CD83 is a maturation marker for dendritic cells. In the B cell lineage, CD83 is expressed especially on activated B cells and on light zone B cells during the germinal center (GC) reaction. The ...function of CD83 during GC responses is unclear. CD83(-/-) mice have a strong reduction of CD4(+) T cells, which makes it difficult to analyze a functional role of CD83 on B cells during GC responses. Therefore, in the present study we generated a B cell-specific CD83 conditional knockout (CD83 B-cKO) model. CD83 B-cKO B cells show defective upregulation of MHC class II and CD86 expression and impaired proliferation after different stimuli. Analyses of GC responses after immunization with various Ags revealed a characteristic shift in dark zone and light zone B cell numbers, with an increase of B cells in the dark zone of CD83 B-cKO mice. This effect was not accompanied by alterations in the level of IgG immune responses or by major differences in affinity maturation. However, an enhanced IgE response was observed in CD83 B-cKO mice. Additionally, we observed a strong competitive disadvantage of CD83-cKO B cells in GC responses in mixed bone marrow chimeras. Furthermore, infection of mice with Borrelia burgdorferi revealed a defect in bacterial clearance of CD83 B-cKO mice with a shift toward a Th2 response, indicated by a strong increase in IgE titers. Taken together, our results show that CD83 is important for B cell activation and modulates GC composition and IgE Ab responses in vivo.
Keloid is an aberrant scarring process of the skin, characterized by excessive extracellular matrix synthesis and deposition. The pathogenesis of this prevalent cutaneous disorder is not fully ...understood; however, a persistent inflammatory process is observed. To obtain more insight into this process, we analyzed lesional, perilesional and healthy tissue using multi-antigen-analysis (MAA) in conjunction with a data mining approach. Here, we demonstrate that monocyte-derived inflammatory dendritic cells (CD1a+, CD11c+, CD14+) and activated CD4+ T lymphocytes (CD45 RO+) dominated the immune infiltration in keloids while associating with fibroblasts. In perilesional tissue, precursor immune cells were dominant in the perivascular area, suggesting that they were attracted by an immune process, potentially in the lesional area. Supporting this hypothesis, only in keloid lesions, high levels of ADAM10/17 and Neprilysin (CD10) were observed in both fibroblasts and leukocytes. The spatial proximity of these two cell types, which could be confirmed by image analysis only in lesional tissue, could be a potential factor leading to the activation of fibroblasts. Our findings provide new insight into the pathogenesis of keloid formation and reveal metalloproteinases as a target for therapeutical intervention.
While the bone marrow attracts tumor cells in many solid cancers leading to poor outcome in affected patients, comprehensive analyses of bone marrow metastases have not been performed on a ...single-cell level. We here set out to capture tumor heterogeneity and unravel microenvironmental changes in neuroblastoma, a solid cancer with bone marrow involvement. To this end, we employed a multi-omics data mining approach to define a multiplex imaging panel and developed DeepFLEX, a pipeline for subsequent multiplex image analysis, whereby we constructed a single-cell atlas of over 35,000 disseminated tumor cells (DTCs) and cells of their microenvironment in the metastatic bone marrow niche. Further, we independently profiled the transcriptome of a cohort of 38 patients with and without bone marrow metastasis. Our results revealed vast diversity among DTCs and suggest that FAIM2 can act as a complementary marker to capture DTC heterogeneity. Importantly, we demonstrate that malignant bone marrow infiltration is associated with an inflammatory response and at the same time the presence of immuno-suppressive cell types, most prominently an immature neutrophil/granulocytic myeloid-derived suppressor-like cell type. The presented findings indicate that metastatic tumor cells shape the bone marrow microenvironment, warranting deeper investigations of spatio-temporal dynamics at the single-cell level and their clinical relevance.
To facilitate the recovery process of chronic and hard-to-heal wounds novel pro-resolving treatment options are urgently needed. We investigated the pro-regenerative properties of soluble CD83 ...(sCD83) on cutaneous wound healing, where sCD83 accelerated wound healing not only after systemic but also after topical application, which is of high therapeutic interest. Cytokine profile analyses revealed an initial upregulation of inflammatory mediators such as TNFα and IL-1β, followed by a switch towards pro-resolving factors, including YM-1 and IL-10, both expressed by tissue repair macrophages. These cells are known to mediate resolution of inflammation and stimulate wound healing processes by secretion of growth factors such as epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF), which promote vascularization as well as fibroblast and keratinocyte differentiation. In conclusion, we have found strong wound healing capacities of sCD83 beyond the previously described role in transplantation and autoimmunity. This makes sCD83 a promising candidate for the treatment of chronic- and hard-to-heal wounds.
The high mortality rate of melanoma is broadly associated with its metastatic potential. Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and ...lymphangiogenesis play an essential role in metastasis. Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients. Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay. Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P‐value = 0.044) and the lymphangiogenic receptor VEGFR‐3 (P‐value = 0.002) were independent prognostic factors of overall survival in melanoma patients. Enhanced reduced representation bisulfite sequencing‐based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells. In patients, VEGFC (P‐value = 0.031), ANGPT2 (P‐value < 0.001), and SIX1 (P‐value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival. Hence, our data suggest that these angio‐ and lymphangiogenesis factors are potential biomarkers of melanoma prognosis. Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.
The identification of new molecular biomarkers is essential for the successful management and treatment of malignant melanoma. Tumor angiogenesis and lymphangiogenesis are strongly associated with tumor progression. Here, we reported that elevated expression of VEGFR‐3 and ANGPT2 genes and hypomethylation of VEGFC, ANGPT2, and SIX1 promoters are independent prognostic factors for shorter overall survival of melanoma patients.
Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25+ cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used ...experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen–specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator–dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25+ cell killing that may affect their clinical use and could be tested for novel indications. This trial was registered at www.clinicaltrials.gov, #NCT00056134.
•ONTAK blocks DC maturation by coreceptor downmodulation and inhibition of Stat3 phosphorylation to induce a tolerogenic phenotype.•ONTAK kills activated CD4 T cells but stimulates antiapoptosis in resting Treg by engagement and stimulation through CD25.
Plasma extracellular vesicles (pEV) can harbor a diverse array of factors including active proteases and the amyloid-precursor-protein (APP) cleavage product Aβ, involved in plaque formation in ...Alzheimer`s diseases (AD). A potential role of such vesicles in AD pathology is unexplored.
In a case-control study of randomly selected patients with AD and other neurological diseases (n = 14), and healthy controls (n = 7), we systematically analyzed the content of pEV, using different assay systems. In addition, we determined their entry path into brain tissue, employing animal (mice) injection experiments with ex vivo generated EV that were similar to AD-pEV, followed by multi antigen analysis (MAA) of brain tissue (n = 4 per condition). The results were compared with an IHC staining of human brain tissue in a small cohort of AD patients (n = 3) and controls with no neurodegenerative diseases (n = 3).
We show that pEV levels are considerably upregulated in AD patients. Besides numerous inflammatory effectors, AD-pEV contained α-, β- and γ-secretases, able to cleave APP in in target cells. In vitro generated EV with similar characteristics as AD-pEV accumulated in the choroid plexus (CP) of injected animals and reached primarily hippocampal neurons. Corroborating findings were made in human brain samples. An inhibitor of hyaluronic-acid-synthetase (HAS) blocked uploading of proteases and Hyaluronan onto EV in vitro and abolished CP targeting in animal injection experiments.
We conclude that protease-containing pEV could be part of a communication axis between the periphery and the brain that could be become detrimental depending on pEV concentration and duration of target cell impact.
See the Acknowledgements section.
Tumor necrosis factor (TNF) is a key cytokine in HIV replication and pathogenesis. For reasons that are not entirely clear, the cytokine remains upregulated despite anti-retroviral therapy (ART). ...Here we demonstrate that HIV Nef induces an alternative TNF secretion mechanism that remains active in chronic infection. Ingestion of Nef-containing plasma extracellular vesicles (pEV) from ART patients by primary immune cells, but also Nef expression, induced intracellular proTNF cleavage and secretion of vesicular TNF endosomes. Key event was the Nef-mediated routing of the TNF-converting enzyme ADAM17 into Rab4+ early endosomes and the Rab27+ secretory pathway. Analysis of lymph-node tissue by multi-epitope-ligand-cartography (MELC) confirmed a vesicular TNF secretion phenotype that co-localized with persistent Nef expression, and implicated Notch1 as an essential co-factor. Surprisingly Notch1 had no transcriptional effect but was required for the endosomal trafficking of ADAM17. We conclude that Nef expression and Nef-containing pEV mobilize TNF from endosomal compartments in acute and chronic infection.
•Nef/ADAM17 containing extracellular vesicles induce an endosomal TNF secretion type in primary target cells.•The mechanism required the shuttling of ADAM17 into Rab4+ endosomal compartments in a Notch1-dependent manner.•The mechanism could be demonstrated in tissue by multi-epitope-ligand-cartography (MELC) technology.
Despite antiviral therapy, plasma levels of TNF remain upregulated and likely play a role in many comorbidities seen in chronic HIV infection. We found that this is due to high levels of HIV-induced plasma extracellular vesicles (pEV) containing the TNF processing ADAM17 protease. Interestingly these vesicles induced a different TNF secretion type. Whereas TNF is usually shed from the plasma membrane, pEV mobilized intracellular TNF storage compartments, secreting endosomal vesicles. We could confirm this mechanism analyzing lymph node tissue sections by a novel immunostaining technology. Our report supports our previous publication implying ongoing viral activity despite successful antiretroviral therapy.
Multi-epitope-ligand cartography (MELC) is an innovative high-throughput fluorescence microscopy–based method. A tissue section is analyzed through a repeated cycling of (1) incubation with a ...fluorophore-labeled antibody, (2) fluorescence imaging, and (3) soft bleaching. This method allows staining of the same tissue section with up to 100 fluorescent markers and to analyze their toponomic expression using further image processing and pixel-precise overlay of the corresponding images. In this study, we adapted this method to identify a large panel of murine leukocyte subpopulations in a whole frozen section of a peripheral lymph node. Using the resulting antibody library, we examined non-inflamed versus inflamed tissues of brain and spinal cord in the experimental autoimmune encephalomyelitis (EAE) model. The presence and activity of specific leukocyte subpopulations (different T cell subpopulations, dendritic cells, macrophages, etc.) could be assessed and the cellular localizations and the corresponding activation status in situ were investigated. The results were then correlated with quantitative RT-PCR.
Summary
Background and objectives
Pyoderma gangrenosum is an ulcerative autoinflammatory disease, lacking distinct histopathological characteristics to differentiate from other ulcerating conditions, ...like venous leg ulcers. The objective of this study was therefore to find histopathological characteristics of pyoderma gangrenosum in a head‐to‐head comparison to venous leg ulcers.
Patients and methods
Eight tissue samples of pyoderma gangrenosum, twelve samples of venous leg ulcers and six samples of healthy skin were stained using an immunohistological multi antigen staining technology. The immune infiltrate and its spatial distribution were analyzed with contextual tissue cytometry software using fluorescence images.
Results
The dense epidermal presence of CD45RO+ memory T cells and the rarefication of CD1a+ Langerhans cells in the epidermis were defining markers for pyoderma gangrenosum, implicating an epidermal immune reaction. In addition, high numbers of CD11c+CD68+ pro‐inflammatory M1 macrophages were detected in the dermis, significantly extending the numbers seen in venous leg ulcers.
Conclusions
The histopathological differences found between pyoderma gangrenosum and venous leg ulcer can be used to distinguish between the two diseases and thus provide an important aid for the rapid initiation of adequate therapy. In addition, our data hint at an antigen‐driven process in the epidermis, possibly involving CD1a+ Langerhans cells.
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