Myeloid cell influx into the colonic epithelium is associated with disease severity and non-response to anti-Tumor Necrosis Factor Therapy in patients with Ulcerative Colitis.

Ulcerative colitis (UC) is an idiopathic chronic inflammatory disease of the colon with sharply rising global prevalence. Dysfunctional epithelial compartment (EC) dynamics are implicated in UC pathogenesis although EC-specific studies are sparse. Applying orthogonal high-dimensional EC profiling to a Primary Cohort (PC; n=222), we detail major epithelial and immune cell perturbations in active UC. Prominently, reduced frequencies of mature BEST4+OTOP2+ absorptive and BEST2+WFDC2+ secretory epithelial enterocytes were associated with the replacement of homeostatic, resident TRDC+KLRD1+HOPX+ γδ+ T cells with RORA+CCL20+S100A4+ TH17 cells and the influx of inflammatory myeloid cells. The EC transcriptome (exemplified by S100A8, HIF1A, TREM1, CXCR1) correlated with clinical, endoscopic, and histological severity of UC in an independent validation cohort (n=649). Furthermore, therapeutic relevance of the observed cellular and transcriptomic changes was investigated in 3 additional published UC cohorts (n=23, 48 and 204 respectively) to reveal that non-response to anti-Tumor Necrosis Factor (anti-TNF) therapy was associated with EC related myeloid cell perturbations. Altogether, these data provide high resolution mapping of the EC to facilitate therapeutic decision-making and personalization of therapy in patients with UC.

TREM2 macrophages drive NK cell paucity and dysfunction in lung cancer.

Natural killer (NK) cells are commonly reduced in human tumors, enabling many to evade surveillance. Here, we sought to identify cues that alter NK cell activity in tumors. We found that, in human lung cancer, the presence of NK cells inversely correlated with that of monocyte-derived macrophages (mo-macs). In a murine model of lung adenocarcinoma, we show that engulfment of tumor debris by mo-macs triggers a pro-tumorigenic program governed by triggering receptor expressed on myeloid cells 2 (TREM2). Genetic deletion of Trem2 rescued NK cell accumulation and enabled an NK cell-mediated regression of lung tumors. TREM2+ mo-macs reduced NK cell activity by modulating interleukin (IL)-18/IL-18BP decoy interactions and IL-15 production. Notably, TREM2 blockade synergized with an NK cell-activating agent to further inhibit tumor growth. Altogether, our findings identify a new axis, in which TREM2+ mo-macs suppress NK cell accumulation and cytolytic activity. Dual targeting of macrophages and NK cells represents a new strategy to boost antitumor immunity.

Single-cell profiling to transform immunotherapy usage and target discovery in immune-mediated inflammatory diseases.

Immunotherapy drugs are transforming the clinical care landscape of major human diseases from cancer, to inflammatory diseases, cardiovascular diseases, neurodegenerative diseases and even aging. In polygenic immune-mediated inflammatory diseases (IMIDs), the clinical benefits of immunotherapy have nevertheless remained limited to a subset of patients. Yet the identification of new actionable molecular candidates has remained challenging, and the use of standard of care imaging and/or histological diagnostic assays has failed to stratify potential responders from non-responders to biotherapies already available. We argue that these limitations partly stem from a poor understanding of disease pathophysiology and insufficient characterization of the roles assumed by candidate targets during disease initiation, progression and treatment. By transforming the resolution and scale of tissue cell mapping, high-resolution profiling strategies offer unprecedented opportunities to the understanding of immunopathogenic events in human IMID lesions. Here we discuss the potential for single-cell technologies to reveal relevant pathogenic cellular programs in IMIDs and to enhance patient stratification to guide biotherapy eligibility and clinical trial design.

IL-22BP production is heterogeneously distributed in Crohn's disease.

Crohn's disease (CD), a form of inflammatory bowel disease (IBD), is characterized by impaired epithelial barrier functions and dysregulated mucosal immune responses. IL-22 binding protein (IL-22BP) is a soluble inhibitor regulating IL-22 bioactivity, a cytokine proposed to play protective roles during CD. We and others have shown that IL-22BP is produced in IBD inflamed tissues, hence suggesting a role in CD. In this work, we extended the characterization of IL-22BP production and distribution in CD tissues by applying enzyme-linked immunosorbent assays to supernatants obtained from the culture of endoscopic biopsies of patients, and reverse transcription-quantitative polymerase chain reaction on sorted immune cell subsets. We reveal that IL-22BP levels are higher in inflamed ileums than colons. We observe that in a cell-intrinsic fashion, populations of mononuclear phagocytes and eosinophils express IL-22BP at the highest levels in comparison to other sources of T cells. We suggest the enrichment of intestinal eosinophils could explain higher IL-22BP levels in the ileum. In inflamed colon, we reveal the presence of increased IL-22/IL22BP ratios compared to controls, and a strong correlation between IL-22BP and CCL24. We identify monocyte-derived dendritic cells (moDC) as a cellular subtype co-expressing both cytokines and validate our finding using in vitro culture systems. We also show that retinoic acid induces the secretion of both IL-22BP and CCL24 by moDC. Finally, we report on higher IL-22BP levels in active smokers. In conclusion, our work provides new information relevant to therapeutic strategies modulating IL-22 bioactivity in CD, especially in the context of disease location.

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.

Ulcerative colitis is characterized by a plasmablast-skewed humoral response associated with disease activity.

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG+ plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin αvß6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis.

Single-cell analysis of human non-small cell lung cancer lesions refines tumor classification and patient stratification.

Immunotherapy is a mainstay of non-small cell lung cancer (NSCLC) management. While tumor mutational burden (TMB) correlates with response to immunotherapy, little is known about the relationship between the baseline immune response and tumor genotype. Using single-cell RNA sequencing, we profiled 361,929 cells from 35 early-stage NSCLC lesions. We identified a cellular module consisting of PDCD1+CXCL13+ activated T cells, IgG+ plasma cells, and SPP1+ macrophages, referred to as the lung cancer activation module (LCAMhi). We confirmed LCAMhi enrichment in multiple NSCLC cohorts, and paired CITE-seq established an antibody panel to identify LCAMhi lesions. LCAM presence was found to be independent of overall immune cell content and correlated with TMB, cancer testis antigens, and TP53 mutations. High baseline LCAM scores correlated with enhanced NSCLC response to immunotherapy even in patients with above median TMB, suggesting that immune cell composition, while correlated with TMB, may be a nonredundant biomarker of response to immunotherapy.

BRAFV600E-induced senescence drives Langerhans cell histiocytosis pathophysiology.

Langerhans cell histiocytosis (LCH) is a potentially fatal condition characterized by granulomatous lesions with characteristic clonal mononuclear phagocytes (MNPs) harboring activating somatic mutations in mitogen-activated protein kinase (MAPK) pathway genes, most notably BRAFV600E. We recently discovered that the BRAFV600E mutation can also affect multipotent hematopoietic progenitor cells (HPCs) in multisystem LCH disease. How the BRAFV600E mutation in HPCs leads to LCH is not known. Here we show that enforced expression of the BRAFV600E mutation in early mouse and human multipotent HPCs induced a senescence program that led to HPC growth arrest, apoptosis resistance and a senescence-associated secretory phenotype (SASP). SASP, in turn, promoted HPC skewing toward the MNP lineage, leading to the accumulation of senescent MNPs in tissue and the formation of LCH lesions. Accordingly, elimination of senescent cells using INK-ATTAC transgenic mice, as well as pharmacologic blockade of SASP, improved LCH disease in mice. These results identify senescent cells as a new target for the treatment of LCH.

An easy and reliable whole blood freezing method for flow cytometry immuno-phenotyping and functional analyses.

BACKGROUND: Immune profiling by flow cytometry is not always possible on fresh blood samples due to time and/or transport constraints. Furthermore, the cryopreservation of peripheral blood mononuclear cells (PBMC) requires on-site specialized lab facilities, thus severely restricting the extent to which blood immune monitoring can be applied to multicenter clinical studies. These major limitations can be addressed through the development of simplified whole blood freezing methods. METHODS: In this report, we describe an optimized easy protocol for rapid whole blood freezing with the CryoStor® CS10 solution. Using flow cytometry, we compared cellular viability and composition on cryopreserved whole blood samples to matched fresh blood, as well as fresh and frozen PBMC. RESULTS: Though partial loss of neutrophils was observed, leucocyte viability was routinely >75% and we verified the preservation of viable T cells, NK cells, monocytes, dendritic cells, and eosinophils in frequencies similar to those observed in fresh samples. A moderate decrease in B cell frequencies was observed. Importantly, we validated the possibility to analyze major intracellular markers, such as FOXP3 and Helios in regulatory T cells. Finally, we demonstrated good functional preservation of CS10-cryopreserved cells through the analysis of intracellular cytokine production in ex vivo stimulated T cells (IFNg, IL-4, IL-17A,) and monocytes (IL-1b, IL-6, TNFa). CONCLUSIONS: In conclusion, our protocol provides a robust method to apply reliable immune monitoring studies to cryopreserved whole blood samples, hence offering new important opportunities for the design of future multicenter clinical trials.

Complex Autoinflammatory Syndrome Unveils Fundamental Principles of JAK1 Kinase Transcriptional and Biochemical Function.

Autoinflammatory disease can result from monogenic errors of immunity. We describe a patient with early-onset multi-organ immune dysregulation resulting from a mosaic, gain-of-function mutation (S703I) in JAK1, encoding a kinase essential for signaling downstream of >25 cytokines. By custom single-cell RNA sequencing, we examine mosaicism with single-cell resolution. We find that JAK1 transcription was predominantly restricted to a single allele across different cells, introducing the concept of a mutational "transcriptotype" that differs from the genotype. Functionally, the mutation increases JAK1 activity and transactivates partnering JAKs, independent of its catalytic domain. S703I JAK1 is not only hypermorphic for cytokine signaling but also neomorphic, as it enables signaling cascades not canonically mediated by JAK1. Given these results, the patient was treated with tofacitinib, a JAK inhibitor, leading to the rapid resolution of clinical disease. These findings offer a platform for personalized medicine with the concurrent discovery of fundamental biological principles.

Why is SARS-CoV-2 infection more severe in obese men? The gut lymphatics - Lung axis hypothesis.

Consistent observations report increased severity of SARS-CoV-2 infection in overweight men with cardiovascular factors. As the visceral fat possesses an intense immune activity, is involved in metabolic syndrome and is at the crossroad between the intestines, the systemic circulation and the lung, we hypothesized that it plays a major role in severe forms of SARS-CoV-2 infection. SARS-CoV2 presents the ability to infect epithelial cells of the respiratory tract as well as the intestinal tract. Several factors may increase intestinal permeability including direct enterocyte damage by SARS-CoV2, systemic inflammatory response syndrome (SIRS) and epithelial ischemia secondary to SARS-CoV2- associated endothelial dysfunction. This increase permeability further leads to translocation of microbial components such as MAMPs (microbial-associated molecular pattern), triggering an inflammatory immune response by TLR-expressing cells of the mesentery fat (mostly macrophages and adipocytes). The pro-inflammatory cytokines produced by the mesentery fat mediates systemic inflammation and aggravate acute respiratory distress syndrome (ARDS) through the mesenteric lymph drainage.

Author Correction: Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages.

The COVID-19 pandemic caused by infection with SARS-CoV-2 has led to more than 200,000 deaths worldwide. Several studies have now established that the hyperinflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. Macrophages are a population of innate immune cells that sense and respond to microbial threats by producing inflammatory molecules that eliminate pathogens and promote tissue repair. However, a dysregulated macrophage response can be damaging to the host, as is seen in the macrophage activation syndrome induced by severe infections, including in infections with the related virus SARS-CoV. Here we describe the potentially pathological roles of macrophages during SARS-CoV-2 infection and discuss ongoing and prospective therapeutic strategies to modulate macrophage activation in patients with COVID-19.

Serum Amyloid A Proteins Induce Pathogenic Th17 Cells and Promote Inflammatory Disease.

Lymphoid cells that produce interleukin (IL)-17 cytokines protect barrier tissues from pathogenic microbes but are also prominent effectors of inflammation and autoimmune disease. T helper 17 (Th17) cells, defined by RORγt-dependent production of IL-17A and IL-17F, exert homeostatic functions in the gut upon microbiota-directed differentiation from naive CD4+ T cells. In the non-pathogenic setting, their cytokine production is regulated by serum amyloid A proteins (SAA1 and SAA2) secreted by adjacent intestinal epithelial cells. However, Th17 cell behaviors vary markedly according to their environment. Here, we show that SAAs additionally direct a pathogenic pro-inflammatory Th17 cell differentiation program, acting directly on T cells in collaboration with STAT3-activating cytokines. Using loss- and gain-of-function mouse models, we show that SAA1, SAA2, and SAA3 have distinct systemic and local functions in promoting Th17-mediated inflammatory diseases. These studies suggest that T cell signaling pathways modulated by the SAAs may be attractive targets for anti-inflammatory therapies.

Single-Cell Analysis of Crohn's Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy.

Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.

IL-7 receptor influences anti-TNF responsiveness and T cell gut homing in inflammatory bowel disease.

It remains unknown what causes inflammatory bowel disease (IBD), including signaling networks perpetuating chronic gastrointestinal inflammation in Crohn's disease (CD) and ulcerative colitis (UC), in humans. According to an analysis of up to 500 patients with IBD and 100 controls, we report that key transcripts of the IL-7 receptor (IL-7R) pathway are accumulated in inflamed colon tissues of severe CD and UC patients not responding to either immunosuppressive/corticosteroid, anti-TNF, or anti-α4ß7 therapies. High expression of both IL7R and IL-7R signaling signature in the colon before treatment is strongly associated with nonresponsiveness to anti-TNF therapy. While in mice IL-7 is known to play a role in systemic inflammation, we found that in humans IL-7 also controlled α4ß7 integrin expression and imprinted gut-homing specificity on T cells. IL-7R blockade reduced human T cell homing to the gut and colonic inflammation in vivo in humanized mouse models, and altered effector T cells in colon explants from UC patients grown ex vivo. Our findings show that failure of current treatments for CD and UC is strongly associated with an overexpressed IL-7R signaling pathway and point to IL-7R as a relevant therapeutic target and potential biomarker to fill an unmet need in clinical IBD detection and treatment.

Breakdown of Immune Tolerance in AIRE-Deficient Rats Induces a Severe Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy-like Autoimmune Disease.

Autoimmune regulator (AIRE) deficiency in humans induces a life-threatening generalized autoimmune disease called autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), and no curative treatments are available. Several models of AIRE-deficient mice have been generated, and although they have been useful in understanding the role of AIRE in central tolerance, they do not reproduce accurately the APECED symptoms, and thus there is still a need for an animal model displaying APECED-like disease. We assessed, in this study, the potential of the rat as an accurate model for APECED. In this study, we demonstrate that in rat, AIRE is expressed by MHC class II (MCH-II)+ and MHC-II- medullary thymic epithelial cells in thymus and by CD4int conventional dendritic cells in periphery. To our knowledge, we generated the first AIRE-deficient rat model using zinc-finger nucleases and demonstrated that they display several of the key symptoms of APECED disease, including alopecia, skin depigmentation, and nail dystrophy, independently of the genetic background. We observed severe autoimmune lesions in a large spectrum of organs, in particular in the pancreas, and identified several autoantibodies in organs and cytokines such as type I IFNs and IL-17 at levels similar to APECED. Finally, we demonstrated a biased Ab response to IgG1, IgM, and IgA isotypes. Altogether, our data demonstrate that AIRE-deficient rat is a relevant APECED animal model, opening new opportunity to test curative therapeutic treatments.

Clinical contribution of myositis-related antibodies detected by immunoblot to idiopathic inflammatory myositis: A one-year retrospective study.

In this study, we aimed at evaluating the contribution of an extended myositis-related antibodies (Abs) determination by immunoblot to the diagnosis, classification, and prognosis of idiopathic inflammatory myositis (IIM). Medical records of all the patients (n = 237) with myositis-related Ab requests addressed to our department over a one-year period were retrospectively analyzed. Patients were classified as IIM, auto-immune disease (AID) other than IIM, and other diagnosis, and examined for their Ab profiles as determined by immunoblot. Ab positivity was qualified semi-quantitatively as low or strong according to the manufacturer's recommendations. Among the 45 Ab-positive patients, 49% were diagnosed an IIM, 22% another AID, and 29% another diagnosis. The clinico-serological patterns of the myositis-related Ab+ patients fully recapitulated those described in the literature. Among non-IIM patients, anti-PM-Scl was the most frequently detected Ab (38%), followed by anti-Mi-2 (15%), and anti-OJ (12%). Importantly, strong Ab positivity was significantly more detected in IIM vs. non-IIM patients (82% vs. 35%; p = .002). This difference was further increased when comparing MSAs only (95% vs. 36%; p = .0004). Accordingly, strong Ab positivity associated with high specificity (96%) and positive likelihood ratio (pLR =12) for IIM. Our data suggest that while myositis-related Ab, including MSA, can be detected by immunoblot in non-IIM patients, strong positivity is nevertheless highly predictive of IIM. In conclusion, this work suggests that relevant clinical contribution to IIM is provided by the immunoblot determination of myositis-related Ab, more especially when considering strong positive detection of MSA.