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ObjectivesGut and joint inflammation commonly co-occur in spondyloarthritis (SpA) which strongly restricts therapeutic modalities. The immunobiology underlying differences between gut and joint immune regulation, however, is poorly understood. We therefore assessed the immunoregulatory role of CD4+FOXP3+regulatory T (Treg) cells in a model of Crohn’s-like ileitis and concomitant arthritis.MethodsRNA-sequencing and flow cytometry was performed on inflamed gut and joint samples and tissue-derived Tregs from tumour necrosis factor (TNF)∆AREmice. In situ hybridisation of TNF and its receptors (TNFR) was applied to human SpA gut biopsies. Soluble TNFR (sTNFR) levels were measured in serum of mice and patients with SpA and controls. Treg function was explored by in vitro cocultures and in vivo by conditional Treg depletion.ResultsChronic TNF exposure induced several TNF superfamily (TNFSF) members (4-1BBL, TWEAK and TRAIL) in synovium and ileum in a site-specific manner. Elevated TNFR2 messenger RNA levels were noted in TNF∆ARE/+mice leading to increased sTNFR2 release. Likewise, sTNFR2 levels were higher in patients with SpA with gut inflammation and distinct from inflammatory and healthy controls. Tregs accumulated at both gut and joints of TNF∆AREmice, yet their TNFR2 expression and suppressive function was significantly lower in synovium versus ileum. In line herewith, synovial and intestinal Tregs displayed a distinct transcriptional profile with tissue-restricted TNFSF receptor and p38MAPK gene expression.ConclusionsThese data point to profound differences in immune-regulation between Crohn’s ileitis and peripheral arthritis. Whereas Tregs control ileitis they fail to dampen joint inflammation. Synovial resident Tregs are particularly maladapted to chronic TNF exposure.

SummaryTissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis.We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from SSc patients deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This can be mimicked in healthy fibroblast stimulated by soluble factors that drive inflammation and fibrosis in SSc and is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling.In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.Abstract Figure

BackgroundPancreatic cancer remains a deadly disease due to difficulties hindering its early diagnosis, giving way to metastasis of the tumor and resulting in poor prognosis. While there are many neoplasms of the pancreas, pancreatic invasive ductal adenocarcinoma (PDAC) is the most common and treatment options are few, with poor overall survival. Aggressive surgeries such as the Whipple procedure coupled to systemic chemotherapy is one of the few treatment options. Recently, several publications have demonstrated improved outcomes with the inclusion of immunotherapy to cytotoxic drug combinations in some patients, however optimally selecting patients as candidates for immunotherapy-chemotherapy combinations remains a critical challenge. The complexities of the tumor microenvironment have been implicated in the failure of chemotherapy, radiation therapy, and immunotherapy. The tumor microenvironment of PDAC is especially rich with multiple interactions between pancreatic epithelial/cancer cells, stromal cells, immune cells and the extracellular matrix (ECM). PDACs are characterized by a complex ECM of desmoplastic reaction consisting of an extensive and dense fibrotic stroma that surrounds and infiltrates clusters of malignant epithelial cells, together with the loss of basement membrane integrity and an abnormal vasculature.MethodsIn the present study we demonstrate a tissue phenotyping workflow combining three complementary methods that can unravel novel insights in the complex tumor microenvironment. This novel translational workflow delivers tissue morphology information, spatial phenotyping of immune cell population on whole slides, and high dimensional imaging in selected regions of interest (ROI), by combining H&E, multiplex immunofluorescence (mIF), and Imaging Mass Cytometry (IMC™).ResultsThe use of the InSituPlex® UltiMapper® I/O PD-L1 kit enabled the streamlined combination and alignment of H&E and mIF data, leading to the strategic selection of relevant ROIs, while utility of IMC technology enabled downstream imaging of 35 protein markers associated with the ECM in the selected ROIs to provide a deeper understanding of the tumor microenvironment.ConclusionsThe incorporation of advanced multiplex imaging platforms such as mIF and IMC with routine H&E workflow in tumor biology can deliver some of the much-needed insight into tumor morphology, cellular composition, cellular functions, and cell-cell interactions and paves the way for potentially improved clinical prognosis and efficacy prediction in patients with cancer.

Pancreatic cancer remains a deadly disease due to difficulties hindering its early diagnosis, giving way to metastasis of the tumor and resulting in poor prognosis. While there are many neoplasms of the pancreas, pancreatic invasive ductal adenocarcinoma (PDAC) is the most common, and treatment options are few, with poor overall survival. Recently, several publications have demonstrated improved outcomes with the inclusion of immunotherapy to cytotoxic drug combinations in some patients. However, optimally selecting patients as candidates for immunotherapy-chemotherapy combinations remains a critical challenge. The complexities of the tumor microenvironment (TME) have been implicated in the failure of chemotherapy, radiation therapy, and immunotherapy. The tumor microenvironment of PDAC is especially rich with multiple interactions between pancreatic epithelial/cancer cells, stromal cells, immune cells, and the extracellular matrix (ECM). PDACs are characterized by a complex ECM of desmoplastic reaction consisting of an extensive and dense fibrotic stroma that surrounds and infiltrates clusters of malignant epithelial cells, together with the loss of basement membrane integrity and an abnormal vasculature. PD-L1 is also expressed in PDACs, and its overexpression has been associated with a poor prognosis. In the present study we demonstrate a unique tissue phenotyping workflow combining complementary methods that can unravel the complexity of the tumor microenvironment. We highlight how a workflow combining multiple elements provides utility, robustness, and an ability to derive biological insights in PDAC samples. An InSituPlex® PD-L1 multiplex immunofluorescence assay (4 markers, CD8, CD68, PD-L1, Pan CK/Sox10) was used on whole slides to identify areas of high, medium, and low PD-L1 expression. Imaging Mass Cytometry™ (IMC™, 40 markers) was performed on selected regions of interest from each slide. Advanced tissue and cell segmentation followed by multiplex cellular phenotyping and spatial analyses were performed on both whole-slide InSituPlex and IMC data. These methods combine to give a detailed readout of the location and bio-distribution of specific cell phenotypes in situ in the TME of PDAC. Four-plex imaging and analysis of whole slides gives an overview of the immune status of the section, while 40-plex imaging and analysis gives a comprehensive and multiparametric exploration of cells present. These methods combine to reveal an exceptional view of the PDAC TME at the single-cell level. Citation Format: Andrew Quong, Jordan Nieto, Derek Quong, Amanda Esch, Kirsteen Maclean, Mark Rees, Devan Fleury, Gourab Chatterjee, Keith Wharton, Jeppe Thagaard, Fabian Schneider, Dan Winkowski, James Mansfield. Improved understanding of the biology and pathophysiology of the tumor microenvironment in PDAC samples revealed by InSituPlex, Imaging Mass Cytometry, and advanced image processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1709.