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

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

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

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

Palmitoylated antigens for the induction of anti-tumor CD8+ T cells and enhanced tumor recognition.

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.

Sialic acids in pancreatic cancer cells drive tumour-associated macrophage differentiation via the Siglec receptors Siglec-7 and Siglec-9.

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.

Tn Antigen Expression Contributes to an Immune Suppressive Microenvironment and Drives Tumor Growth in Colorectal Cancer.

Expression of the tumor-associated glycan Tn antigen (αGalNAc-Ser/Thr) has been correlated to poor prognosis and metastasis in multiple cancer types. However, the exact mechanisms exerted by Tn antigen to support tumor growth are still lacking. One emerging hallmark of cancer is evasion of immune destruction. Although tumor cells often exploit the glycosylation machinery to interact with the immune system, the contribution of Tn antigen to an immunosuppressive tumor microenvironment has scarcely been studied. Here, we explored how Tn antigen influences the tumor immune cell composition in a colorectal cancer (CRC) mouse model. CRISPR/Cas9-mediated knock out of the C1galt1c1 gene resulted in elevated Tn antigen levels on the cell surface of the CRC cell line MC38 (MC38-Tnhigh). RNA sequencing and subsequent GO term enrichment analysis of our Tnhigh glycovariant not only revealed differences in MAPK signaling and cell migration, but also in antigen processing and presentation as well as in cytotoxic T cell responses. Indeed, MC38-Tnhigh tumors displayed increased tumor growth in vivo, which was correlated with an altered tumor immune cell infiltration, characterized by reduced levels of cytotoxic CD8+ T cells and enhanced accumulation of myeloid-derived suppressor cells. Interestingly, no systemic differences in T cell subsets were observed. Together, our data demonstrate for the first time that Tn antigen expression in the CRC tumor microenvironment affects the tumor-associated immune cell repertoire.

FUT9-Driven Programming of Colon Cancer Cells towards a Stem Cell-Like State.

Cancer stem cells (CSCs) are located in dedicated niches, where they remain inert to chemotherapeutic drugs and drive metastasis. Although plasticity in the CSC pool is well appreciated, the molecular mechanisms implicated in the regulation of cancer stemness are still elusive. Here, we define a fucosylation-dependent reprogramming of colon cancer cells towards a stem cell-like phenotype and function. De novo transcriptional activation of Fut9 in the murine colon adenocarcinoma cell line, MC38, followed by RNA seq-based regulon analysis, revealed major gene regulatory networks related to stemness. Lewisx, Sox2, ALDH and CD44 expression, tumorsphere formation, resistance to 5-FU treatment and in vivo tumor growth were increased in FUT9-expressing MC38 cells compared to the control cells. Likewise, human CRC cell lines highly expressing FUT9 displayed phenotypic features of CSCs, which were significantly impaired upon FUT9 knock-out. Finally, in primary CRC FUT9+ tumor cells pathways related to cancer stemness were enriched, providing a clinically meaningful annotation of the complicity of FUT9 in stemness regulation and may open new avenues for therapeutic intervention.

Adaptable antigen matrix platforms for peptide vaccination strategies and T cell-mediated anti-tumor immunity.

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.

Monocyte-derived APCs are central to the response of PD1 checkpoint blockade and provide a therapeutic target for combination therapy.

BACKGROUND: PD1 immune checkpoint blockade (αPD1 ICB) has shown unparalleled success in treating many types of cancer. However, response to treatment does not always lead to tumor rejection. While αPD1 ICB relies on cytotoxic CD8+ T cells, antigen-presenting cells (APCs) at the tumor site are also needed for costimulation of tumor-infiltrating lymphocytes (TILs). It is still unclear how these APCs develop and function before and during αPD1 ICB or how they are associated with tumor rejection. METHODS: Here, we used B16 mouse melanoma and MC38 colorectal carcinoma tumor models, which show differential responses to αPD1 ICB. The immune composition of ICB insensitive B16 and sensitive MC38 were extensively investigated using multi-parameter flow cytometry and unsupervised clustering and trajectory analyses. We additionally analyzed existing single cell RNA sequencing data of the myeloid compartment of patients with melanoma undergoing αPD1 ICB. Lastly, we investigated the effect of CD40 agonistic antibody on the tumor-infiltrating monocyte-derived cells during αPD1 ICB. RESULTS: We show that monocyte-derived dendritic cells (moDCs) express high levels of costimulatory molecules and are correlated with effector TILs in the tumor microenvironment (TME) after αPD1 ICB only in responding mouse tumor models. Tumor-resident moDCs showed distinct differentiation from monocytes in both mouse and human tumors. We further confirmed significant enrichment of tumor-resident differentiated moDCs in patients with melanoma responding to αPD1 ICB therapy compared with non-responding patients. Moreover, moDCs could be targeted by agonistic anti-CD40 antibody, supporting moDC differentiation, effector T-cell expansion and anti-tumor immunity. CONCLUSION: The combined analysis of myeloid and lymphoid populations in the TME during successful and non-successful PD1 ICB led to the discovery of monocyte-to-DC differentiation linked to expanding T-cell populations. This differentiation was found in patients during ICB, which was significantly higher during successful ICB. The finding of tumor-infiltrating monocytes and differentiating moDCs as druggable target for rational combination therapy opens new avenues of anti-tumor therapy design.

Immune involvement of the contralateral hemisphere in a glioblastoma mouse model.

BACKGROUND: Glioblastoma (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. METHODS: Here, 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. RESULTS: We 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. CONCLUSION: Whole-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.

Immunological dynamics after subcutaneous immunization with a squalene-based oil-in-water adjuvant.

The clinically successful adjuvant MF59 is used in seasonal influenza vaccines, which is proposed to enhance immunity by creating an immune-competent microenvironment in the muscle that allows recruitment of immune cells that drive adaptive immune responses. Here, we examined whether the clinically successful adjuvants MF59/AddaVax could be used for subcutaneous use and how antigen delivery can be synergized with cellular dynamics at the vaccination site. Subcutaneous injection of AddaVax leads to thickening of the skin, characterized by a neutrophil-monocyte recruitment sequence. Skin-infiltrating CCR2+Ly6Chigh monocytes showed differentiation to CD11b+Ly6C+MHCII+CD11c+CD64+ monocyte-derived DCs over time in the hypodermal layers of the skin, expressing high levels of CD209a/mDC-SIGN. Surprisingly, skin thickening was accompanied with increased white adipose tissue highly enriched with monocytes. Analysis of the skin-draining lymph nodes revealed early increases in neutrophils and moDCs at 12 hours after injection and later increases in migratory cDC2s. Subcutaneous vaccination with AddaVax enhanced antigen-specific CD8+ and CD4+ T cell responses, while moDC targeting using antigen-coupled CD209a antibody additionally boosted humoral responses. Hence, oil-in-water emulsions provide an attractive immune modulatory adjuvants aimed at increasing cellular responses, as well as antibody responses when combined with moDC targeting.

Non-digestible oligosaccharides scFOS/lcFOS facilitate safe subcutaneous immunotherapy for peanut allergy.

BACKGROUND: Improving the safety of subcutaneous immunotherapy (SCIT) for food allergy is necessary to reduce side effects and achieve long-term tolerance. We determined the effect of dietary supplementation with 1% non-digestible short- and long-chain fructo-oligosaccharides (scFOS/lcFOS) on safety and efficacy of SCIT using a peanut allergy mouse model. METHODS: After sensitization, mice received a scFOS/lcFOS or control diet for the rest of the study. To study safety of SCIT, mice were dosed with a single subcutaneous injection of peanut extract (PE) or PBS. To study efficacy, mice were dosed subcutaneously (SCIT, 3 times/week) with PE or PBS for 3 weeks. Hereafter, acute allergic skin responses, anaphylactic shock symptoms and body temperature were assessed. To study the mechanism in vitro, the human IgE receptor (FcεRI)-transfected rat mast cell (RBL) line was sensitized with an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) and incubated with the oligosaccharides before exposure to BLG to assess direct the effect on degranulation. RESULTS: scFOS/lcFOS reduced anaphylaxis caused by a single PE SCIT dose. scFOS/lcFOS alone also reduced the acute allergic skin response. Moreover, scFOS/lcFOS supplementation resulted in lower MMCP-1 levels in serum after PE SCIT dose compared to control diet, while antibody levels were not affected by the diet. In vitro incubation with scFOS/lcFOS at 0.5% suppressed the degranulation of IgE-sensitized RBL cells. However, dietary supplementation with scFOS/lcFOS did not improve the efficacy of SCIT. CONCLUSIONS: We show that scFOS/lcFOS diet improves the safety of SCIT, as evidenced by lower anaphylactic responses without compromising the efficacy in a mouse model for peanut allergy. This effect is likely to result from the suppression of mast cell effector function.

Outer membrane vesicles engineered to express membrane-bound antigen program dendritic cells for cross-presentation to CD8+ T cells.

Outer membrane vesicles (OMVs) are vesicular nano-particles produced by Gram-negative bacteria that are recently being explored as vaccine vector. The fact that OMVs can be efficiently produced by a hypervesiculating Salmonella typhimurium strain, are packed with naturally-occurring adjuvants like lipopolysaccharides (LPS), and can be engineered to express any antigen of choice, makes them ideal candidates for vaccinology. However, it is unclear whether OMVs induce dendritic cell (DC)-mediated antigen-specific T cell responses and how immune activation is coordinated. Here, we show that OMVs induce maturation of human monocyte-derived DCs, murine bone marrow-derived DCs and CD11c+ splenic DCs. OMV-induced DC maturation was dependent on the presence of LPS and the myeloid differentiation primary response 88 (MyD88) adapter protein downstream of toll-like receptor signaling. Importantly, OMVs did not induce pyroptosis/cell death, but instead provided a significant survival benefit in DCs over non-stimulated DCs. OMVs displaying a sizeable ovalbumin fragment at the vesicle surface induce potent cross-presentation in BMDCs and splenic CD11c+ DCs to OTI CD8+ T cells, dependent on MyD88. Interestingly, the OMV-induced preference to cross-presentation was only partly dependent on the BATF3-dependent CD8a+ professional cross-presenting DC subset. Hence, an OMV-specific programming of DCs that induces maturation and provides a survival benefit for antigen presentation to T cells is identified. Additionally, for the first time, antigen-specific and potent cross-presentation of antigen-loaded OMVs to CD8+ T cells is demonstrated. These data provide mechanistical insight into the processes needed for the DC-mediated cross-presentation of OMV-derived antigens to CD8+ T cells with implications for therapeutic strategies. STATEMENT OF SIGNIFICANCE: Bacteria are primarily known to cause disease. However, recent research has focused on using engineered bacteria and its byproducts as vaccine agents. In particular, outer membrane vesicles (OMVs) have shown promise in eliciting potent immunity against a variety of pathogens. While most vaccines rely on the generation of antibodies, the control of viral replication and tumor growth is driven by cytotoxic CD8+ T cells induced by dendritic cells (DCs). As such, there is a dire need for vaccines that use DCs to elicit CD8+ T cell responses. Studying OMVs as engineered biomaterial and its interaction with DCs allows tailored induction of immunity. This study includes important findings on OMV-dendritic cell interactions and for the first time supports OMVs as vehicles for the induction of antigen-specific CD8+ T cell responses. Additionally, important mechanistical insight into the molecular pathways needed for the cross-presentation of OMV-derived antigens to CD8+ T cells is provided.

Disruption of sialic acid metabolism drives tumor growth by augmenting CD8+ T cell apoptosis.

Sialylated glycan structures are known for their immunomodulatory capacities and their contribution to tumor immune evasion. However, the role of aberrant sialylation in colorectal cancer and the consequences of complete tumor desialylation on anti-tumor immunity remain unstudied. Here, we report that CRISPR/Cas9-mediated knock out of the CMAS gene, encoding a key enzyme in the sialylation pathway, in the mouse colorectal cancer MC38 cell line completely abrogated cell surface expression of sialic acids (MC38-Sianull ) and, unexpectedly, significantly increased in vivo tumor growth compared to the control MC38-MOCK cells. This enhanced tumor growth of MC38-Sianull cells could be attributed to decreased CD8+ T cell frequencies in the tumor microenvironment only, as immune cell frequencies in tumor-draining lymph nodes remained unaffected. In addition, MC38-Sianull cells were able to induce CD8+ T cell apoptosis in an antigen-independent manner. Moreover, low CMAS gene expression correlated with reduced recurrence-free survival in a human colorectal cancer cohort, supporting the clinical relevance of our work. Together, these results demonstrate for the first time a detrimental effect of complete tumor desialylation on colorectal cancer tumor growth, which greatly impacts the design of novel cancer therapeutics aimed at altering the tumor glycosylation profile.

Trovafloxacin-Induced Liver Injury: Lack in Regulation of Inflammation by Inhibition of Nucleotide Release and Neutrophil Movement.

The fluoroquinolone trovafloxacin (TVX) is associated with a high risk of drug-induced liver injury (DILI). Although part of the liver damage by TVX+TNF relies on neutrophils, we have recently demonstrated that liver recruitment of monocytes and neutrophils is delayed by TVX. Here we show that the delayed leukocyte recruitment is caused by a combination of effects which are linked to the capacity of TVX to block the hemichannel pannexin 1. TVX inhibited find-me signal release in apoptotic HepG2 hepatocytes, decelerated freshly isolated human neutrophils toward IL-8 and f-MLF, and decreased the liver expression of ICAM-1. In blood of TVX+TNF-treated mice, we observed an accumulation of activated neutrophils despite an increased MIP-2 release by the liver. Depletion of monocytes and neutrophils caused increased serum concentrations of TNF, IL-6, and MIP-2 in TVX-treated mice as well as in mice treated with the fluoroquinolone levofloxacin, known to have a lower DILI-inducing profile. This supports the idea that early leukocyte recruitment regulates inflammation. In conclusion, disrupted regulation by leukocytes appears to constitute a fundamental step in the onset of TVX-induced liver injury, acting in concert with the capability of TVX to induce hepatocyte cell death. Interference of leukocyte-mediated regulation of inflammation represents a novel mechanism to explain the onset of DILI.

Mouse DC-SIGN/CD209a as Target for Antigen Delivery and Adaptive Immunity.

The efficacy of vaccination studies aimed at targeting antigens to human DC-SIGN (hDC-SIGN) have been notoriously difficult to study in vivo, as eight dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) homologs have been described in mice. CD209a/SIGNR5 has been coined as the mouse DC-SIGN (mDC-SIGN) ortholog, based on its expression and location in the genome. Nonetheless, which properties of hDC-SIGN are covered by mDC-SIGN is poorly investigated. One of the most important functions of DC-SIGN is the induction of adaptive immunity. As such, the aim of this study is to determine the capability of mDC-SIGN to induce adaptive immune responses. Here, we show that mDC-SIGN is expressed on GM-CSF cultured bone marrow-derived dendritic cells (BMDCs) and macrophages. However, mDC-SIGN is an internalizing receptor which, unlike hDC-SIGN, quickly resurfaces after internalization. Binding of OVA-coupled anti-mDC-SIGN antibody by BMDCs leads to quick internalization, processing, and presentation to antigen-specific CD8+ and CD4+ T cells, which can be boosted using the TLR4 ligand, monophosphoryl lipid A. In the homeostatic condition, mDC-SIGN is mostly expressed on myeloid cells in the skin and spleen. A subcutaneous injection of fluorescent anti-mDC-SIGN reveals specific targeting to mDC-SIGN+ skin dendritic cells (DCs) and monocyte-derived DCs in situ. A subcutaneous vaccination strategy containing OVA-coupled anti-mDC-SIGN antibody generated antigen-specific polyfunctional CD8+ T cell and CD4+ T cell responses and a strong isotype-switched OVA-specific antibody response in vivo. We conclude that mDC-SIGN shows partly overlapping similarities to hDC-SIGN and that targeting mDC-SIGN provides a valuable approach to investigate the immunological function of DC-SIGN in vivo.

Tissue influx of neutrophils and monocytes is delayed during development of trovafloxacin-induced tumor necrosis factor-dependent liver injury in mice.

Idiosyncratic drug-induced liver injury (iDILI) has a poorly understood pathogenesis. However, iDILI is often associated with inflammatory stress signals in human patients as well as animal models. Tumor necrosis factor (TNF) and neutrophils play a key role in onset of trovafloxacin (TVX)-induced iDILI, but the exact role of neutrophils and other leukocytes remains to be defined. We therefore set out to study the kinetics of immunological changes during the development of TVX-induced iDILI in the established murine model of acute liver injury induced by administration of TVX and TNF. Initially, TNF stimulated the appearance of leukocytes, in particular neutrophils, into the liver of TVX-treated mice, but even more so in control mice treated with the non-DILI inducing analogue levofloxacin (LVX) or saline as vehicle (Veh). This difference was apparent at 2 hours after TNF administration, but at 4 hours, the relative neutrophil amounts were reduced again in Veh- and LVX-treated mice whereas the amounts in TVX-treated mice remained at the same increased level as at 2 hours. The influx of monocytes/macrophages, which was unaffected in Veh- and LVX-treated mice was markedly reduced or even absent in TVX-treated mice. Unlike controls, mice receiving TVX + TNF display severe hepatotoxicity with clear pathology and apoptosis, coagulated hepatic vessels and increased alanine aminotransferase levels and interleukin 6/10 ratios. Findings indicate that TVX delays the acute influx of neutrophils and monocytes/macrophages. Considering their known anti-inflammatory functions, the disruption of influx of these innate immune cells may hamper the resolution of initial cytotoxic effects of TVX and thus contribute to liver injury development.

Immune responses induced by diclofenac or carbamazepine in an oral exposure model using TNP-Ficoll as reporter antigen.

Immune-mediated drug hypersensitivity reactions (IDHR) may result from immuno-sensitization to a drug-induced neo-antigen. They rarely occur in patients and are usually not predicted preclinically using standard toxicity studies. To assess the potential of a drug to induce T-cell sensitization, trinitrophenyl (TNP)-Ficoll was used here as a bystander antigen in animal experiments. TNP-Ficoll will only elicit TNP-specific IgG antibodies in the presence of non-cognate T-cell help. Therefore, the presence of TNP-specific IgG antibodies after co-injection of drug and TNP-Ficoll was indicative of T-cell sensitization potential. This TNP-Ficoll-approach was used here to characterize T-cell help induced by oral exposure to diclofenac (DF) or carbamazepine (CMZ). DF or CMZ was administered orally to BALB/c mice and after 3 w, the mice were challenged in a hind paw with TNP-Ficoll and a dose of the drug that by itself does only elicit a sub-optimal popliteal lymph node assay (PLNA) response. T-cell-dependent responses were then evaluated in paw-draining popliteal lymph nodes (PLN). Also, shortly after oral exposure, mesenteric lymph nodes (MLN) were excised for evaluation of local responses. Both drugs were able to increase PLN cellularity and TNP-specific IgG1 production after challenge. Both DF and CMZ stimulated CD4+ and CD8+ T-cells and caused shifts of the subsets toward an effector phenotype. DF, but not CMZ, appeared to stimulate interferon (IFN)-γ production. Remarkably, depletion of CD8+, but not CD4+, T-cells reduced TNP-specific IgG1 production, and was more pronounced in CMZ- than in DF-exposed animals. Local responses in the MLN caused by DF or CMZ also showed shifts of CD4+ and CD8+-cells toward a memory phenotype. Together, the data indicate that oral exposure to CMZ and DF differentially induced neo-antigen-specific T-cell reactions in the PLNA.

Oral exposure to immunostimulating drugs results in early changes in innate immune parameters in the spleen.

The development of immune-dependent drug hypersensitivity reactions (IDHR) is likely to involve activation of the innate immune system to stimulate neo-antigen specific T-cells. Previously it has been shown that, upon oral exposure to several drugs with immune-adjuvant capacity, mice developed T-cell-dependent responses to TNP-OVA. These results were indicative of the adjuvant potential of these drugs. The present study set out to evaluate the nature of this adjuvant potential by focusing on early immune changes in the spleen, by testing several drugs in the same experimental model. Mice were exposed to one or multiple oral doses of previously-tested drugs: the non-steroidal-anti-inflammatory drug (NSAID) diclofenac (DF), the analgesic acetaminophen (APAP), the anti-epileptic drug carbamazepine (CMZ) or the antibiotic ofloxacin (OFLX). Within 24 h after the final dosing, early innate and also adaptive immune parameters in the spleen were examined. In addition, liver tissue was also evaluated for damage. Exposure to APAP resulted in severe liver damage, increased levels of serum alanine aminotransferase (ALT) and local MIP-2 expression. DF exposure did not cause visible liver damage, but did increase liver weight. DF also elicited clear effects on splenic innate and adaptive immune cells, i.e. increased levels of NK cells and memory T-cells. Furthermore, an increase in plasma MIP-2 levels combined with an influx of neutrophils into the spleen was observed. OFLX and CMZ exposure resulted in increased liver weights, MIP-2 expression and up-regulation of co-stimulatory molecules on antigen-presenting cells (APC). The data suggested that multiple immune parameters were altered upon exposure to drugs known to elicit immunosensitization and that broad evaluation of immune changes in straightforward short-term animal models is needed to determine whether a drug may harbor the hazard to induce IDHR. The oral exposure approach as used here may be applied in the future as an immunotoxicological research tool in this type of evaluation.

Intra- and inter-laboratory validation of an innovative huFcεRIα-RBL-2H3 degranulation assay for in vitro allergenicity assessment of whey hydrolysates.

Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine ß-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models.