Human skin CD141+ dendritic cells regulate cutaneous immunity via the neuropeptide urocortin 2.

Skin immune homeostasis is a multi-faceted process where dermal dendritic cells (DDCs) are key in orchestrating responses to environmental stressors. We have previously identified CD141+CD14+ DDCs as a skin-resident immunoregulatory population that is vitamin-D3 (VitD3) inducible from monocyte-derived DCs (moDCs), termed CD141hi VitD3 moDCs. We demonstrate that CD141+ DDCs and CD141hi VitD3 moDCs share key immunological features including cell surface markers, reduced T cell stimulation, IL-10 production, and a common transcriptomic signature. Bioinformatic analysis identified the neuroactive ligand receptor pathway and the neuropeptide, urocortin 2 (UCN2), as a potential immunoregulatory candidate molecule. Incubation with VitD3 upregulated UCN2 in CD141+ DCs and UVB irradiation induced UCN2 in CD141+ DCs in healthy skin in vivo. Notably, CD141+ DDC generation of suppressive Tregs was dependent upon the UCN2 pathway as in vivo administration of UCN2 reversed skin inflammation in humanized mice. We propose the neuropeptide UCN2 as a novel skin DC-derived immunoregulatory mediator with a potential role in UVB and VitD3-dependent skin immune homeostasis.

Molecular Features and Stages of Pulmonary Fibrosis Driven by Type 2 Inflammation.

Systemic sclerosis (SSc) is a progressive, multiorgan disease with limited treatment options. Although a recent proof-of-concept study using romilkimab or SAR156597, a bispecific IL-4/IL-13 antibody, suggests a direct role of these cytokines in the pathophysiology of SSc, their contributions to the balance between inflammation and fibrosis are unclear. Here, we determine the roles of type 2 inflammation in fibrogenesis using FRA2-Tg (Fos-related antigen 2-overexpressing transgenic) mice, which develop spontaneous, age-dependent progressive lung fibrosis. We defined the molecular signatures of inflammation and fibrosis at three key stages in disease progression, corresponding to preonset, inflammatory dominant, and fibrosis dominant biology, and revealed an early increase in cytokine-cytokine receptor interactions and antigen-processing and presentation pathways followed by enhanced Th2- and M2 macrophage-driven type 2 responses. This type 2 inflammation progressed to extensive fibrotic pathology by 14-18 weeks of age, with these gene signatures overlapping significantly with those seen in the lungs of patients with SSc with interstitial lung disease (ILD). These changes were also evident in the histopathology, which showed perivascular and peribronchiolar inflammation with prominent eosinophilia and accumulation of profibrotic M2-like macrophages followed by rapid progression to fibrosis with thickened alveolar walls with multifocal fibrotic bands and signs of interstitial pneumonia. Critically, treatment with a bispecific antibody targeting IL-4 and IL-13 during the inflammatory phase abrogated the Th2 and M2 responses and led to near-complete abrogation of lung fibrosis. These data recapitulate important features of fibrotic progression in the lungs of patients with SSc-ILD and enhance our understanding of the progressive pathobiology of SSc. This study also further establishes FRA2-Tg mice as a valuable tool for testing future therapeutic agents in SSc-ILD.

Anti-cancer pro-inflammatory effects of an IgE antibody targeting the melanoma-associated antigen chondroitin sulfate proteoglycan 4.

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma.

Additive efficacy of a bispecific anti-TNF/IL-6 nanobody compound in translational models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is one of the most common autoimmune diseases affecting primarily the joints. Despite successful therapies including antibodies against tumor necrosis factor (TNF) and interleukin-6 (IL-6) receptor, only 20 to 30% of patients experience remission. We studied whether inhibiting both TNF and IL-6 would result in improved efficacy. Using backtranslation from single-cell RNA sequencing (scRNA-seq) data from individuals with RA, we hypothesized that TNF and IL-6 act synergistically on fibroblast-like synoviocytes (FLS) and T cells. Coculture of FLS from individuals with RA and T cells supported this hypothesis, revealing effects on both disease-driving pathways and biomarkers. Combining anti-TNF and anti-IL-6 antibodies in collagen-induced arthritis (CIA) mouse models resulted in sustained long-term remission, improved histology, and effects on bone remodeling pathways. These promising data initiated the development of an anti-TNF/IL-6 bispecific nanobody compound 1, with similar potencies against TNF and IL-6. We observed additive efficacy of compound 1 in a FLS/T cell coculture affecting arthritis and T helper 17 (TH17) pathways. This nanobody compound transcript signature inversely overlapped with described RA endotypes, indicating a potential efficacy in a broader patient population. In summary, we showed superiority of a bispecific anti-TNF/IL-6 nanobody compound or combination treatment over monospecific treatments in both in vitro and in vivo models. We anticipate improved efficacy in upcoming clinical studies.

Influence of FLG loss-of-function mutations in host-microbe interactions during atopic skin inflammation.

BACKGROUND: Loss-of-function mutations in the filaggrin (FLG) gene directly alter skin barrier function and critically influence atopic inflammation. While skin barrier dysfunction, Th2-associated inflammation and bacterial dysbiosis are well-known characteristics of atopic dermatitis (AD), the mechanisms interconnecting genotype, transcriptome and microbiome remain largely elusive. OBJECTIVE: In-depth analysis of FLG genotype-associated skin gene expression alterations and host-microbe interactions in AD. METHODS: Multi-omics characterization of a cohort of AD patients carrying heterozygous loss-of-function mutations in the FLG gene (ADMut) (n = 15), along with matched wild-type (ADWt) patients and healthy controls. Detailed clinical characterization, microarray gene expression and 16 S rRNA-based microbial marker gene data were generated and analyzed. RESULTS: In the context of filaggrin dysfunction, the transcriptome was characterized by dysregulation of barrier function and water homeostasis, while the lesional skin of ADWt demonstrated the specific upregulation of pro-inflammatory cytokines and T-cell proliferation. S. aureus dominated the microbiome in both patient groups, however, shifting microbial communities could be observed when comparing healthy with non-lesional ADWt or ADMut skin, offering the opportunity to identify microbe-associated transcriptomic signatures. Moreover, an AD core signature with 28 genes, including CCL13, CCL18, BTC, SCIN, RAB31 and PCLO was identified. CONCLUSIONS: Our integrative approach provides molecular insights for the concept that FLG loss-of-function mutations are a genetic shortcut to atopic inflammation and unravels the complex interplay between genotype, transcriptome and microbiome in the human holobiont.

Current and Emerging Strategies to Inhibit Type 2 Inflammation in Atopic Dermatitis.

Type 2 immunity evolved to combat helminth infections by orchestrating a combined protective response of innate and adaptive immune cells and promotion of parasitic worm destruction or expulsion, wound repair, and barrier function. Aberrant type 2 immune responses are associated with allergic conditions characterized by chronic tissue inflammation, including atopic dermatitis (AD) and asthma. Signature cytokines of type 2 immunity include interleukin (IL)-4, IL-5, IL-9, IL-13, and IL-31, mainly secreted from immune cells, as well as IL-25, IL-33, and thymic stromal lymphopoietin, mainly secreted from tissue cells, particularly epithelial cells. IL-4 and IL-13 are key players mediating the prototypical type 2 response; IL-4 initiates and promotes differentiation and proliferation of naïve T-helper (Th) cells toward a Th2 cell phenotype, whereas IL-13 has a pleiotropic effect on type 2 inflammation, including, together with IL-4, decreased barrier function. Both cytokines are implicated in B-cell isotype class switching to generate immunoglobulin E, tissue fibrosis, and pruritus. IL-5, a key regulator of eosinophils, is responsible for eosinophil growth, differentiation, survival, and mobilization. In AD, IL-4, IL-13, and IL-31 are associated with sensory nerve sensitization and itch, leading to scratching that further exacerbates inflammation and barrier dysfunction. Various strategies have emerged to suppress type 2 inflammation, including biologics targeting cytokines or their receptors, and Janus kinase inhibitors that block intracellular cytokine signaling pathways. Here we review type 2 inflammation, its role in inflammatory diseases, and current and future therapies targeting type 2 pathways, with a focus on AD. INFOGRAPHIC.

Enhanced NF-κB signaling in type-2 dendritic cells at baseline predicts non-response to adalimumab in psoriasis.

Biologic therapies have transformed the management of psoriasis, but clinical outcome is variable leaving an unmet clinical need for predictive biomarkers of response. Here we perform in-depth immunomonitoring of blood immune cells of 67 patients with psoriasis, before and during therapy with the anti-TNF drug adalimumab, to identify immune mediators of clinical response and evaluate their predictive value. Enhanced NF-κBp65 phosphorylation, induced by TNF and LPS in type-2 dendritic cells (DC) before therapy, significantly correlates with lack of clinical response after 12 weeks of treatment. The heightened NF-κB activation is linked to increased DC maturation in vitro and frequency of IL-17+ T cells in the blood of non-responders before therapy. Moreover, lesional skin of non-responders contains higher numbers of dermal DC expressing the maturation marker CD83 and producing IL-23, and increased numbers of IL-17+ T cells. Finally, we identify and clinically validate LPS-induced NF-κBp65 phosphorylation before therapy as a predictive biomarker of non-response to adalimumab, with 100% sensitivity and 90.1% specificity in an independent cohort. Our study uncovers important molecular and cellular mediators underpinning adalimumab mechanisms of action in psoriasis and we propose a blood biomarker for predicting clinical outcome.

CYP1A1 Enzymatic Activity Influences Skin Inflammation Via Regulation of the AHR Pathway.

The AHR is an environmental sensor and transcription factor activated by a variety of man-made and natural ligands, which has recently emerged as a critical regulator of homeostasis at barrier organs such as the skin. Activation of the AHR pathway downmodulates skin inflammatory responses in animal models and psoriasis clinical samples. In this study, we identify CYP1A1 enzymatic activity as a critical regulator of beneficial AHR signaling in the context of skin inflammation. Mice constitutively expressing Cyp1a1 displayed increased CYP1A1 enzymatic activity in the skin, which resulted in exacerbated immune cell activation and skin pathology, mirroring that observed in Ahr-deficient mice. Inhibition of CYP1A1 enzymatic activity ameliorated the skin immunopathology by restoring beneficial AHR signaling. Importantly, patients with psoriasis displayed reduced activation of the AHR pathway and increased CYP1A1 enzymatic activity compared with healthy donors, suggesting that dysregulation of the AHR/CYP1A1 axis may play a role in inflammatory skin disease. Thus, modulation of CYP1A1 activity may represent a promising alternative strategy to harness the anti-inflammatory effect exerted by activation of the AHR pathway in the skin.

Microbial and transcriptional differences elucidate atopic dermatitis heterogeneity across skin sites.

It is well established that different sites in healthy human skin are colonized by distinct microbial communities due to different physiological conditions. However, few studies have explored microbial heterogeneity between skin sites in diseased skin, such as atopic dermatitis (AD) lesions. To address this issue, we carried out deep analysis of the microbiome and transcriptome in the skin of a large cohort of AD patients and healthy volunteers, comparing two physiologically different sites: upper back and posterior thigh. Microbiome samples and biopsies were obtained from both lesional and nonlesional skin to identify changes related to the disease process. Transcriptome analysis revealed distinct disease-related gene expression profiles depending on anatomical location, with keratinization dominating the transcriptomic signatures in posterior thigh, and lipid metabolism in the upper back. Moreover, we show that relative abundance of Staphylococcus aureus is associated with disease severity in the posterior thigh, but not in the upper back. Our results suggest that AD may select for similar microbes in different anatomical locations-an "AD-like microbiome," but distinct microbial dynamics can still be observed when comparing posterior thigh to upper back. This study highlights the importance of considering the variability across skin sites when studying the development of skin inflammation.

Publisher Correction: An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples.

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

An optimized workflow for single-cell transcriptomics and repertoire profiling of purified lymphocytes from clinical samples.

Establishing clinically relevant single-cell (SC) transcriptomic workflows from cryopreserved tissue is essential to move this emerging immune monitoring technology from the bench to the bedside. Improper sample preparation leads to detrimental cascades, resulting in loss of precious time, money and finally compromised data. There is an urgent need to establish protocols specifically designed to overcome the inevitable variations in sample quality resulting from uncontrollable factors in a clinical setting. Here, we explore sample preparation techniques relevant to a range of clinically relevant scenarios, where SC gene expression and repertoire analysis are applied to a cryopreserved sample derived from a small amount of blood, with unknown or partially known preservation history. We compare a total of ten cell-counting, viability-improvement, and lymphocyte-enrichment methods to highlight a number of unexpected findings. Trypan blue-based automated counters, typically recommended for single-cell sample quantitation, consistently overestimate viability. Advanced sample clean-up procedures significantly impact total cell yield, while only modestly increasing viability. Finally, while pre-enrichment of B cells from whole peripheral blood mononuclear cells (PBMCs) results in the most reliable BCR repertoire data, comparable T-cell enrichment strategies distort the ratio of CD4+ and CD8+ cells. Furthermore, we provide high-resolution analysis of gene expression and clonotype repertoire of different B cell subtypes. Together these observations provide both qualitative and quantitative sample preparation guidelines that increase the chances of obtaining high-quality single-cell transcriptomic and repertoire data from human PBMCs in a variety of clinical settings.

Translational drug discovery and development with the use of tissue-relevant biomarkers: Towards more physiological relevance and better prediction of clinical efficacy.

Due to the clinical development of drugs such as secukinumab, ustekinumab and dupilumab, major changes have been achieved in the treatment of patients diagnosed with psoriasis and atopic dermatitis. In academia and the pharmaceutical industry, research is increasingly moving towards the development of bispecific antibodies and multi-specific nanobodies, as there is a compelling need for new treatment modalities for patients suffering from autoimmune or malignant disease. The purpose of this review is to discuss aspects of translational drug development with a particular emphasis on indications such as psoriasis and atopic dermatitis. The identification of biomarkers, the assessment of target organ pharmacokinetic and pharmacodynamics interactions and a wide range of in vitro, ex vivo and in vivo models should contribute to an appropriate prediction of a biological effect in the clinical setting. As human biology may not be perfectly reflected by approaches such as skin equivalents or animal models, novel approaches such as the use of human skin and dermal microperfusion assays in healthy volunteers and patients appear both reasonable and mandatory. These models may indeed generate highly translationally relevant data that have the potential to reduce the failure rate of drugs currently undergoing clinical development.

Microbe-host interplay in atopic dermatitis and psoriasis.

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment.

ROCK-Myosin II drives fast rounded-amoeboid migration in cancer cells during metastatic dissemination. Analysis of human melanoma biopsies revealed that amoeboid melanoma cells with high Myosin II activity are predominant in the invasive fronts of primary tumors in proximity to CD206+CD163+ tumor-associated macrophages and vessels. Proteomic analysis shows that ROCK-Myosin II activity in amoeboid cancer cells controls an immunomodulatory secretome, enabling the recruitment of monocytes and their differentiation into tumor-promoting macrophages. Both amoeboid cancer cells and their associated macrophages support an abnormal vasculature, which ultimately facilitates tumor progression. Mechanistically, amoeboid cancer cells perpetuate their behavior via ROCK-Myosin II-driven IL-1α secretion and NF-κB activation. Using an array of tumor models, we show that high Myosin II activity in tumor cells reprograms the innate immune microenvironment to support tumor growth. We describe an unexpected role for Myosin II dynamics in cancer cells controlling myeloid function via secreted factors.

Anti-Folate Receptor Alpha-Directed Antibody Therapies Restrict the Growth of Triple-negative Breast Cancer.

Purpose: Highly aggressive triple-negative breast cancers (TNBCs) lack validated therapeutic targets and have high risk of metastatic disease. Folate receptor alpha (FRα) is a central mediator of cell growth regulation that could serve as an important target for cancer therapy.Experimental Design: We evaluated FRα expression in breast cancers by genomic (n = 3,414) and IHC (n = 323) analyses and its association with clinical parameters and outcomes. We measured the functional contributions of FRα in TNBC biology by RNA interference and the antitumor functions of an antibody recognizing FRα (MOv18-IgG1), in vitro, and in human TNBC xenograft models.Results: FRα is overexpressed in significant proportions of aggressive basal like/TNBC tumors, and in postneoadjuvant chemotherapy-residual disease associated with a high risk of relapse. Expression is associated with worse overall survival. TNBCs show dysregulated expression of thymidylate synthase, folate hydrolase 1, and methylenetetrahydrofolate reductase, involved in folate metabolism. RNA interference to deplete FRα decreased Src and ERK signaling and resulted in reduction of cell growth. An anti-FRα antibody (MOv18-IgG1) conjugated with a Src inhibitor significantly restricted TNBC xenograft growth. Moreover, MOv18-IgG1 triggered immune-dependent cancer cell death in vitro by human volunteer and breast cancer patient immune cells, and significantly restricted orthotopic and patient-derived xenograft growth.Conclusions: FRα is overexpressed in high-grade TNBC and postchemotherapy residual tumors. It participates in cancer cell signaling and presents a promising target for therapeutic strategies such as ADCs, or passive immunotherapy priming Fc-mediated antitumor immune cell responses. Clin Cancer Res; 24(20); 5098-111. ©2018 AACR.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation.

The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of Tregs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs.