different dendritic cell (DC) subpopulations. By using LPSs from bacteria that share some of Brucella LPS structural features, we demonstrated that the core component of B. melitensis wild-type (Bm-wt) LPS accounts for the low activation potential of Brucella LPS in mouse GM-CSF-derived (GM-) DCs. Contrary to the accepted dogma considering Brucella LPS a poor TLR4 agonist and DC activator, Bm-wt LPS selectively induced expression of surface activation markers and cytokine secretion from Flt3-Ligand-derived (FL-) DCs in a TLR4-dependent manner. It also primed in vitro T cell proliferation by FL-DCs. In contrast, modified LPS with a defective core purified from Brucella carrying a mutated wadC gene (Bm-wadC), efficiently potentiated mouse and human DC activation and T cell proliferation in vitro. In vivo, Bm-wt LPS promoted scant activation of splenic DC subsets and limited recruitment of monocyte- DC like cells in the spleen, conversely to Bm-wadC LPS. Bm-wadC live bacteria drove high cytokine secretion levels in sera of infected mice. Altogether, these results illustrate the immunomodulatory properties of Brucella LPS and the enhanced DC activation ability of the wadC mutation with potential for vaccine development targeting Brucella core LPS structure.

International audience; Background The adaptor protein Linker for activation of T cell (LAT) is a key signaling hub used by the T cell antigen receptor. Mutant mice expressing loss-of-function mutations affecting LAT and including a mutation in which tyrosine 136 is replaced by a phenylalanine (Lat(Y136F)) develop lymphoproliferative disorder involving T helper type 2 effector cells capable of triggering a massive polyclonal B cell activation that leads to hypergammaglobulinemia G1 and E and to non-resolving inflammation and autoimmunity. The purpose of this study was to evaluate whether the phenotypes of Lat(Y136F) knock-in mice resemble the immunohistopathological features of immunoglobulin G4-related disease (IgG4-RD).Methods Lat(Y136F) knock-in mice were sacrificed at 4-20 weeks of age, and pancreas, kidney, salivary gland and lung were obtained. All organs were stained with hematoxylin-eosin and with Azan for estimation of collagen in fibrosis, and the severity scores of inflammation and fibrosis were evaluated. Immunostainings were performed to analyze the types of infiltrating cells. In addition, the effects of corticosteroid treatment on the development of tissue lesions and serum levels of IgG1 were assessed.Results Tissue lesions characterized by inflammatory mononuclear cell infiltration and fibrosis were detected in pancreas, kidney, and salivary gland starting from 6 weeks of age. Immunostainings showed pronounced infiltration of plasma cells, CD4-positive T cells, and macrophages. Infiltrating plasma cells predominantly expressed IgG1. The extent of inflammation in pancreas and salivary glands was markedly reduced by corticosteroid treatment.Conclusions Lat(Y136F) knock-in mice displayed increased production of Th2-type IgG1 (a homologue of human IgG4) and developed multiple organ tissue lesions reminiscent of those seen in patients with IgG4-RD. Moreover, the development of these tissue lesions was highly sensitive to corticosteroid treatment like in IgG4-RD. For these reasons we consider the Lat(Y136F) knock-in mouse strain to represent a promising model for human IgG4-RD.

Propagation of retrotransposons induces genomic instability. Their roles in HSCs remain poorly studied. Barbieri et al. show that retrotransposon expression and mobilization are involved in long-lasting HSC impairment upon irradiation. These effects are counteracted by the self-renewal cytokine THPO through induction of interferon-like response., Maintenance of genomic integrity is crucial for the preservation of hematopoietic stem cell (HSC) potential. Retrotransposons, spreading in the genome through an RNA intermediate, have been associated with loss of self-renewal, aging, and DNA damage. However, their role in HSCs has not been addressed. Here, we show that mouse HSCs express various retroelements (REs), including long interspersed element-1 (L1) recent family members that further increase upon irradiation. Using mice expressing an engineered human L1 retrotransposition reporter cassette and reverse transcription inhibitors, we demonstrate that L1 retransposition occurs in vivo and is involved in irradiation-induced persistent γH2AX foci and HSC loss of function. Thus, RE represents an important intrinsic HSC threat. Furthermore, we show that RE activity is restrained by thrombopoietin, a critical HSC maintenance factor, through its ability to promote a potent interferon-like, antiviral gene response in HSCs. This uncovers a novel mechanism allowing HSCs to minimize irradiation-induced injury and reinforces the links between DNA damage, REs, and antiviral immunity., Graphical Abstract