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Rationale: Bacterial lung microbiota are correlated with lung inflammation and acute respiratory distress syndrome (ARDS) and altered in severe coronavirus disease (COVID-19). However, the association between lung microbiota (including fungi) and resolution of ARDS in COVID-19 remains unclear. We hypothesized that increased lung bacterial and fungal burdens are related to nonresolving ARDS and mortality in COVID-19. Objectives: To determine the relation between lung microbiota and clinical outcomes of COVID-19-related ARDS. Methods: This observational cohort study enrolled mechanically ventilated patients with COVID-19. All patients had ARDS and underwent bronchoscopy with BAL. Lung microbiota were profiled using 16S rRNA gene sequencing and quantitative PCR targeting the 16S and 18S rRNA genes. Key features of lung microbiota (bacterial and fungal burden, α-diversity, and community composition) served as predictors. Our primary outcome was successful extubation adjudicated 60 days after intubation, analyzed using a competing risk regression model with mortality as competing risk. Measurements and Main Results: BAL samples of 114 unique patients with COVID-19 were analyzed. Patients with increased lung bacterial and fungal burden were less likely to be extubated (subdistribution hazard ratio, 0.64 [95% confidence interval, 0.42-0.97]; P = 0.034 and 0.59 [95% confidence interval, 0.42-0.83]; P = 0.0027 per log10 increase in bacterial and fungal burden, respectively) and had higher mortality (bacterial burden, P = 0.012; fungal burden, P = 0.0498). Lung microbiota composition was associated with successful extubation (P = 0.0045). Proinflammatory cytokines (e.g., tumor necrosis factor-α) were associated with the microbial burdens. Conclusions: Bacterial and fungal lung microbiota are related to nonresolving ARDS in COVID-19 and represent an important contributor to heterogeneity in COVID-19-related ARDS.

Background and AimsHistone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD].MethodsCES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn’s disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter.ResultsCES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis.ConclusionsWe demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.

Background: CD11c +Tbet + B cells are enriched in autoimmunity and chronic infections and also expand on immune challenge in healthy individuals. CD11c +Tbet + B cells remain an enigmatic B-cell population because of their intrinsic heterogeneity. Objectives: We investigated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-specific development and differentiation properties of 3 separate CD11c + B-cell subsets—age-associated B cells (ABCs), double-negative 2 (DN2) B cells, and activated naive B cells—and compared them to their canonical CD11c − counterparts. Methods: Dynamics of the response of the 3 CD11c + B-cell subsets were assessed at SARS-CoV-2 vaccination in healthy donors by spectral flow cytometry. Distinct CD11c + B-cell subsets were functionally characterized by optimized in vitro cultures. Results: In contrast to a durable expansion of antigen-specific CD11c − memory B cells over time, both ABCs and DN2 cells were strongly expanded shortly after second vaccination and subsequently contracted. Functional characterization of antibody-secreting cell differentiation dynamics revealed that CD11c +Tbet + B cells were primed for antibody-secreting cell differentiation compared to relevant canonical CD11c − counterparts. Conclusion: Overall, CD11c +Tbet + B cells encompass heterogeneous subpopulations, of which primarily ABCs as well as DN2 B cells respond early to immune challenge and display a pre–antibody-secreting cell phenotype.

Changes in glycosylation patterns have been associated with malignant transformation and clinical outcome in several types of cancer, although no comprehensive analysis has been performed in a pan-cancer setting. Here, we performed an extensive transcriptomic analysis of glycosylation related genes (such as enzymes involved in synthesis and degradation of glycoconjugates, transporters, mucins and galectins), using publicly available bulk and single cell transcriptomic data sets from tumor samples and cancer cell lines. We identified genes and pathways associated with different tumor types, which may represent novel diagnostic biomarkers as α2-3 sialylation for Melanoma, MUC21 for Lung adenocarcinoma and Galectin-7 for Squamous cell carcinomas (SCC). Accordingly, serum levels of Galectin-7 in patients with lung cancer were elevated in SCC respect to adenocarcinomas, supporting its biomarker potential. Moreover, we characterized the contribution of different cell types to the overall glycosylation profiles observed by performing the integration and analysis of 14 single cell RNA-seq datasets. This led us to identify that cancer cells are responsible for the specific tumor glyco-codes identified in bulk transcriptomics, while stromal and immune cells contribute in a conserved manner across various malignancies. Furthermore, our results suggest that the glycosylation-related genes and pathways expressed by cancer cells are influenced by the cell of origin and the oncogenic pathways that led to malignant transformation. Lastly, we described the association of different glycosylation-related genes and pathways with the clinical outcome of patients. Interestingly, while the expression of genes associated to some pathways (as proteoglycan biosynthesis) are consistently associated with a more aggressive disease, the correlation of others pathways with the survival of patients depends on the particular tumor type. Remarkably, the expression of genes associated with the synthesis of CMP-sialic acid was correlated with lower survival of patients in Uveal Melanoma and PDAC, while the opposite was observed for colorectal cancer. The extensive transcriptomic analysis of glycosylation pathways in cancer that we report here can serve as a resource for future research aimed to unravel the glyco-code in cancer related to clinical outcome or biomarker development.

Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of pathologies, including haemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, haemolytic activity can greatly debilitate victims and contributes to systemic haemotoxicity. Current assays designed for studying haemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput haemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving haemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for haemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The haemolytic venom toxins identified were mainly phospholipase A2s and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralisation of venom cytotoxins.

Induction of tumor-specific cytotoxic CD8+ T cells (CTLs) via immunization relies on the presentation of tumor-associated peptides in major histocompatibility complex (MHC) class I molecules by dendritic cells (DCs). To achieve presentation of exogenous peptides into MHC class I, cytosolic processing and cross-presentation are required. Vaccination strategies aiming to induce tumor-specific CD8+ T cells via this exogenous route therefore pose a challenge. In this study, we describe improved CD8+ T cell induction and in vivo tumor suppression of mono-palmitic acid-modified (C16:0) antigenic peptides, which can be attributed to their unique processing route, efficient receptor-independent integration within lipid bilayers, and continuous intracellular accumulation and presentation through MHC class I. We propose that this membrane-integrating feature of palmitoylated peptides can be exploited as a tool for quick and efficient antigen enrichment and MHC class I loading. Importantly, both DCs and non-professional antigen-presenting cells (APCs), similar to tumor cells, facilitate anti-tumor immunity by efficient CTL priming via DCs and effective recognition of tumors through enhanced presentation of antigens., Graphical abstract, A dual loading vaccination strategy with palmitoylated peptides is developed to induce robust anti-tumor immunity. Dendritic cells are loaded with palmitoylated peptides to activate antigen-specific T cells, while tumor cells are loaded with palmitoylated peptides to present epitopes for enhanced recognition by antigen-specific T cells.

Undoubtedly, the global pandemic caused by the SARS‐CoV‐2 virus has had a significant impact on Shared Resource Laboratories (SRL) operations worldwide. Unlike other crises (e.g., natural disasters, acts of war, or terrorism) which often result in a sudden and sustained cessation of scientific research usually affecting one or two cities at a time, this impact is being seen simultaneously in every SRL worldwide albeit to a varying degree. The alterations to SRL operations caused by the COVID‐19 pandemic can generally be divided into three categories: (1) complete shutdown, (2) partial shutdown, and (3) uninterrupted operations. In many cases, SRLs that remained partially or fully operational during the initial wave of global infections saw a concurrent increase in COVID‐19‐related research coming through their facilities. This forced SRLs to make rapid adjustments to core operations at the same time as infectious disease experts were still developing recommendations for the safety of frontline medical workers. Although many SRLs already had contingency plans in place, this pandemic has highlighted the importance of having such plans for continuity of service, if possible, during a crisis. Immediate changes have occurred in the way SRLs operate due to potential virus transmission and in line with this new “Best Practices” have been established, that is,social distancing, remote working, and technology‐based meetings and training. Many of these changes are likely to be in place for some time with the threat of further waves of infections toward the end of 2020 and into 2021. Some of these best practices, such as having many training resources recorded and available online, are likely to remain long‐term. Although many changes have been made in haste, these will alter the future operations of SRLs. In addition, we have learnt how to deal with future crises that may be encountered in the workplace. © 2020 The Authors. Cytometry Part A published by Wiley Periodicals LLC. on behalf of International Society for Advancement of Cytometry.

BackgroundPrimary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease affecting the intra- and extrahepatic bile ducts, and is strongly associated with ulcerative colitis (UC). In this study, we explored the peripheral blood DNA methylome and its immune cell composition in patients with PSC-UC, UC, and healthy controls (HC) with the aim to develop a predictive assay in distinguishing patients with PSC-UC from those with UC alone.MethodsThe peripheral blood DNA methylome of male patients with PSC and concomitant UC, UC and HCs was profiled using the Illumina HumanMethylation Infinium EPIC BeadChip (850K) array. Differentially methylated CpG position (DMP) and region (DMR) analyses were performed alongside gradient boosting classification analyses to discern PSC-UC from UC patients. As observed differences in the DNA methylome could be the result of differences in cellular populations, we additionally employed mass cytometry (CyTOF) to characterize the immune cell compositions.ResultsGenome wide methylation analysis did not reveal large differences between PSC-UC and UC patients nor HCs. Nonetheless, using gradient boosting we were capable of discerning PSC-UC from UC with an area under the receiver operator curve (AUROC) of 0.80. Four CpG sites annotated to the NINJ2 gene were found to strongly contribute to the predictive performance. While CyTOF analyses corroborated the largely similar blood cell composition among patients with PSC-UC, UC and HC, a higher abundance of myeloid cells was observed in UC compared to PSC-UC patients.ConclusionDNA methylation enables discerning PSC-UC from UC patients, with a potential for biomarker development.

Contains fulltext : 290668.pdf (Publisher’s version ) (Open Access) BACKGROUND: Myelodysplastic syndromes (MDS) at risk of transformation to acute myeloid leukemia (AML) are difficult to identify. The bone marrows of MDS patients harbor specific hematopoietic stem and progenitor cell (HSPC) abnormalities that may be associated with sub-types and risk-groups. Leukemia-associated characteristics of such cells may identify MDS patients at risk of progression to AML and provide insight in the pathobiology of MDS. METHODS: Bone marrow samples from healthy donors (n = 10), low risk (n = 12) and high risk (n = 13) MDS patients were collected, in addition, AML samples for 5 out of 6 MDS patients that progressed. Mass cytometry was applied to assess expression of stem cell subset and leukemia-associated immunophenotype markers. RESULTS: We analyzed the data using FlowSOM to cluster cells with similar expression of 10 commonly used stem cell markers. Metaclusters (n = 20) of these clusters represented populations of cells with a related phenotype, largely resembling known stem cell subsets. Within specific subsets, intra-cellular expression levels of pCREB, IkBα, or pS6 differed significantly between healthy bone marrow (HBM) and MDS or consecutive secondary AML samples. CD34, CD44, and CD49f expression was significantly increased in high risk MDS and AML-associated metaclusters. We identified MDS/sAML cells with aberrant phenotypes when compared to HBM. Such cells were observed in clusters of both primary MDS and secondary AML samples. CONCLUSIONS: High-dimensional mass cytometry and computational data analyses enabled characterization of HSPC subsets in MDS and identification of leukemia stem cell populations based on their immunophenotype. Stem cells in MDS that display leukemia-associated features may predict the risk of developing AML.

Deconvolution of bulk gene expression profiles into the cellular components is pivotal to portraying tissue’s complex cellular make-up, such as the tumor microenvironment. However, the inherently variable nature of gene expression requires a comprehensive statistical model and reliable prior knowledge of individual cell types that can be obtained from single-cell RNA sequencing. We introduce BLADE (Bayesian Log-normAl Deconvolution), a unified Bayesian framework to estimate both cellular composition and gene expression profiles for each cell type. Unlike previous comprehensive statistical approaches, BLADE can handle > 20 types of cells due to the efficient variational inference. Throughout an intensive evaluation with > 700 simulated and real datasets, BLADE demonstrated enhanced robustness against gene expression variability and better completeness than conventional methods, in particular, to reconstruct gene expression profiles of each cell type. In summary, BLADE is a powerful tool to unravel heterogeneous cellular activity in complex biological systems from standard bulk gene expression data., Deconvolution methods reveal individual cell types in complex tissues profiled by bulk methods. Here the authors present a Bayesian deconvolution method that outperforms existing methods when benchmarked on >700 datasets, especially in estimating cell-type-specific gene expression profiles.

Contains fulltext : 244589.pdf (Publisher’s version ) (Open Access) An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcgamma receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.

Contains fulltext : 215802.pdf (Publisher’s version ) (Open Access) The human skin is an attractive anti-tumor vaccination site due to the vast network of dendritic cell (DC) subsets that carry antigens to the draining lymph nodes and stimulate tumor specific CD4(+) and CD8(+) T cells in. Specific vaccine delivery to skin DC can be accomplished by targeting glycan coated antigens to C-type lectin receptors (CLRs) such as DC-SIGN expressed by human dermal DCs and Langerin expressed by Langerhans cells (LCs), which facilitate endocytosis and processing for antigen presentation and T cell activation. Although there are multiple human skin DC subsets, targeting individual DC subsets and receptors has been a focus in the past. However, the simultaneous targeting of multiple human skin DC subsets that mobilize the majority of the skin antigen presenting cells (APC) is preferred to accomplish more robust and efficient T cell stimulation. Dual CLR targeting using a single tumor vaccine has been difficult, as we previously showed Langerin to favor binding and uptake of monovalent glyco-peptides whereas DC-SIGN favors binding of larger multivalent glyco-particles such as glyco-liposomes. Methods: We used branched polyamidoamine (PAMAM) dendrimers as scaffold for melanoma specific gp100 synthetic long peptides and the common DC-SIGN and Langerin ligand Lewis Y (Le(Y)), to create multivalent glyco-dendrimers with varying molecular weights for investigating dual DC-SIGN and Langerin targeting. Using DC-SIGN(+) monocyte derived DC (moDC) and Langerin(+) primary LC we investigated glyco-dendrimer CLR targeting properties and subsequent gp100 specific CD8(+) T cell activation in vitro. In situ targeting ability to human dermal DC and LC through intradermal injection in a human skin explant model was elucidated. Results: Dual DC-SIGN and Langerin binding was achieved using glyco-dendrimers of approximately 100kD, thereby fulfilling our criteria to simultaneously target LCs and CD1a(+) and CD14(+) dermal DC in situ. Both DC-SIGN and Langerin targeting by glyco-dendrimers resulted in enhanced internalization and gp100 specific CD8(+) T cell activation. Conclusion: We designed the first glyco-vaccine with dual CLR targeting properties, thereby reaching multiple human skin DC subsets in situ for improved anti-tumor CD8(+) T cell responses.

Diffuse midline gliomas (DMG) are highly aggressive pediatric brain tumors with a grim prognosis. A lack of effective treatment options highlights the critical need to investigate new therapeutic strategies. This includes the use of immunotherapy, which has shown promise in other hard-to-treat tumors. To facilitate immunotherapeutic research in this field, and to complement the existing immunodeficient patient-derived DMG models, we developed three distinct immunocompetent mouse models representing different DMG subtypes, i.e., histone 3 wildtype and histone 3 K27M mutant DMG (H3.3K27M or H3.1K27M), that can be used for preclinical testing of new therapies. We first established primary tumor cell cultures from murine DMG tumors that were generated by brainstem-targeted intra-uterine electroporation (IUE). This method enabled the introduction of DMG-associated mutations within the intact developing brainstem, thereby generating DMG tumors in a spatially and temporally defined manner, while maintaining a genetically identical (isogenic) background. We then created allograft DMG mouse models by orthotopically implanting the established primary cell cultures into syngeneic (C57BL/6) mice. Herewith, we provide an allograft tool that is better suitable for large-scale therapeutic studies and more accessible to the scientific community. Importantly, we demonstrated that these allograft models recapitulate the histopathologic phenotype of human DMG, including their diffuse infiltrative growth and expression of DMG-associated antigens. Furthermore, CyTOF mass cytometry analysis indicated that these murine pontine tumors exhibit a tumor immune microenvironment (TIME) similar to human DMG, characterized by considerable myeloid cell infiltration and a paucity of T-lymphocytes and NK cells. As such, we provide a representative model to further delineate the immune landscape in DMG and to preclinically investigate novel (immuno)therapies. Currently, we are using these immunocompetent models to study the interaction between DMG cells and microglia, and we are investigating how we can modify the immune microenvironment to improve checkpoint inhibition in DMG.

Changes in glycosylation during tumour progression are a key hallmark of cancer. One of the glycan moieties generally overexpressed in cancer are sialic acids, which can induce immunomodulatory properties via binding to Siglec receptors. We here show that Pancreatic Ductal Adenocarcinoma (PDAC) tumour cells present an increased sialylation that can be recognized by Siglec-7 and Siglec-9 on myeloid cells. We identified the expression of the α2,3 sialyltransferases ST3GAL1 and ST3GAL4 as main contributor to the synthesis of ligands for Siglec-7 and Siglec-9 in tumour cells. Analysing the myeloid composition in PDAC, using single cell and bulk transcriptomics data, we identified monocyte-derived macrophages as contributors to the poor clinical outcome. Tumour-derived sialic acids dictate monocyte to macrophage differentiation via signalling through Siglec-7 and Siglec-9. Moreover, triggering of Siglec-9 in macrophages reduce inflammatory programmes, while increasing PD-L1 and IL-10 expression, illustrating that sialic acids modulate different myeloid cells. This work highlights a critical role for sialylated glycans in controlling immune suppression and provides new potential targets for cancer immunotherapy in PDAC., Alterations in glycosylation in tumours facilitate tumour progression. Here, the authors show that pancreatic ductal adenocarcinomas present increased sialylation, which stimulates the polarisation of monocytes via Siglec receptors, resulting in the generation of immune suppressive tumour associated macrophages.

Background The sentinel lymph node (SLN) in melanoma represents the crossroads of the initiation of effector T cell responses and of lymphatic metastasis of the primary tumor. As such, alterations in the human LN immune cell network during melanoma progression are of particular interest for the development of effective immunotherapeutic approaches for each stage of disease. Methods We used mass cytometry (CyTOF) and multiparameter flow cytometry to characterize the alterations in the major immune populations in the human LN. We included LN derived from healthy donors (n=10), tumor-negative (SLN-, n=7) and tumor-positive SLN (SLN+, n=3) and LN metastatic samples (n=4). Results Our results show that melanoma progression in the LN is accompanied by increased relative frequencies of myeloid cells, B cells and NK cells whereas T cell rates are significantly decreased. More specifically, for the myeloid cells we observed a decrease in frequencies of migratory cDC subsets and of LN-resident cDC and macrophage subsets in the SLN accompanying early melanoma development and metastasis. In fully metastatic LN from patients with advanced melanoma, a clear predominance of inflammatory, monocyte-derived subsets was observed. Simultaneously with this shift in myeloid subsets, an increase in CD4+ Tregs and CD8+ effector T cell subsets became apparent with metastatic progression in the LN. Both Tregs and CD8+ effector T cells in LN metastases were further characterized by relatively high expression of PD-1 and TIGIT immune checkpoints. Conclusions The changes observed in the myeloid compartment accompanying metastatic progression in melanoma-draining LN, were found to be related to the shifts in lymphocytic subsets and their differentiation and activation state. Our results provide insights into the steady-state immune characteristics of the healthy human LN and identify all the changes that accompany melanoma progression through the different stages and give important clues about possible therapeutic interventions, aiming at immune potentiation of the SLN.

Our understanding of the coronavirus disease-19 (COVID-19) immune response is almost exclusively derived from studies that examined blood. To gain insight in the pulmonary immune response we analysed BALF samples and paired blood samples from 17 severe COVID-19 patients. Macrophages and T cells were the most abundant cells in BALF. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells and expressed higher levels of the exhaustion marker PD-1 than in peripheral blood. Prolonged ICU stay associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma. In conclusion, the bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood.SummaryThe bronchoalveolar immune response in severe COVID-19 strongly differs from the peripheral blood immune profile. Fatal COVID-19 associated with T cell activation blood, but not in BALF.

BackgroundKnowledge of the pathophysiology of COVID-19 is almost exclusively derived from studies that examined the immune response in blood. We here aimed to analyse the pulmonary immune response during severe COVID-19 and to compare this with blood responses.MethodsThis was an observational study in patients with COVID-19 admitted to the intensive care unit (ICU). Mononuclear cells were purified from bronchoalveolar lavage fluid (BALF) and blood, and analysed by spectral flow cytometry; inflammatory mediators were measured in BALF and plasma.FindingsPaired blood and BALF samples were obtained from 17 patients, four of whom died in the ICU. Macrophages and T cells were the most abundant cells in BALF, with a high percentage of T cells expressing the ƴδ T cell receptor. In the lungs, both CD4 and CD8 T cells were predominantly effector memory cells (87·3% and 83·8%, respectively), and these cells expressed higher levels of the exhaustion marker programmad death-1 than in peripheral blood. Prolonged ICU stay (>14 days) was associated with a reduced proportion of activated T cells in peripheral blood and even more so in BALF. T cell activation in blood, but not in BALF, was higher in fatal COVID-19 cases. Increased levels of inflammatory mediators were more pronounced in BALF than in plasma.InterpretationThe bronchoalveolar immune response in COVID-19 has a unique local profile that strongly differs from the immune profile in peripheral blood. Fully elucidating COVID-19 pathophysiology will require investigation of the pulmonary immune response.

BackgroundThe likelihood of a tumor recurrence in patients with T3-4N0–1 non-small cell lung cancer following multimodality treatment remains substantial, mainly due distant metastases. As pathological complete responses (pCR) in resected specimens are seen in only a minority (28–38%) of patients following chemoradiotherapy, we designed the INCREASE trial (EudraCT-Number: 2019–003454-83; Netherlands Trial Register number: NL8435) to assess if pCR rates could be further improved by adding short course immunotherapy to induction chemoradiotherapy. Translational studies will correlate changes in loco-regional and systemic immune status with patterns of recurrence.Methods/designThis single-arm, prospective phase II trial will enroll 29 patients with either resectable, or borderline resectable, T3-4N0–1 NSCLC. The protocol was approved by the institutional ethics committee. Study enrollment commenced in February 2020.On day 1 of guideline-recommended concurrent chemoradiotherapy (CRT), ipilimumab (IPI, 1 mg/kg IV) and nivolumab (NIVO, 360 mg flat dose IV) will be administered, followed by nivolumab (360 mg flat dose IV) after 3 weeks. Radiotherapy consists of once-daily doses of 2 Gy to a total of 50 Gy, and chemotherapy will consist of a platinum-doublet. An anatomical pulmonary resection is planned 6 weeks after the last day of radiotherapy. The primary study objective is to establish the safety of adding IPI/NIVO to pre-operative CRT, and its impact on pathological tumor response. Secondary objectives are to assess the impact of adding IPI/NIVO to CRT on disease free and overall survival. Exploratory objectives are to characterize tumor inflammation and the immune contexture in the tumor and tumor-draining lymph nodes (TDLN), and to explore the effects of IPI/NIVO and CRT and surgery on distribution and phenotype of peripheral blood immune subsets.DiscussionThe INCREASE trial will evaluate the safety and local efficacy of a combination of 4 modalities in patients with resectable, T3-4N0–1 NSCLC. Translational research will investigate the mechanisms of action and drug related adverse events.Trial registrationNetherlands Trial Registration (NTR):NL8435, Registered 03 March 2020.

Cytoreductive surgery (CRS) followed by hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment with curative intent for peritoneal metastasis of colorectal cancer (CRC). Currently, there is no standardized HIPEC protocol: choice of drug, perfusate temperature, and duration of treatment vary per institute. We investigated the temperature-dependent effectiveness of drugs often used in HIPEC. Methods: The effect of temperature on drug uptake, DNA damage, apoptosis, cell cycle distribution, and cell growth were assessed using the temperature-dependent IC50 and Thermal Enhancement Ratio (TER) values of the chemotherapeutic drugs cisplatin, oxaliplatin, carboplatin, mitomycin-C (MMC), and 5-fluorouracil (5-FU) on 2D and 3D CRC cell cultures at clinically relevant hyperthermic conditions (38&ndash, 43 &deg, C/60 min). Results: Hyperthermia alone decreased cell viability and clonogenicity of all cell lines. Treatment with platinum-based drugs and MMC resulted in G2-arrest. Platinum-based drugs display a temperature-dependent synergy with heat, with increased drug uptake, DNA damage, and apoptosis at elevated temperatures. Apoptotic levels increased after treatment with MMC or 5-FU, without a synergy with heat. Conclusion: Our in vitro results demonstrate that a 60-min exposure of platinum-based drugs and MMC are effective in treating 2D and 3D CRC cell cultures, where platinum-based drugs require hyperthermia (&gt, 41 &deg, C) to augment effectivity, suggesting that they are, in principle, suitable for HIPEC.

BackgroundGlioblastoma (GBM) is the most common and deadliest form of brain cancer in adults. Standard treatment, consisting of surgery and radiochemotherapy, only provides a modest survival benefit and is incapable of combating infiltrating GBM cells in other parts of the brain. New therapies in clinical trials, such as anti-programmed cell death 1 immunotherapy, have so far shown limited success in GBM. Moreover, it is unclear how the growth of GBM suppresses the immune system locally at the site of the brain tumor or if distant sites of tumor cell migration are also involved. Invasive GBM cells in brain tissue beyond the primary tumor limit the use of surgery, thus immunotherapy could be beneficial if activated/suppressed immune cells are present in the contralateral hemisphere.MethodsHere, we used a syngeneic orthotopic GL26 GBM mouse model and multiparameter fluorescence-activated cell sorting analysis to study the phenotype of resident and infiltrating immune cells in both the brain tumor hemisphere and contralateral hemisphere.ResultsWe show that lymphoid cells, including tumor antigen-specific CD8+tumor-infiltrating lymphocytes (TILs) are present in the tumor and are characterized by a tolerogenic phenotype based on high immune checkpoint expression. Massive infiltration of myeloid cells is observed, expressing immune checkpoint ligands, suggesting an immune-dependent coinhibitory axis limiting TIL responses. Surprisingly, these phenotypes are paralleled in the contralateral hemisphere, showing that infiltrating immune cells are also present at distant sites, expressing key immune checkpoints and immune checkpoint ligands.ConclusionWhole-brain analysis indicates active immune involvement throughout the brain, both at the site of the primary tumor and in the contralateral hemisphere. Using the right combination and timing, immune checkpoint blockade could have the potential to activate immune cells at the site of the brain tumor and at distant sites, thereby also targeting diffusely infiltrating GBM cells.

Glioblastoma is the most aggressive brain malignancy, for which immunotherapy has failed to prolong survival. Glioblastoma-associated immune infiltrates are dominated by tumor-associated macrophages and microglia (TAMs), which are key mediators of immune suppression and resistance to immunotherapy. We and others demonstrated aberrant expression of glycans in different cancer types. These tumor-associated glycans trigger inhibitory signaling in TAMs through glycan-binding receptors. We investigated the glioblastoma glycocalyx as a tumor-intrinsic immune suppressor. We detected increased expression of both tumor-associated truncated O-linked glycans and their receptor, macrophage galactose-type lectin (MGL), on CD163+ TAMs in glioblastoma patient-derived tumor tissues. In an immunocompetent orthotopic glioma mouse model overexpressing truncated O-linked glycans (MGL ligands), high-dimensional mass cytometry revealed a wide heterogeneity of infiltrating myeloid cells with increased infiltration of PD-L1+ TAMs as well as distant alterations in the bone marrow (BM). Our results demonstrate that glioblastomas exploit cell surface O-linked glycans for local and distant immune modulation. Fil: Dusoswa, Sophie A.. Vrije Universiteit Amsterdam; Países Bajos Fil: Verhoeff, Jan. Vrije Universiteit Amsterdam; Países Bajos Fil: Abels, Erik. Vrije Universiteit Amsterdam; Países Bajos Fil: Mendez Huergo, Santiago Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Croci Russo, Diego Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; Argentina Fil: Kuijper, Lisan H.. Vrije Universiteit Amsterdam; Países Bajos Fil: de Miguel, Elena. Vrije Universiteit Amsterdam; Países Bajos Fil: Wouters, Valerie M. C. J.. Vrije Universiteit Amsterdam; Países Bajos Fil: Best, Myron G.. Vrije Universiteit Amsterdam; Países Bajos Fil: Rodriguez, Ernesto. Vrije Universiteit Amsterdam; Países Bajos Fil: Cornelissen, Lenneke A.M.. Vrije Universiteit Amsterdam; Países Bajos Fil: van Vliet, Sandra J.. Vrije Universiteit Amsterdam; Países Bajos Fil: Wesseling, Pieter. Vrije Universiteit Amsterdam; Países Bajos Fil: Breakefield, Xandra O.. Vrije Universiteit Amsterdam; Países Bajos Fil: Noske, David P.. Vrije Universiteit Amsterdam; Países Bajos Fil: Würdinger, Thomas. Harvard Medical School; Estados Unidos Fil: Broekman, Marike L.D.. Harvard Medical School; Estados Unidos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: van Kooyk, Yvette. Vrije Universiteit Amsterdam; Países Bajos Fil: Garcia Vallejo, Juan J.. Vrije Universiteit Amsterdam; Países Bajos

Injection of antigenic peptides has been widely used as a vaccine strategy to boost T cell immunity. However, the poor immunogenicity of single peptides can potentially be strengthened through modification of the tertiary structure and the selection of the accompanying adjuvant. Here, we generated antigenic peptides into non-linear trimers by solid phase peptide synthesis, thereby enhancing antigen presentation by dendritic cells to CD8+ T cells in vitro and in vivo. CD8+ T cells from mice vaccinated with trimers showed an KLRG1+ effector phenotype and were able to recognize and kill antigen-expressing tumor cells ex vivo. Importantly, trimers outperformed synthetic long peptide in terms of T cell response even when equal number of epitopes were used for immunization. To improve the synthesis of trimers containing difficult peptide sequences, we developed a novel small molecule that functions as conjugation platform for synthetic long peptides. This platform, termed Antigen MAtriX (AMAX) improved yield, purity and solubility of trimers over conventional solid phase synthesis strategies. AMAX outperformed synthetic long peptides in terms of both CD8+ and CD4+ T cell responses and allowed functionalization with DC-SIGN-binding carbohydrates for in vivo dendritic cell targeting strategies, boosting T cell responses even further. Moreover, we show that agonistic CD40 antibody combined with MF59 (AddaVax) emulsion synergistically improves the antigen-specific T cell response of the AMAX in vivo. Also, tumor-associated antigens and neo-antigens could be incorporated in AMAX for tumor-specific CD8+ T cell responses. Importantly, immunization with a mix of neoantigen AMAX could reduce tumor growth in a pre-clinical syngeneic mouse model. Hence, we provide pre-clinical support for the induction of effector CD8+ T cells through the adaptable AMAX platform as easy implementable peptidic vaccination strategy against any antigen of choice, including neoantigens for anti-tumor immunity.

Contains fulltext : 215800.pdf (Publisher’s version ) (Open Access) Glioblastoma is the most prevalent and aggressive primary brain tumour for which total tumour lysate-pulsed dendritic cell vaccination is currently under clinical evaluation. Glioblastoma extracellular vesicles (EVs) may represent an enriched cell-free source of tumour-associated (neo-) antigens to pulse dendritic cells (DCs) for the initiation of an anti-tumour immune response. Capture and uptake of EVs by DCs could occur in a receptor-mediated and presumably glycan-dependent way, yet the glycan composition of glioblastoma EVs is unknown. Here, we set out to characterize the glycocalyx composition of glioblastoma EVs by lectin-binding ELISA and comprehensive immunogold transmission electron microscopy (immuno-TEM). The surface glycan profile of human glioblastoma cell line-derived EVs (50-200 nm) was dominated by alpha-2,3- and alpha-2,6 linked sialic acid-capped complex N-glycans and bi-antennary N-glycans. Since sialic acids can trigger immune inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, we screened for Siglec ligands on the EVs. Glioblastoma EVs showed significant binding to Siglec-9, which is highly expressed on DCs. Surprisingly, however, glioblastoma EVs lack glycans that could bind Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN, CD209), a receptor that mediates uptake and induction of CD4(+) and CD8(+) T cell activation. Therefore, we explored whether modification of the EV glycan surface could reduce immune inhibitory Siglec binding, while enhancing EV internalization by DCs in a DC-SIGN dependent manner. Desialylation with a pan-sialic acid hydrolase led to reduction of sialic acid expression on EVs. Moreover, insertion of a high-affinity ligand (Lewis(Y)) for DC-SIGN resulted in a four-fold increase of uptake by monocyte-derived DCs. In conclusion, we show that the glycocalyx composition of EVs is a key factor of efficient DC targeting and that modification of the EV glycocalyx potentiates EVs as anti-cancer vaccine.

Background Multiple sclerosis (MS) involves a misdirected immune attack against myelin in the brain and spinal cord, leading to profound neuroinflammation and neurodegeneration. While the mechanisms of disease pathogenesis have been widely studied, the suppression mechanisms that lead to the resolution of the autoimmune response are still poorly understood. Here, we investigated the role of the C-type lectin receptor macrophage galactose-type lectin (MGL), usually expressed on tolerogenic antigen-presenting cells (APCs), as a negative regulator of autoimmune-driven neuroinflammation. Methods We used in silico, immunohistochemical, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry analysis to explore the expression and functionality of MGL in human macrophages and microglia, as well as in MS post-mortem tissue. In vitro, we studied the capacity of MGL to mediate apoptosis of experimental autoimmune encephalomyelitis (EAE)-derived T cells and mouse CD4+ T cells. Finally, we evaluated in vivo and ex vivo the immunomodulatory potential of MGL in EAE. Results MGL plays a critical role in the resolution phase of EAE as MGL1-deficient (Clec10a−/−) mice showed a similar day of onset but experienced a higher clinical score to that of WT littermates. We demonstrate that the mouse ortholog MGL1 induces apoptosis of autoreactive T cells and diminishes the expression of pro-inflammatory cytokines and inflammatory autoantibodies. Moreover, we show that MGL1 but not MGL2 induces apoptosis of activated mouse CD4+ T cells in vitro. In human settings, we show that MGL expression is increased in active MS lesions and on alternatively activated microglia and macrophages which, in turn, induces the secretion of the immunoregulatory cytokine IL-10, underscoring the clinical relevance of this lectin. Conclusions Our results show a new role of MGL-expressing APCs as an anti-inflammatory mechanism in autoimmune neuroinflammation by dampening pathogenic T and B cell responses, uncovering a novel clue for neuroprotective therapeutic strategies with relevance for in MS clinical applications. Electronic supplementary material The online version of this article (10.1186/s12974-019-1522-4) contains supplementary material, which is available to authorized users.

Tumors that lack T cell infiltration are less likely to respond to immune checkpoint inhibition and could benefit from cancer vaccination for the initiation of anti-tumor T cell responses. An attractive vaccine strategy is in vivo targeting of dendritic cells (DCs), key initiators of antigen-specific T cell responses. In this study we generated tumor-derived apoptotic extracellular vesicles (ApoEVs), which are potentially an abundant source of tumor-specific neo-antigens and other tumor-associated antigens (TAAs), and which can be manipulated to express DC-targeting ligands for efficient antigen delivery. Our data demonstrates that by specifically modifying the glycocalyx of tumor cells, high-mannose glycans can be expressed on their cell surface and on extracellular vesicles derived after the induction of apoptosis. High-mannose glycans are the natural ligands of dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), a dendritic cell associated C-type lectin receptor (CLR), which has the ability to efficiently internalize its cargo and direct it to both major histocompatibility complex (MHC)-I and MHC-II pathways for the induction of CD8+ and CD4+ T cell responses, respectively. Compared to unmodified ApoEVs, ApoEVs carrying DC-SIGN ligands are internalized to a higher extent, resulting in enhanced priming of tumor-specific CD8+ T cells. This approach thus presents a promising vaccination strategy in support of T cell-based immunotherapy of cancer.

Glioblastoma is the most common primary brain tumor with a dismal prognosis and a lack of effective treatment options after relapse. Despite striking successes that were achieved in the last decade in other types of cancer, evidence of sustainable responses to T cell directed immunotherapy in glioblastoma remains limited. This might be explained by an emerging hypothesis that glioblastomas are cold tumours with poor lymphocyte infiltration. Therefore, some recent efforts have been focused on increasing cytotoxic lymphocyte infiltration. Here we present our workflow for subpopulation frequency, marker expression, and correlation network (INFERENCE) analysis, in this study applied to screen for possible new targets to enhance anti-glioblastoma immune responses. Using high-dimensional, single-cell mass cytometry (CyTOF) we profiled peripheral blood mononuclear cells derived from 160 patients with glioblastoma, lower grade glioma, or metastasis, meningioma and epilepsy as controls. Based on a CyTOF panel composed mainly of lymphocyte markers and check-point inhibition markers we could effectively dissect blood leukocytes into 100 phenotypically separate subsets of CD4 T cells (35 subsets), CD8 T cells (17 subsets), monocytes (12 subsets), NK cells (14 subsets), and B cells (15 subsets), and map their frequencies, phenotypes and correlation statistics. We thereby created an in depth human atlas of peripheral blood mononuclear cells in patients with different types of intracranial tumors. Interestingly, a subset of B cells characterized by high expression of the Interleukin-2 receptor alpha chain (CD25, a typical marker of regulatory T cells) was significantly increased in glioblastoma patients. This immune suppressive B cell subset has previously been associated with enhancing the suppressive effect of regulatory T cells. Further research is warranted to investigate the role of this aberrant B cell subset in the context of glioblastoma and its potential as target for immunotherapy.

Recent clinical trials in glioblastoma have yet to show any benefit of immune checkpoint inhibitors and are mostly driven by the fact that these inhibitors have efficacy in other cancer types. For immunotherapy to succeed in glioblastoma patients, we need to understand the immunological subsets and phenotypes involved to target and manipulate them. To this end, we set up different glioblastoma mouse models and used multiparameter flow cytometry and t-SNE unsupervised clustering of immune subsets including co-expression of immune checkpoints and their ligands. When comparing the brain tumor microenvironment with immune cells present in the contralateral hemisphere and the systemic compartment, we identified unique immune subsets. Different populations of infiltrating CD4 T cells were characterized by expression of TIGIT, PD-1 and HVEM. PD-1 expression was also significantly increased on infiltrating CD8 T cells. Furthermore, analysis of myeloid subsets showed massive infiltration of macrophages in the brain tumor microenvironment, which is also apparent in clinical glioblastoma samples. Co-expression of PD-L1, CD155 and BTLA was observed on both infiltrating macrophages and brain-resident microglia. Together, these results suggest a glioblastoma-induced ‘tolerogenic’ microenvironment and, while T cells are present, myeloid cells might prove to be a better target for combination immunotherapy.

Glioblastoma is the most aggressive brain malignancy, for which conventional therapy has failed to achieve major improvements in survival since 2005. Glioblastoma-associated immune infiltrates are dominated by tumour-associated macrophages (TAM), which could be the key mediators of immune suppression. Recently we proposed the tumour “glyco-code” as a novel target for immunotherapy, and source for biomarkers. Malignant transformation is associated with aberrant expression of cell surface glycans, which play crucial roles in processes such as migration, angiogenesis and immune modulation. Here we investigated the glioblastoma glycocalyx as a tumour-intrinsic immune suppressive mechanism via tumour infiltrating macrophages. We detected increased expression of tumour-associated truncated O-linked glycans in patient-derived glioblastoma- versus control tissues by immunofluorescence and ELISA based assays. In concert, the macrophage galactose lectin (MGL) receptor, which interacts with truncated O-linked glycans, was overexpressed on glioblastoma infiltrating TAM, also detected by immunofluorescence, and confirmed on RNA level in TCGA data. Unsupervised, high-dimensional mass cytometry (CyTOF) analysis of a murine immunocompetent orthotopic glioblastoma model overexpressing truncated O-linked glycans revealed increased tumor infiltration by CCR2+, PD-L1+ macrophages. At a systemic level, we discovered increased frequencies of CCR2+ peripheral dendritic cells (pDC), and a decrease in CCR2+ monocyte frequencies in the bone marrow of these mice. Moreover, we observed a drastic increase in correlations between glioblastoma infiltrating CCR2+ macrophages and several myeloid cell subsets in the bone marrow using network analysis of frequency correlation statistics. Our results suggest that glioblastomas overexpress truncated O-linked glycans and exploit cell surface glycosylation- mediated immune regulatory circuits for systemic immune modulation and recruitment of suppressive TAM.

Interleukin (IL)-1β has proven to be crucial in the differentiation of human and mouse Th17 cells. Although it has become evident that IL-1β has potent IL-17-inducing effects on CD4(+) T cells directly, it has not yet been explored whether IL-1β can also prime dendritic cells (DCs) for a Th17 instruction program. Here, we show that human immature DCs exposed to IL-1β promote IL-17 production in human memory CD4(+) T cells. IL-1β-primed DCs express high levels of CD14 that mediate IL-17 production through direct interaction with T cells. Moreover, culturing human CD4(+)CD45RO(+) memory T cells with soluble CD14 is sufficient for the upregulation of retinoic acid-related orphan receptor-γ thymus and IL-17 production. In addition, in a human in situ model using tissue-resident skin DCs, upregulation of CD14 expression induced by IL-1β on skin residents DCs promotes IL-17 production in memory T cells; strongly suggesting the in vivo relevance of this mechanism. Our findings uncover new roles for IL-1β and CD14, and may therefore have important consequences for the development of new therapies for Th17-mediated autoimmune diseases and bacterial and fungal pathogenic infections