Mast cells, cortistatin, and its receptor, MRGPRX2, are linked to the pathogenesis of chronic prurigo.

BACKGROUND: Chronic prurigo (CPG) is characterized by intensive itch and interactions among nerves, neuropeptides, and mast cells (MCs). The role of some neuropeptides such as cortistatin (CST) and its receptor, Mas-related G protein-coupled receptor X2 (MRGPRX2), in CPG remains poorly investigated. OBJECTIVES: We evaluated first whether CST activates human skin MCs, and second whether CST and MRGPRX2 are expressed in the skin of CPG patients, and by which cells. METHODS: Skin prick tests and microdialysis with CST were performed in 6 and 1 healthy volunteers, respectively. Degranulation of human skin MCs was assessed using ß-hexosaminidase and histamine release assays. Skin samples from 10 patients with CPG and 10 control subjects were stained for CST, MCs, and MRGPRX2 (protein and mRNA) using immunohistochemistry, immunofluorescence, and/or in situ hybridization. Flow cytometry was used to assess CST in human skin MCs. MRGPRX2 levels were measured in serum by ELISA. RESULTS: CST induced concentration-dependent degranulation of human skin MCs in vivo and ex vivo. Skin lesions of CPG patients exhibited markedly higher numbers of CST-expressing cells, CST-expressing MCs, MRGPRX2-expressing cells, and MRGPRX2 mRNA-expressing cells than nonlesional skin. MCs were the main MRGPRX2 mRNA-expressing cells in the lesions of most CPG patients (70%). Stimulation of human skin MCs with anti-IgE led to a release of CST. The number of MRGPRX2-expressing cells correlated with disease severity (r = 0.649, P = .04). MRGPRX2 serum levels in CPG patients correlated with disease severity (r = 0.704, P = .023) and quality-of-life impairment (r = 0.687, P = .028). CONCLUSIONS: CST and MRGPRX2 may contribute to the pathogenesis of CPG and should be evaluated in further studies as potential biomarkers and novel therapeutic targets.

Novel intragraft regulatory lymphoid structures in kidney allograft tolerance.

Intragraft events thought to be relevant to the development of tolerance are here subjected to a comprehensive mechanistic study during long-term spontaneous tolerance that occurs in C57BL/6 mice that receive life sustaining DBA/2 kidneys. These allografts rapidly develop periarterial Treg-rich organized lymphoid structures (TOLS) that form in response to class II but not to class I MHC disparity and form independently of lymphotoxin α and lymphotoxin ß receptor pathways. TOLS form in situ in the absence of lymph nodes, spleen, and thymus. Distinctive transcript patterns are maintained over time in TOLS including transcripts associated with Treg differentiation, T cell checkpoint signaling, and Th2 differentiation. Pathway transcripts related to inflammation are expressed in early stages of accepted grafts but diminish with time, while B cell transcripts increase. Intragraft transcript patterns at one week posttransplant distinguish those from kidneys destined to be rejected, that is, C57BL/6 allografts into DBA/2 recipients, from those that will be accepted. In contrast to inflammatory tertiary lymphoid organs (iTLOs) that form in response to chronic viral infection and transgenic Lta expression, TOLS lack high endothelial venules and germinal centers. TOLS represent a novel, pathogenetically important type of TLO that are in situ markers of regulatory tolerance.

Anti-PD-1/PD-L1 therapy augments lenvatinib's efficacy by favorably altering the immune microenvironment of murine anaplastic thyroid cancer.

Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite multimodal therapy with surgery and chemoradiation. Lenvatinib, a multi-targeted tyrosine kinase inhibitor, as well as checkpoint inhibitors targeting the programmed cell death pathway, have proven effective in some patients with advanced thyroid cancer. Combination of these therapies is a potential means to boost effectiveness and minimize treatment resistance in ATC. We utilized our novel immunocompetent murine model of orthotopic ATC to demonstrate that lenvatinib led to significant tumor shrinkage and increased survival, while combination therapy led to dramatic improvements in both. Lenvatinib monotherapy increased tumor-infiltrating macrophages, CD8+ T-cells, regulatory T-cells, and most notably, polymorphonuclear myeloid derived suppressor cells (PMN-MDSCs). While both combination therapies led to further increases in CD8+ T-cells, only the lenvatinib and anti-PD-1 combination decreased PMN-MDSCs. PMN-MDSC expansion was also seen in the blood of mice and one patient receiving lenvatinib therapy for ATC. RNA-Seq of the ATC cell line used in our mouse model demonstrated that lenvatinib has multifaceted effects on angiogenesis, response to hypoxia, the epithelial-to-mesenchymal transition, and on multiple pathways implicated in inflammation and host immunity. Combination of lenvatinib with anti-Gr-1 antibody ameliorated lenvatinib's expansion of MDSCs and significantly improved lenvatinib's anti-tumor effect. These data suggest that MDSCs play a negative role in ATC's response to lenvatinib and support future study of their role as a potential biomarker and treatment target.

Combinations of BRAF inhibitor and anti-PD-1/PD-L1 antibody improve survival and tumour immunity in an immunocompetent model of orthotopic murine anaplastic thyroid cancer.

BACKGROUND: Patients with anaplastic thyroid cancer (ATC) have an extremely poor prognosis despite aggressive multimodal therapy. ATC has a high prevalence of BRAFV600E mutations and is associated with an immunosuppressive microenvironment; we previously demonstrated that the combination of BRAF inhibitor and checkpoint inhibitor immunotherapy synergistically reduce tumour volume in an immunocompetent mouse model of orthotopic ATC. METHODS: We again utilised our mouse model of ATC to assess the combination of BRAFV600E inhibitor PLX4720 and anti-PD-L1 or anti-PD-1 antibody on survival, and performed immune cell profiling of lymphoid and myeloid-lineage cells during maximal treatment response and tumour regrowth. RESULTS: Combination therapy dramatically improved mouse survival. Maximal tumour reduction was associated with increases in the number and cytotoxicity of CD8+ T cells and NK cells, as well as increases in mostly M1-polarised tumour-associated macrophages (TAM) and decreases in myeloid-derived suppressor-like cells. Regrowth of tumour occurred after 2-3 weeks of ongoing combination therapy, and was most significantly associated with decreased TAMs and a dramatic increase in M2-polarisation. CONCLUSIONS: Combination of PLX4720 and anti-PD-L1/PD-1 antibody dramatically reduced tumour volume, prolonged survival and improved the anti-tumour immune profile in murine ATC. Tumour growth inevitably recurred and demonstrated re-emergence of an immunosuppressive tumour microenvironment.

The Number of MRGPRX2-Expressing Cells Is Increased in Skin Lesions of Patients With Indolent Systemic Mastocytosis, But Is Not Linked to Symptom Severity.

Background: Recently, the expression of the mast cell (MC) receptor Mas-related G protein-coupled receptor X2 (MRGPRX2) has been detected in lesional skin of adult patients with cutaneous mastocytosis. As of yet, little is known about the clinical relevance of MRGPRX2 and its agonists in patients with mastocytosis, including indolent systemic mastocytosis (ISM). Methods: MRGPRX2 and MRGPRX2 agonists, cortistatin (CST), and major basic protein (MBP) were analyzed in lesional and non-lesional skin of patients with ISM and skin of healthy controls by immunohistochemistry. Co-localization of MRGPRX2 and MRGPRX2-mRNA with the MC marker tryptase was assessed by immunofluorescence microscopy and in situ hybridization, respectively. We assessed clinical, demographic, and laboratory data, including mastocytosis activity score (MAS), serum tryptase, and KIT D816V allele burden. Results: The number of MRGPRX2-expressing (MRGPRX2+) cells, MRGPRX2-mRNA+ MCs, and CST-expressing (CST+) and MBP-expressing (MBP+) cells was significantly higher in lesional skin as compared to non-lesional skin and/or skin of healthy controls (all p < 0.05). Increased numbers of MRGPRX2+ cells, MRGPRX2-mRNA+ MCs, and CST+ and MBP+ cells were not associated with clinical and laboratory features of ISM, including disease burden, symptom severity, evidence of anaphylaxis, and tryptase levels. Conclusions: Skin lesions of patients with ISM showed high numbers of MRGPRX2+ cells, although they were not linked to symptom severity. Clinical relevance of the MRGPRX2-mediated pathway of MC activation in ISM remains unclear and should be investigated in further studies.