Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions.

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

In-depth immune profiling of peripheral blood mononuclear cells in patients with pancreatic ductal adenocarcinoma reveals discriminative immune subpopulations.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time-of-flight to undertake an in-depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro-inflammatory cells, as CD86+ classical monocytes and memory T cells expressing CCR6+ and CXCR3+, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39+ regulatory T cells and CCR4+CCR6-CXCR3- memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms.

The role of CD56bright NK cells in neurodegenerative disorders.

Emerging evidence suggests a potential role for natural killer (NK) cells in neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise function of NK cells in these diseases remains ambiguous. The existence of two NK cell subsets, CD56bright and CD56dim NK cells, complicates the understanding of the contribution of NK cells in neurodegeneration as their functions within the context of neurodegenerative diseases may differ significantly. CD56bright NK cells are potent cytokine secretors and are considered more immunoregulatory and less terminally differentiated than their mostly cytotoxic CD56dim counterparts. Hence, this review focusses on NK cells, specifically on CD56bright NK cells, and their role in neurodegenerative diseases. Moreover, it explores the mechanisms underlying their ability to enter the central nervous system. By consolidating current knowledge, we aim to provide a comprehensive overview on the role of CD56bright NK cells in neurodegenerative diseases. Elucidating their impact on neurodegeneration may have implications for future therapeutic interventions, potentially ameliorating disease pathogenesis.

Cancer-on-chip models for metastasis: importance of the tumor microenvironment.

Cancer-on-chip (CoC) models, based on microfluidic chips harboring chambers for 3D tumor-cell culture, enable us to create a controlled tumor microenvironment (TME). CoC models are therefore increasingly used to systematically study effects of the TME on the various steps in cancer metastasis. Moreover, CoC models have great potential for developing novel cancer therapies and for predicting patient-specific response to cancer treatments. We review recent developments in CoC models, focusing on three main TME components: (i) the anisotropic extracellular matrix (ECM) architectures, (ii) the vasculature, and (iii) the immune system. We aim to provide guidance to biologists to choose the best CoC approach for addressing questions about the role of the TME in metastasis, and to inspire engineers to develop novel CoC technologies.

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC is characterized by a desmoplastic, hypoxic and highly immunosuppressive tumor microenvironment (TME), where T-cell infiltration is often lacking (immune desert), or where T cells are located distant from the tumor islands (immune excluded). Converting the TME to an immune-inflamed state, allowing T-cell infiltration, could increase the success of immunotherapy in PDAC. METHOD: In this study, we use the KPC3 subcutaneous PDAC mouse model to investigate the role of tumor-derived sialic acids in shaping the tumor immune landscape. A sialic acid deficient KPC3 line was generated by genetic knock-out of the CMAS (cytidine monophosphate N-acetylneuraminic acid synthetase) enzyme, a critical enzyme in the synthesis of sialic acid-containing glycans. The effect of sialic acid-deficiency on immunotherapy efficacy was assessed by treatment with anti-programmed cell death protein 1 (PD-1) and agonistic CD40. RESULT: The absence of sialic acids in KPC3 tumors resulted in increased numbers of CD4+ and CD8+ T cells in the TME, and reduced frequencies of CD4+ regulatory T cells (Tregs) within the T-cell population. Importantly, CD8+ T cells were able to infiltrate the tumor islands in sialic acid-deficient tumors. These favorable alterations in the immune landscape sensitized sialic acid-deficient tumors to immunotherapy, which was ineffective in sialic acid-expressing KPC3 tumors. In addition, high expression of sialylation-related genes in human pancreatic cancer correlated with decreased CD8+ T-cell infiltration, increased presence of Tregs, and poorer survival probability. CONCLUSION: Our results demonstrate that tumor-derived sialic acids mediate T-cell exclusion within the PDAC TME, thereby impairing immunotherapy efficacy. Targeting sialic acids represents a potential strategy to enhance T-cell infiltration and improve immunotherapy outcomes in PDAC.

Spatial immune composition of tumor microenvironment in patients with pancreatic cancer.

This study examined the composition of the immune microenvironment at different sites within resected pancreas specimens from patients with pancreatic ductal adenocarcinoma (PDAC). Therefore, single-cell suspensions were made from fresh tumor and non-tumorous tissue. Fourteen patients were included from whom twelve PDAC and five non-tumorous samples were obtained. These samples were analyzed with a nineteen marker panel on the Aurora spectral flow cytometer. Furthermore, slides from formalin-fixed paraffine PDACs of eight additional patients were stained with eight markers and analyzed by multispectral imaging. These corresponded to central tumor, periphery of the tumor, i.e., invasive front and resected lymph node and were divided into tumor and adjacent tissue. In the single-cell suspension, a decreased ratio between lymphoid and myeloid cells and between M1 and M2 macrophages was observed in the tumor tissue compared to non-tumorous tissue. Furthermore, an increase in CD169 + macrophages in patients undergoing neoadjuvant therapy was found. Using immunofluorescence, more macrophages compared to T cells were observed, as well as a lower ratio of CD8 to M2 macrophage, a higher ratio of CD4-CD8 T cells and a higher ratio of immune-suppressive cells to pro-inflammatory cells in the PDAC area compared to the adjacent non-tumorous tissue. Finally, there were more immune-suppressive cells in the central tumor area compared to the invasive front. In conclusion, we show a gradient in the immune-suppressive environment in PDAC from most suppressive in the central tumor to least suppressive in distant non-tumorous tissue.

The immune microenvironment after neoadjuvant therapy compared to upfront surgery in patients with pancreatic cancer.

BACKGROUND: Patients with resectable and borderline resectable pancreatic ductal adenocarcinoma increasingly receive neoadjuvant therapy prior to surgery. However, the effect of neoadjuvant therapy on the immune microenvironment remains largely unknown. We analyzed the immune microenvironment in pancreatic cancer tumor tissue samples from patients treated with neoadjuvant therapy compared to patients after upfront surgery to gain knowledge about the immunological environment after therapy. METHODS: Multispectral imaging was performed on tissue from resected specimens from patients with PDAC who underwent upfront surgery (n = 10), neoadjuvant FOLFIRINOX (n = 10) or gemcitabine + radiotherapy (gem-RT) (n = 9) followed by surgery. The samples were selected by a dedicated pancreas pathologist from both the central part and the invasive front of the tumor (by the resected vein or venous surface) and subsequently analyzed using the Vectra Polaris. RESULTS: Patients receiving neoadjuvant FOLFIRINOX display a more pro-inflammatory immune profile, with less regulatory T cells and more CD8 T cells in the tumor tissue compared to patients receiving neoadjuvant gem-RTgem-RT or undergoing upfront surgery. Furthermore, CD163+ macrophages were decreased, and a higher CD163- macrophages versus CD163+ macrophages ratio was found in patients with neoadjuvant FOLFIRINOX. In all treatment groups, percentage of FoxP3+ B cells was significantly higher in tumor tissue compared to adjacent tissue. Furthermore, an increase in regulatory T cells in the tumor tissue was found in patients undergoing upfront surgery or receiving neoadjuvant gem-RT. In the gem-RT group, less CD8 T cells and a higher CD163+ macrophages to CD8 ratio were noted in the tumor tissue, suggesting a more immune suppressive profile in the tumor tissue. CONCLUSION: Patients receiving neoadjuvant FOLFIRINOX display a more pro-inflammatory immune profile compared to patients receiving neoadjuvant gem-RT or undergoing upfront surgery. Furthermore, in all treatment groups, a more immune suppressive microenvironment was found in the tumor tissue compared to the adjacent non-tumorous tissue.

Factors that influence the pancreatic and duodenal microbiome in patients undergoing pancreatic surgery.

BACKGROUND/OBJECTIVES: This study examined the correlation between pancreatic microbiome and patients characteristics. Furthermore, we compared different duodenal materials to examine their reflection of the pancreatic microbiome. METHODS: Patients undergoing pancreatic surgery were included in the study. Characteristics of those patients were prospectively registered and sterile pancreatic biopsies were collected during surgery. After completion of the resection, duodenal fluid, -tissue and -swab were collected. Bacterial DNA was extracted and analyzed with IS-pro assay. RESULTS: Paired samples of 51 patients were available for evaluation, including pancreatic biopsies from all patients, 22 duodenal fluids, 21 duodenal swabs and 11 duodenal tissues. The pancreatic microbiome consisted mostly of Proteobacteria followed by Firmicutes, Actinobacteria, Fusobacteria and Verrucomicrobia (FAFV) and Bacteroidetes. On species level, Enterococcus faecalis, Escherichia coli, and Enterobacter-Klebsiella were most abundant. In pancreatic biopsies, the total bacterial load and Proteobacteria load were significantly higher in patients with biliary drainage (54618.0 vs 5623.5; 9119.0 vs 2067.1). Patients who used proton pump inhibitors had a significantly higher total bacterial load (115964.7 vs 8495.8), more FAFV (66862.9 vs 1890.1), more Proteobacteria (24245.9 vs 2951.4) and more Bacteroidetes (542.5 vs 25.8). The head of the pancreas contained significantly more bacteria (21193.4 vs 2096.8) and more FAFV (5225.7 vs 19.0) compared to the tail, regardless of biliary drainage. Furthermore, the microbiome of all duodenal materials showed a weak correlation with the pancreatic microbiome. CONCLUSION: Biliary drainage, use of proton pump inhibitors, and anatomic location of the pancreatic biopsy influence the pancreatic microbiome. Furthermore, the duodenal microbiome does not suffice as a surrogate for the pancreatic microbiome.

Fcα Receptor-1-Activated Monocytes Promote B Lymphocyte Migration and IgA Isotype Switching.

Patients with inflammatory bowel disease (IBD) produce enhanced immunoglobulin A (IgA) against the microbiota compared to healthy individuals, which has been correlated with disease severity. Since IgA complexes can potently activate myeloid cells via the IgA receptor FcαRI (CD89), excessive IgA production may contribute to IBD pathology. However, the cellular mechanisms that contribute to dysregulated IgA production in IBD are poorly understood. Here, we demonstrate that intestinal FcαRI-expressing myeloid cells (i.e., monocytes and neutrophils) are in close contact with B lymphocytes in the lamina propria of IBD patients. Furthermore, stimulation of FcαRI-on monocytes triggered production of cytokines and chemokines that regulate B-cell differentiation and migration, including interleukin-6 (IL6), interleukin-10 (IL10), tumour necrosis factor-α (TNFα), a proliferation-inducing ligand (APRIL), and chemokine ligand-20 (CCL20). In vitro, these cytokines promoted IgA isotype switching in human B cells. Moreover, when naïve B lymphocytes were cultured in vitro in the presence of FcαRI-stimulated monocytes, enhanced IgA isotype switching was observed compared to B cells that were cultured with non-stimulated monocytes. Taken together, FcαRI-activated monocytes produced a cocktail of cytokines, as well as chemokines, that stimulated IgA switching in B cells, and close contact between B cells and myeloid cells was observed in the colons of IBD patients. As such, we hypothesize that, in IBD, IgA complexes activate myeloid cells, which in turn can result in excessive IgA production, likely contributing to disease pathology. Interrupting this loop may, therefore, represent a novel therapeutic strategy.

A Straightforward Method for 3D Visualization of B Cell Clusters and High Endothelial Venules in Lymph Nodes Highlights Differential Roles of TNFRI and -II.

Whole mount tissue immunolabeling and imaging of complete organs has tremendous benefits in characterizing organ morphology. Here, we present a straightforward method for immunostaining, clearing and imaging of whole murine peripheral lymph nodes (PLNs) for detailed analysis of their architecture and discuss all procedures in detail in a step-by-step approach. Given the importance of tumor necrosis factor receptor (TNFR) signaling in development of PLNs we used TNFRI-/- and TNFRII-/- mice models as proof-of-concept for this technique by visualizing and analyzing structural changes in PLN B cell clusters and high endothelial venules (HEVs). Samples were subjected to de- and rehydration with methanol, labeled with antibodies for B cells, T cells and high endothelial venules (HEVs) and optically cleared using benzyl alcohol-benzyl benzoate. Imaging was done using LaVision light sheet microscope and analysis with Imaris software. Using these techniques, we confirmed previous findings that TNFRI signaling is essential for formation of individual B cell clusters. In addition, Our data suggest that TNFRII signaling is also to some extent involved in this process as TNFRII-/- PLNs had a B cell cluster morphology reminiscent of TNFRI-/- PLNs. Moreover, visualization and objective quantification of the complete PLN high endothelial vasculature unveiled reduced volume, length and branching points of HEVs in TNFRI-/- PLNs, revealing an earlier unrecognized contribution of TNFRI signaling in HEV morphology. Together, these results underline the potential of whole mount tissue staining and advanced imaging techniques to unravel even subtle changes in lymphoid tissue architecture.

Clickable Vitamins as a New Tool to Track Vitamin A and Retinoic Acid in Immune Cells.

The vitamin A derivative, retinoid acid (RA) is key player in guiding adaptive mucosal immune responses. However, data on the uptake and metabolism of vitamin A within human immune cells has remained largely elusive because retinoids are small, lipophilic molecules which are difficult to detect. To overcome this problem and to be able to study the effect of vitamin A metabolism in human immune cell subsets, we have synthesized novel bio-orthogonal retinoid-based probes (clickable probes), which are structurally and functionally indistinguishable from vitamin A. The probes contain a functional group (an alkyne) to conjugate to a fluorogenic dye to monitor retinoid molecules in real-time in immune cells. We demonstrate, by using flow cytometry and microscopy, that multiple immune cells have the capacity to internalize retinoids to varying degrees, including human monocyte-derived dendritic cells (DCs) and naïve B lymphocytes. We observed that naïve B cells lack the enzymatic machinery to produce RA, but use exogenous retinoic acid to enhance CD38 expression. Furthermore, we showed that human DCs metabolize retinal into retinoic acid, which in co-culture with naïve B cells led to of the induction of CD38 expression. These data demonstrate that in humans, DCs can serve as an exogenous source of RA for naïve B cells. Taken together, through the use of clickable vitamins our data provide valuable insight in the mechanism of vitamin A metabolism and its importance for human adaptive immunity.

De Novo Carcinoma after Solid Organ Transplantation to Give Insight into Carcinogenesis in General-A Systematic Review and Meta-Analysis.

Immunosuppressive therapy after solid organ transplantation leads to the development of cancer in many recipients. Analysis of the occurrence of different types of de novo carcinomas in relation to specific immunosuppressive drugs may give insight into their carcinogenic process and carcinogenesis in general. Therefore, a systematic search was performed in Embase and PubMed. Studies describing over five de novo carcinomas in patients using immunosuppressive drugs after solid organ transplantation were included. Incidence per 1000 person-years was calculated with DerSimonian-Laird random effects model and odds ratio for developing carcinomas with the Mantel-Haenszel test. Following review of 5606 papers by title and abstract, a meta-analysis was conducted of 82 studies. The incidence rate of de novo carcinomas was 8.41. Patients receiving cyclosporine developed more de novo carcinomas compared to tacrolimus (OR1.56, 95%CI 1.00-2.44) and mycophenolate (OR1.26, 95%CI 1.03-1.56). Patients receiving azathioprine had higher odds to develop de novo carcinomas compared to mycophenolate (OR3.34, 95%CI 1.29-8.65) and head and neck carcinoma compared to tacrolimus (OR3.78, 95%CI 1.11-12.83). To conclude, patients receiving immunosuppressive drugs after solid organ transplantation have almost a 20-fold increased likelihood of developing carcinomas, with the highest likelihood for patients receiving cyclosporine A and azathioprine. Looking into altered immune pathways affected by immunosuppressive drugs might lead to better understanding of carcinogenesis in general.

Tertiary Lymphoid Structures: Diversity in Their Development, Composition, and Role.

Lymph node stromal cells coordinate the adaptive immune response in secondary lymphoid organs, providing both a structural matrix and soluble factors that regulate survival and migration of immune cells, ultimately promoting Ag encounter. In several inflamed tissues, resident fibroblasts can acquire lymphoid-stroma properties and drive the formation of ectopic aggregates of immune cells, named tertiary lymphoid structures (TLSs). Mature TLSs are functional sites for the development of adaptive responses and, consequently, when present, can have an impact in both autoimmunity and cancer conditions. In this review, we go over recent findings concerning both lymph node stromal cells and TLSs function and formation and further describe what is currently known about their role in disease, particularly their potential in tolerance.

Phenotypical Characterization of Spleen Remodeling in Murine Experimental Visceral Leishmaniasis.

Background: Visceral leishmaniasis (VL) is caused by Leishmania infantum or L. donovani infection. One of the main problems related to this disease is the emergence of severe clinical forms with a lethality of 5-20%, even while under specific treatment. In humans and other species susceptible to fatal VL, such as dogs and hamsters, the disruption of splenic white pulp (WP) is accompanied by disease progression. Control of VL progression is seen in BALB/c mice, as evidenced by a mild clinical presentation and controlled parasite replication in the liver and spleen. In this study, we investigated the features involved in the morphological remodeling of splenic compartments associated with the control of VL progression to death. Methods: We evaluated cohorts of BALB/c mice after 30, 60, and 90 days of infection by L. infantum. Spleen morphology, cell population subsets and cytokine production were studied in the spleen using flow- and histo-cytometry. Results: Intraperitoneal infection with 108 promastigotes of L. infantum led to progressive increases in spleen size at 60 and 90 days after infection. Splenomegaly was the only clinical sign of disease observed. At 30 days after infection, hyperplasia in the WP and decreased numbers of plasmacytoid dendritic cells were observed. The WP hyperplasia subsided at 60 days post-infection. However, the splenomegaly remained in association with increased numbers of macrophages, B and T lymphocytes and plasma cells. An increased number of lymphoid tissue inducer (LTi) cells was observed; these were distributed around the periarteriolar lymphoid sheath in control mice and scattered throughout the red pulp in the Leishmania-infected mice. After 90 days of infection, increased IL-6 and IFN-γ production was seen in the spleen, as well as higher frequencies of follicular and plasmacytoid dendritic cells. Conclusion: The data presented herein emphasizes the potential role of spleen remodeling in the control of severe forms of VL and highlights features potentially involved in this process.

Intestinal Macrophages Balance Inflammatory Expression Profiles via Vitamin A and Dectin-1-Mediated Signaling.

Tissue resident intestinal macrophages are known to exhibit an anti-inflammatory phenotype and produce little pro-inflammatory cytokines upon TLR ligation, allowing symbiotic co-existence with the intestinal microbiota. However, upon acute events such as epithelial damage and concomitant influx of microbes, these macrophages must be able to quickly mount a pro-inflammatory response while more inflammatory macrophages are recruited from the blood stream simultaneously. Here, we show that dietary intake of vitamin A is required for the maintenance of the anti-inflammatory state of tissue resident intestinal macrophages. Interestingly, these anti-inflammatory macrophages were characterized by high levels of Dectin-1 expression. We show that Dectin-1 expression is enhanced by the vitamin A metabolite retinoic acid and our data suggests that Dectin-1 triggering might provide a switch to induce a rapid production of pro-inflammatory cytokines. In addition, Dectin-1 stimulation resulted in an altered metabolic profile which is linked to a pro-inflammatory response. Together, our data suggests that presence of vitamin A in the small intestine enhances an anti-inflammatory phenotype as well as Dectin-1 expression by macrophages and that this anti-inflammatory phenotype can rapidly convert toward a pro-inflammatory state upon Dectin-1 signaling.

The Role of Endothelial Cells and TNF-Receptor Superfamily Members in Lymphoid Organogenesis and Function During Health and Inflammation.

Lymph nodes (LNs) are crucial for the orchestration of immune responses. LN reactions depend on interactions between incoming and local immune cells, and stromal cells. To mediate these cellular interactions an organized vascular network within the LN exists. In general, the LN vasculature can be divided into two components: blood vessels, which include the specialized high endothelial venules that recruit lymphocytes from the bloodstream, and lymphatic vessels. Signaling via TNF receptor (R) superfamily (SF) members has been implicated as crucial for the development and function of LNs and the LN vasculature. In recent years the role of cell-specific signaling of TNFRSF members in different endothelial cell (EC) subsets and their roles in development and maintenance of lymphoid organs has been elucidated. Here, we discuss recent insights into EC-specific TNFRSF member signaling and highlight its importance in different EC subsets in LN organogenesis and function during health, and in lymphocyte activation and tertiary lymphoid structure formation during inflammation.

CD62L Is a Functional and Phenotypic Marker for Circulating Innate Lymphoid Cell Precursors.

Innate lymphoid cells (ILCs) guard epithelial tissue integrity during homeostasis, but can be potent immune effector cells during inflammation. Precursors to all ILC subsets (ILC precursors [ILCP]) have been identified in human peripheral blood (PB). We found that during homeostasis, ILCP in PB of mouse and human expressed homing receptors for secondary lymphoid organs, mainly CD62L. These ILCP entered mouse lymph nodes in a CD62L-dependent way and relied on S1P receptors for their exit. Importantly, CD62L expression was absent on human ILCs expressing NKp44 in tonsils and PB of Crohn disease patients, and relatively fewer CD62L+ ILCP were present in PB of Crohn disease patients. These data are in agreement with selective expression of CD62L on nonactivated ILCP. As such, we conclude that CD62L not only serves as a functional marker of ILCP, but has potential to be used in the clinic as a diagnostic marker in inflammatory disorders.

Development of a Retinal-Based Probe for the Profiling of Retinaldehyde Dehydrogenases in Cancer Cells.

Retinaldehyde dehydrogenases belong to a superfamily of enzymes that regulate cell differentiation and are responsible for detoxification of anticancer drugs. Chemical tools and methods are of great utility to visualize and quantify aldehyde dehydrogenase (ALDH) activity in health and disease. Here, we present the discovery of a first-in-class chemical probe based on retinal, the endogenous substrate of retinal ALDHs. We unveil the utility of this probe in quantitating ALDH isozyme activity in a panel of cancer cells via both fluorescence and chemical proteomic approaches. We demonstrate that our probe is superior to the widely used ALDEFLUOR assay to explain the ability of breast cancer (stem) cells to produce all-trans retinoic acid. Furthermore, our probe revealed the cellular selectivity profile of an advanced ALDH1A1 inhibitor, thereby prompting us to investigate the nature of its cytotoxicity. Our results showcase the application of substrate-based probes in interrogating pathologically relevant enzyme activities. They also highlight the general power of chemical proteomics in driving the discovery of new biological insights and its utility to guide drug discovery efforts.

The Microenvironment in Barrett's Esophagus Tissue Is Characterized by High FOXP3 and RALDH2 Levels.

Metaplasia in Barrett's esophagus (BE) is characterized by the transition of squamous epithelium into intestinal-type columnar epithelium. The immune response in BE shares many similarities with the response found in the gut, which is different from the response found in a normal-looking esophagus. Here, we investigated the role of the genes associated with the retinoic acid (RA) pathway in BE, as RA is important not only in shaping the gut's immune response but also in the induction of metaplasia in vitro. mRNA was isolated from esophageal and duodenal biopsies from BE (n = 14), reflux esophagitis patients (n = 9), and controls (n = 12). cDNA was made and qPCR was performed. The expression of RALDH1, CYP26A1, MAdCAM1 were similar for both the BE and duodenum, but different when compared to squamous esophageal epithelium. BE was characterized by a higher expression of RALDH2 and FOXP3, compared to the duodenum. In BE, RALDH2 correlated with expression of the myeloid dendritic cell-specific genes: CD11c and CD1c. Also, RALDH2 expression correlated with RAR-ß and FOXP3. Hierarchical clustering on the expression of multiple relevant genes demonstrated that BE, duodenum, and SQ tissues are clustered as three different groups. The differential expression of RA-specific genes and dendritic cell (DC)-subsets indicates that BE resembles duodenal tissue. The higher expression of RALDH2 and FOXP3 in BE points at a mechanism associated with a possible anti-inflammatory microenvironment. This aberrant immune regulation might contribute to the altered tissue and immune responses found in BE.

Dendritic Cell Migration to Skin-Draining Lymph Nodes Is Controlled by Dermatan Sulfate and Determines Adaptive Immunity Magnitude.

For full activation of naïve adaptive lymphocytes in skin-draining lymph nodes (LNs), presentation of peptide:MHC complexes by LN-resident and skin-derived dendritic cells (DCs) that encountered antigens (Ags) is an absolute prerequisite. To get to the nearest draining LN upon intradermal immunization, DCs need to migrate from the infection site to the afferent lymphatics, which can only be reached by traversing a collagen-dense network located in the dermis of the skin through the activity of proteolytic enzymes. Here, we show that mice with altered collagen fibrillogenesis resulting in thicker collagen fibers in the skin display a reduced DC migration to the draining LN upon immune challenge. Consequently, the initiation of the cellular and humoral immune response was diminished. Ag-specific CD8+ and CD4+ T cells as well as Ag-specific germinal center B cells and serum immunoglobulin levels were significantly decreased. Hence, we postulate that alterations to the production of extracellular matrix, as seen in various connective tissue disorders, may in the end affect the qualitative outcome of adaptive immunity.