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Abstract Background Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Standardized treatment options are lacking for patients with unresectable or multifocal follicular dendritic cell sarcoma (FDCS) and disease-related mortality is as high as 20%. Applying whole-genome sequencing (WGS) in one case and whole-exome sequencing (WES) in additional twelve cases, this study adds information on the molecular landscape of FDCS, expanding knowledge on pathobiological mechanisms and identifying novel markers of potential theragnostic significance. Massive parallel sequencing showed high frequency of mutations on oncosuppressor genes, particularly in RB1, CARS and BRCA2 and unveiled alterations on homologous recombination DNA damage repair-related genes in 70% (9/13) of cases. This indicates that patients with high-stage FDCS may be eligible for poly ADP ribose polymerase inhibition protocols. Low tumor mutational burden was confirmed in this study despite common PDL1 expression in FDCS arguing on the efficacy of immune checkpoint inhibitors. CDKN2A deletion, detected by WGS and confirmed by fluorescence in situ hybridization in 41% of cases (9/22) indicates that impairment of cell cycle regulation may sustain oncogenesis in FDCS. Absence of mutations in the RAS/RAF/MAPK pathway and lack of clonal hematopoiesis-related mutations in FDCS sanction its differences from dendritic cell-derived neoplasms of hematopoietic derivation. WGS and WES in FDCS provides additional information on the molecular landscape of this rare tumor, proposing novel candidate genes for innovative therapeutical approaches to improve survival of patients with multifocal disease.

BackgroundPsoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood.MethodsIn this study, we utilized the model of psoriasiform dermatitis, caused by the repeated topical application of an imiquimod containing cream, in neutrophil-depleted mice or in mice carrying impairment in neutrophil functions, including p47phox -/- mice (lacking a cytosolic subunit of the phagocyte nicotinamide adenine dinucleotide phosphate - NADPH - oxidase) and Sykfl/fl MRP8-cre+ mice (carrying the specific deletion of the Syk kinase in neutrophils only), to elucidate the specific contribution of neutrophils to psoriasis development.ResultsBy analyzing disease development/progression in neutrophil-depleted mice, we now report that neutrophils act as negative modulators of disease propagation and exacerbation by inhibiting gammadelta T cell effector functions via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production. We also report that Syk functions as a crucial molecule in determining the outcome of neutrophil and γδ T cell interactions. Accordingly, we uncover that a selective impairment of Syk-dependent signaling in neutrophils is sufficient to reproduce the enhancement of skin inflammation and γδ T cell infiltration observed in neutrophil-depleted mice.ConclusionsOverall, our findings add new insights into the specific contribution of neutrophils to disease progression in the IMQ-induced mouse model of psoriasis, namely as negative regulatory cells.

Macrophages are the most abundant immune cells of the tumor microenvironment (TME) and have multiple important functions in cancer. During tumor growth, both tissue-resident macrophages and newly recruited monocyte-derived macrophages can give rise to tumor-associated macrophages (TAMs), which have been associated with poor prognosis in most cancers. Compelling evidence indicate that the high degree of plasticity of macrophages and their ability to self-renew majorly impact tumor progression and resistance to therapy. In addition, the microenvironmental factors largely affect the metabolism of macrophages and may have a major influence on TAMs proliferation and subsets functions. Thus, understanding the signaling pathways regulating TAMs self-renewal capacity may help to identify promising targets for the development of novel anticancer agents. In this review, we focus on the environmental factors that promote the capacity of macrophages to self-renew and the molecular mechanisms that govern TAMs proliferation. We also highlight the impact of tumor-derived factors on macrophages metabolism and how distinct metabolic pathways affect macrophage self-renewal.

Abstract Objectives The role of tumor‐associated neutrophils (TANs) in the nodal spread of cancer cells remains unexplored. The present study evaluates the occurrence and clinical significance of human nodal TANs. Methods The relevance, derivation, phenotype and interactions of nodal TANs were explored via a large immunohistochemical analysis of carcinoma‐draining lymph nodes, and their clinical significance was evaluated on a retrospective cohort of oral squamous cell carcinomas (OSCC). The tumor‐promoting function of nodal TAN was probed in the OSCC TCGA dataset combining TAN and epithelial‐to‐mesenchymal transition (EMT) signatures. Results The pan‐carcinoma screening identified a consistent infiltration (59%) of CD66b+ TANs in tumor‐draining lymph nodes (TDLNs). Microscopic findings, including the occurrence of intra‐lymphatic conjugates of TANs and cancer cells, indicate that TANs migrate through lymphatic vessels. In vitro experiments revealed that OSCC cell lines sustain neutrophil viability and activation via release of GM‐CSF. Moreover, by retrospective analysis, a high CD66b+ TAN density in M‐TDLNs of OSCC (n = 182 patients) predicted a worse prognosis. The analysis of the OSCC‐TCGA dataset unveiled that the expression of a set of neutrophil‐specific genes in the primary tumor (PT) is highly associated with an EMT signature, which predicts nodal spread. Accordingly, in the PT of OSCC cases, CD66b+TANs co‐localised with PDPN+S100A9− EMT‐switched tumor cells in areas of lymphangiogenesis. The pro‐EMT signature is lacking in peripheral blood neutrophils from OSCC patients, suggesting tissue skewing of TANs. Conclusion Our findings are consistent with a novel pro‐tumoral TAN compartment that may promote nodal spread via EMT, through the lymphatics.

Interferon-γ receptor 1 (IFNγR1) deficiency is one of the inborn errors of IFN-γ immunity underlying Mendelian Susceptibility to Mycobacterial Disease (MSMD). This molecular circuit plays a crucial role in regulating the interaction between dendritic cells (DCs) and T lymphocytes, thus affecting DCs activation, maturation, and priming of T cells involved in the immune response against intracellular pathogens. We studied a girl who developed at the age of 2.5 years a Mycobacterium avium infection characterized by disseminated necrotizing granulomatous lymphadenitis, and we compared her findings with other patients with the same genetic condition. The patient carried a heterozygous 818del4 mutation in the IFNGR1 gene responsible of autosomal dominant (AD) partial IFNγR1 deficiency. During the acute infection blood cells immunophenotyping showed a marked reduction in DCs counts, including both myeloid (mDCs) and plasmacytoid (pDCs) subsets, that reversed after successful prolonged antimicrobial therapy. Histology of her abdomen lymph node revealed a profound depletion of tissue pDCs, as compared to other age-matched granulomatous lymphadenitis of mycobacterial origin. Circulating DCs depletion was also observed in another patient with AD partial IFNγR1 deficiency during mycobacterial infection. To conclude, AD partial IFNγR1 deficiency can be associated with a transient decrease in both circulating and tissular DCs during acute mycobacterial infection, suggesting that DCs counts monitoring might constitute a useful marker of treatment response.

Oral cavity squamous cell carcinoma (OSCC) is a common head and neck cancer characterized by a poor prognosis associated with locoregional or distant failure. Among the predictors of prognosis, a dense infiltration of adaptive immune cells is protective and associated with improved clinical outcomes. However, few tools are available to integrate immune contexture variables into clinical settings. By using digital microscopy analysis of a large retrospective OSCC cohort (n = 182), we explored the clinical significance of tumor-infiltrating CD8+ T-cells. To this end, CD8+ T-cells counts were combined with well-established clinical variables and peripheral blood immune cell parameters. Through variable clustering, five metavariables (MV) were obtained and included descriptors of nodal (NODALMV) and primary tumor (TUMORMV) involvement, the frequency of myeloid (MYELOIDMV) or lymphoid (LYMPHOIDMV) peripheral blood immune cell populations, and the density of tumor-infiltrating CD8+ T-cells (TI-CD8MV). The clinical relevance of the MV was evaluated in the multivariable survival models. The NODALMV was significantly associated with all tested outcomes (p < 0.001), the LYMPHOIDMV showed a significant association with the overall, disease-specific and distant recurrence-free survival (p < 0.05) and the MYELOIDMV with the locoregional control only (p < 0.001). Finally, TI-CD8MV was associated with distant recurrence-free survival (p = 0.029). Notably, the performance in terms of survival prediction of the combined effect of NODALMV and immune metavariables (LYMPHOIDMV, MYELOIDMV and TI-CD8MV) was superior to the TNM stage for most of the outcomes analyzed. These findings indicate that the analysis of the baseline host immune features are promising tools to complement clinical features, in stratifying the risk of recurrences.

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E2 (PGE2). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c+) are a small percentage of BM cells, we demonstrated colocalization of CD271+ MSCs with CD1c+ DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c+ cells in the proximity of CD271+ MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.

Neutrophils are known to perform a series of effector functions that are crucial for the innate and adaptive responses, including the synthesis and secretion of a variety of cytokines. In light of the controversial data in the literature, the main objective of this study was to more in-depth reevaluate the capacity of human neutrophils to express and produce cytokines of the IL-17 family in vitro. By reverse transcription quantitative real-time PCR, protein measurement via commercial ELISA, immunohistochemistry (IHC) and immunofluorescence (IF), flow cytometry, immunoblotting, chromatin immunoprecipitation (ChIP), and ChIP-seq experiments, we found that highly pure (>99.7%) populations of human neutrophils do not express/produce IL-17A, IL-17F, IL-17AF, or IL-17B mRNA/protein upon incubation with a variety of agonists. Similar findings were observed by analyzing neutrophils isolated from active psoriatic patients. In contrast with published studies, IL-17A and IL-17F mRNA expression/production was not even found when neutrophils were incubated with extremely high concentrations of IL-6 plus IL-23, regardless of their combination with inactivated hyphae or conidia from Aspergillus fumigatus. Consistently, no deposition of histone marks for active (H3K27Ac) and poised (H3K4me1) genomic regulatory elements was detected at the IL-17A and IL-17F locus of resting and IL-6 plus IL-23-stimulated neutrophils, indicating a closed chromatin conformation. Concurrent experiments revealed that some commercial anti-IL-17A and anti-IL-17B antibodies (Abs), although staining neutrophils either spotted on cytospin slides or present in inflamed tissue samples by IHC/IF, do not recognize intracellular protein having the molecular weight corresponding to IL-17A or IL-17B, respectively, in immunoblotting experiments of whole neutrophil lysates. By contrast, the same Abs were found to more specifically recognize other intracellular proteins of neutrophils, suggesting that their ability to positively stain neutrophils in cytospin preparations and, eventually, tissue samples derives from IL-17A- or IL-17B-independent detections. In sum, our data confirm and extend, also at epigenetic level, previous findings on the inability of highly purified populations of human neutrophils to express/produce IL-17A, IL-17B, and IL-17F mRNAs/proteins in vitro, at least under the experimental conditions herein tested. Data also provide a number of justifications explaining, in part, why it is possible to false positively detect IL-17A+-neutrophils.

Helicobacter pylori (Hp) is a Gram-negative bacterium that infects the human gastric mucosa, leading to chronic inflammation. If not eradicated with antibiotic treatment, the bacterium persists in the human stomach for decades increasing the risk to develop chronic gastritis, gastroduodenal ulcer, and gastric adenocarcinoma. The lifelong persistence of Hp in the human stomach suggests that the host response fails to clear the infection. It has been recently shown that during Hp infection phagocytic cells promote high Hp loads rather than contributing to bacterial clearance. Within these cells Hp survives in “megasomes,” large structures arising from homotypic fusion of phagosomes, but the mechanism that Hp employs to avoid phagocytic killing is not completely understood. Here, we show that Hp infection induces the downregulation of specific microRNAs involved in the regulation of transcripts codifying for inflammatory proteins. miR-4270 targets the most upregulated gene: the immune receptor CD300E, whose expression is strictly dependent on Hp infection. CD300E engagement enhances the pro-inflammatory potential of macrophages, but in parallel it affects their ability to express and expose MHC class II molecules on the plasma membrane, without altering phagocytosis. This effect compromises the possibility for effector T cells to recognize and activate the killing potential of macrophages, which, in turn would become a survival niche for the bacterium. Taken together, our data add another piece to the complicate puzzle represented by the long-life coexistence between Hp and the human host and contribute with new insights toward understanding the regulation and function of the immune receptor CD300E.

Summary: Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells. : Roccaro et al. show that stromal-cell-derived factor-1 (SDF-1) is highly expressed in active multiple myeloma (MM), as well as in bone marrow (BM) sites of tumor metastasis, and report on a high-affinity PEGylated mirror-image l-oligonucleotide (olaptesed pegol) that specifically binds and neutralizes SDF-1 in vitro and in vivo. Using in vivo murine and xenograft mouse models, the authors document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces clonal plasma cell homing and growth, thereby inhibiting MM disease progression.

Background: Celiac disease (CD) is an immune-mediated enteropathy triggered by the ingestion of gluten in genetically susceptible subjects. Although the small intestinal mucosa is the main site of the gut's involvement in CD, other mucosal surfaces belonging to the gastrointestinal tract and the gut-associated lymphoid tissue are known to be affected. Aim: Assuming that the oral mucosa could reflect the histopathological inflammatory alterations of the intestine in CD patients, this study wishes to assess the pattern of T-cell subsets in the oral mucosa of young adults with CD. Methods: A group of 37 patients (age range 20-38 years; female: male ratio 28:9) with CD were enrolled. Out of 37 patients, 19 patients (group A) followed a gluten free diet (GFD) -2 patients from less than one year; 6 patients between 1 and 5 years; 11 patients more than 5 years- while 18 patients (group B) were still untreated. Fifteen healthy volunteers (age range 18-35 years, female: Male ratio 11:4) served as controls for the CD patients. Ethical approval for the research was granted by the Ethics Committee. Biopsy specimens were taken from normal looking oral mucosa. The immunohistochemical investigation was performed with monoclonal antibodies to CD3, CD4, CD8, and γδ-chains T cell receptor (TCR). Results: The T-lymphocytic inflammatory infiltrate was significantly (p < 0.0001) increased in group B (both compared with group A and with the control group). Conclusion: This study confirms the oral cavity to be a site of involvement of CD and its possible diagnostic potentiality in this disease.

Hermansky Pudlak type 2 syndrome (HPS2) is a rare autosomal recessive primary immune deficiency caused by mutations on β3A gene (AP3B1 gene). The defect results in the impairment of the adaptor protein 3 (AP-3) complex, responsible for protein sorting to secretory lysosomes leading to oculo-cutaneous albinism, bleeding disorders and immunodeficiency. We have studied peripheral blood and lymph node biopsies from two siblings affected by HPS2. Lymph node histology showed a nodular lymphocyte predominance type Hodgkin lymphoma (NLPHL) in both HPS2 siblings. By immunohistochemistry, CD8 T-cells from HPS2 NLPHL contained an increased amount of perforin (Prf) + suggesting a defect in the release of this granules-associated protein. By analyzing peripheral blood immune cells we found a significant reduction of circulating NKT cells and of CD56(bright)CD16(-) Natural Killer (NK) cells subset. Functionally, NK cells were defective in their cytotoxic activity against tumor cell lines including Hodgkin Lymphoma as well as in IFN-γ production. This defect was associated with increased baseline level of CD107a and CD63 at the surface level of unstimulated and IL-2-activated NK cells. In summary, these results suggest that a combined and profound defect of innate and adaptive effector cells might explain the susceptibility to infections and lymphoma in these HPS2 patients.

Abstract  We evaluated the usefulness of the tight-junction associated protein Claudin 4 (CL-4) in the diagnosis of mesothelioma and mimickers, analyzing biopsies from 454 tumors, including 82 mesotheliomas, 336 carcinomas of different origin (278 primary, 58 metastatic to serosae), 36 nonepithelial spindle cell neoplasms, as well as 97 cytological samples from reactive effusions (12), mesothelioma (23) and metastatic carcinomas (62). CL-4 was consistently negative in normal and reactive mesothelium, as well as in all 82 mesotheliomas. In contrast, strong reactivity was found in 57/58 serosal metastasis, and in 245/278 primary carcinomas, with uppermost expression (150/153) in those most frequently involved in the differential with mesothelioma (lung, breast, gastrointestinal tract, pancreas, ovary, primary serous papillary carcinoma of peritoneum). On effusions, reactive and neoplastic mesothelial cells were regularly negative, while metastatic tumor cells stained positively in 60/62 (96.8%) cases. Among spindle cell neoplasms, only 2/9 biphasic synovial sarcomas and 4/4 follicular dendritic cell sarcomas stained positively. Results indicate that CL-4 reacts with the majority of epithelial neoplasms that often metastasize to serous membranes, representing a pancarcinoma marker with extremely high sensitivity and specificity. CL-4 may be considered a primary immunohistochemical reagent to rule out the diagnosis of mesothelioma. [ABSTRACT FROM AUTHOR]