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Background Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson’s disease (PD) pathogenesis, α-synuclein is a known culprit. Evidence for α-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these α-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of α-synuclein-specific immune responses in PD in a mouse model. Methods We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-α-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)\(^{-/-}\) mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4\(^{+}\)/CD8\(^{-}\), CD4\(^{-}\)/CD8\(^{+}\), or CD4\(^{+}\)/CD8\(^{+}\) (JHD\(^{-/-}\)) mice into the RAG-1\(^{-/-}\) mice was also employed. In addition to the in vivo studies, a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay was utilized. Results AAV-based overexpression of pathogenic human A53T-α-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a pro-inflammatory gene profile. T cell responses were directed against A53T-α-synuclein-peptides in the vicinity of position 53 (68–78) and surrounding the pathogenically relevant S129 (120–134). T cells were required for α-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration. Conclusions Using T cell and/or B cell deficient mice and a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic α-synuclein peptide-specific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology.

Multiple myeloma remains a largely incurable disease of clonally expanding malignant plasma cells. The bone marrow microenvironment harbors treatment-resistant myeloma cells, which eventually lead to disease relapse in patients. In the bone marrow, CD4+FoxP3+ regulatory T cells (Tregs) are highly abundant amongst CD4+ T cells providing an immune protective niche for different long-living cell populations, e.g., hematopoietic stem cells. Here, we addressed the functional role of Tregs in multiple myeloma dissemination to bone marrow compartments and disease progression. To investigate the immune regulation of multiple myeloma, we utilized syngeneic immunocompetent murine multiple myeloma models in two different genetic backgrounds. Analyzing the spatial immune architecture of multiple myeloma revealed that the bone marrow Tregs accumulated in the vicinity of malignant plasma cells and displayed an activated phenotype. In vivo Treg depletion prevented multiple myeloma dissemination in both models. Importantly, short-term in vivo depletion of Tregs in mice with established multiple myeloma evoked a potent CD8 T cell- and NK cell-mediated immune response resulting in complete and stable remission. Conclusively, this preclinical in-vivo study suggests that Tregs are an attractive target for the treatment of multiple myeloma.

European journal of immunology (2022). doi:10.1002/eji.202249816, Published by Wiley-VCH, Weinheim

The original concept stated that immature dendritic cells (DC) act tolerogenically whereas mature DC behave strictly immunogenically. Meanwhile, it is also accepted that phenotypically mature stages of all conventional DC subsets can promote tolerance as steady-state migratory DC by transporting self-antigens to lymph nodes to exert unique functions on regulatory T cells. We propose that in vivo 1) there is little evidence for a tolerogenic function of immature DC during steady state such as CD4 T cell anergy induction, 2) all tolerance as steady-state migratory DC undergo common as well as subset-specific molecular changes, and 3) these changes differ by quantitative and qualitative markers from immunogenic DC, which allows one to clearly distinguish tolerogenic from immunogenic migratory DC.

The critical role of suppressive myeloid cells in immune regulation has come to the forefront in cancer research, with myeloid-derived suppressor cells (MDSCs) as a main oncology immunotherapeutic target. Recent improvement and standardization of criteria classifying tumor-induced MDSCs have led to unified descriptions and also promoted MDSC research in tuberculosis (TB) and AIDS. Despite convincing evidence on the induction of MDSCs by pathogen-derived molecules and inflammatory mediators in TB and AIDS, very little attention has been given to their therapeutic modulation or roles in vaccination in these diseases. Clinical manifestations in TB are consequences of complex host-pathogen interactions and are substantially affected by HIV infection. Here we summarize the current understanding and knowledge gaps regarding the role of MDSCs in HIV and Mycobacterium tuberculosis (co)infections. We discuss key scientific priorities to enable application of this knowledge to the development of novel strategies to improve vaccine efficacy and/or implementation of enhanced treatment approaches. Building on recent findings and potential for cross-fertilization between oncology and infection biology, we highlight current challenges and untapped opportunities for translating new advances in MDSC research into clinical applications for TB and AIDS.

The development of two conventional dendritic cells (DC) subsets (cDC1 and cDC2) and the plasmacytoid DC (pDC) in vivo and in cultures of bone marrow (BM) cells is mediated by the growth factor Flt3L. However, little is known about the factors that direct the development of the individual DC subsets. Here, we describe the selective in vitro generation of murine ESAM

IL-4 and IL-13 cytokines have been associated with a non-healing phenotype in murine leishmaniasis in

Myeloid-derived suppressor cells (MDSC) appear at relatively low frequencies in diseased organs such as tumors or infection sites, but accumulate systemically in the spleen. So far MDSC have been reported in humans and experimental animals such as mice, rats, and nonhuman primates. Therefore, methods to generate MDSC in large amounts in vitro can serve as an additional tool to study their biology. Here, we describe in detail the generation of murine MDSC with GM-CSF from bone marrow (BM). Both subsets of granulocytic (G-MDSC) and monocytic MDSC (M-MDSC) are generated by this cytokine. We provide panels of phenotypic markers to distinguish them from non-suppressive cells and define developmental stages of monocytes developing into M-MDSC by two subsequent steps in vitro.

Myeloid-derived suppressor cells (MDSC) appear at relatively low frequencies in diseased organs such as tumors or infection sites, but accumulate systemically in the spleen. So far MDSC have been reported in humans and experimental animals such as mice, rats, and nonhuman primates. Therefore, methods to generate MDSC in large amounts in vitro can serve as an additional tool to study their biology. Here, we describe in detail the generation of murine MDSC with GM-CSF from bone marrow (BM). Both subsets of granulocytic (G-MDSC) and monocytic MDSC (M-MDSC) are generated by this cytokine. We provide panels of phenotypic markers to distinguish them from non-suppressive cells and define developmental stages of monocytes developing into M-MDSC by two subsequent steps in vitro.

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ+ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4+/CD8+ T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to > 40% reduction of regulatory T cells (Treg). In parallel to Treg-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8+VLA-4+ T-cells with CNS-homing properties, but not of CD4+ VLA-4+ T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses. Electronic supplementary material The online version of this article (10.1007/s00262-018-2214-0) contains supplementary material, which is available to authorized users.

Myeloid-derived suppressor cells (MDSC) are induced during active TB disease to restore immune homeostasis but instead exacerbate disease outcome due to chronic inflammation. Autophagy, in conventional phagocytes, ensures successful clearance of M.tb. However, autophagy has been demonstrated to induce prolonged MDSC survival. Here we investigate the relationship between autophagy mediators and MDSC in the context of active TB disease and during anti-TB therapy. We demonstrate a significant increase in MDSC frequencies in untreated active TB cases with these MDSC expressing TLR4 and significantly more mTOR and IL-6 than healthy controls, with mTOR levels decreasing during anti-TB therapy. Finally, we show that HMGB1 serum concentrations decrease in parallel with mTOR. These findings suggest a complex interplay between MDSC and autophagic mediators, potentially dependent on cellular localisation and M.tb infection state.

Myeloid-derived suppressor cells (MDSC) are important immune-regulatory cells but their identification remains difficult. Here, we provide a critical view on selected surface markers, transcriptional and translational pathways commonly used to identify MDSC by specific, their developmental origin and new possibilities by transcriptional or proteomic profiling. Discrimination of MDSC from their non-suppressive counterparts is a prerequisite for the development of successful therapies. Understanding the switch mechanisms that direct granulocytic and monocytic development into a pro-inflammatory or anti-inflammatory direction will be crucial for therapeutic strategies. Manipulation of these myeloid checkpoints are exploited by tumors and pathogens, such as M. tuberculosis (Mtb), HIV or SARS-CoV-2, that induce MDSC for immune evasion. Thus, specific markers for MDSC identification may reveal also novel molecular candidates for therapeutic intervention at the level of MDSC.

Increased disease susceptibility during early life has been linked to immune immaturity, regulatory T-cell/TH2 immune biasing and hyporesponsiveness. The contribution of myeloid derived suppressor cells (MDSCs) remains uninvestigated. Here, we assessed peripheral MDSC in HIV-infected and -uninfected children with tuberculosis (TB) disease before, during and after TB treatment, along with matched household contacts (HHCs), HIV-exposed, -infected and -uninfected children without recent TB exposure. Serum analytes and enzymes associated with MDSC accumulation/activation/function were measured by colorimetric- and fluorescence arrays. Peripheral frequencies of cells phenotypically resembling MDSCs were significantly increased in HIV-exposed uninfected (HEU) and M.tb-infected children, but peaked in children with TB disease and remained high following treatment. MDSC in HIV-infected (HI) children were similar to unexposed uninfected controls; however, HAART-mediated MDSC restoration to control levels could not be disregarded. Increased MDSC frequencies in HHC coincided with enhanced indoleamine-pyrrole-2,3-dioxygenase (IDO), whereas increased MDSC in TB cases were linked to heightened IDO and arginase-1. Increased MDSC were paralleled by reduced plasma IP-10 and thrombospondin-2 levels in HEU and significantly increased plasma IL-6 in HI HHC. Current investigations into MDSC-targeted treatment strategies, together with functional analyses of MDSCs, could endorse these cells as novel innate immune regulatory mechanism of infant HIV/TB susceptibility.

Success of DC vaccines relies on the quality of antigen presentation, costimulation, lymph node migration, and the release of IL-12, in case of Th1 priming. Here, we provide evidence for interaction between the injected vaccine DCs with endogenous lymph node–resident DCs for Th1 induction. While migration of the injected DCs was essential for antigen delivery to the lymph node, the injected DCs contributed only partially to Th0 priming and were unable to instruct Th1 generation. Instead, we provide evidence that the lymph node–resident XCR1+ DCs are activated by the injected DCs to present the cognate antigen and release IL-12 for Th1 polarization. The timing of interactions in the draining lymph nodes appeared step-wise as (a) injected DCs with cognate T cells, (b) injected DCs with bystander DCs, and (c) bystander DCs with T cells. The transcriptome of the bystander DCs showed a downregulation of Treg- and Th2/Th9-inducing genes and self-antigen presentation, as well as upregulation of MHC class II and genes required for Th1 instruction. Together, these data show that injected mature lymph node migratory DCs direct T cell priming and bystander DC activation, but not Th1 polarization, which is mediated by endogenous IL-12p70+XCR1+ resident bystander DCs. Our results are of importance for clinical DC-based vaccinations against tumors where endogenous DCs may be functionally impaired by chemotherapy., Successful Th1 priming by DC vaccines in mice depends on IL-12 from endogenous and XCR1+ cDC1 population.

Mycobacterium tuberculosis (M.tb) is likely the most successful human pathogen, capable of evading protective host immune responses and driving metabolic changes to support its own survival and growth. Ineffective innate and adaptive immune responses inhibit effective clearance of the bacteria from the human host, resulting in the progression to active TB disease. Many regulatory mechanisms exist to prevent immunopathology, however, chronic infections result in the overproduction of regulatory myeloid cells, like myeloid-derived suppressor cells (MDSC), which actively suppress protective host T lymphocyte responses among other immunosuppressive mechanisms. The mechanisms of M.tb internalization by MDSC and the involvement of host-derived lipid acquisition, have not been fully elucidated. Targeted research aimed at investigating MDSC impact on phagocytic control of M.tb, would be advantageous to our collective anti-TB arsenal. In this review we propose a mechanism by which M.tb may be internalized by MDSC and survive via the manipulation of host-derived lipid sources. Keywords: Lipid metabolism, Caveolin, Mycobacterium tuberculosis, Myeloid-derived suppressor cells, Internalization

Background Alveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a chronic zoonosis associated with significant modulation of the host immune response. A role of regulatory T-cells (Treg) in generating an immunosuppressive environment around the metacestode during chronic disease has been reported, but the molecular mechanisms of Treg induction by E. multilocularis, particularly parasite immunoregulatory factors involved, remain elusive so far. Methodology/Principal Findings We herein demonstrate that excretory/secretory (E/S) products of the E. multilocularis metacestode promote the formation of Foxp3+ Treg from CD4+ T-cells in vitro in a TGF-β-dependent manner, given that this effect was abrogated by treatment with antibody to mammalian TGF-β. We also show that host T-cells secrete elevated levels of the immunosuppressive cytokine IL-10 in response to metacestode E/S products. Within the E/S fraction of the metacestode we identified an E. multilocularis activin A homolog (EmACT) that displays significant similarities to mammalian Transforming Growth Factor-β (TGF-β/activin subfamily members. EmACT obtained from heterologous expression failed to directly induce Treg expansion from naïve T cells but required addition of recombinant host TGF-β to promote CD4+ Foxp3+ Treg conversion in vitro. Furthermore, like in the case of metacestode E/S products, EmACT-treated CD4+ T-cells secreted higher levels of IL-10. These observations suggest a contribution of EmACT to in vitro expansion of Foxp3+ Treg by the E. multilocularis metacestode. Using infection experiments we show that intraperitoneally injected metacestode tissue expands host Foxp3+ Treg, confirming the expansion of this cell type in vivo during parasite establishment. Conclusion/Significance In conclusion, we herein demonstrate that E. multilocularis larvae secrete factors that induce the secretion of IL-10 by T-cells and contribute to the expansion of TGF-b-driven Foxp3+ Treg, a cell type that has been reported crucial for generating a tolerogenic environment to support parasite establishment and proliferation. Among the E/S factors of the parasite we identified a factor with structural and functional homologies to mammalian activin A which might play an important role in these activities.

Tuberculosis patients and mice infected with live Mycobacterium tuberculosis accumulate high numbers of myeloid-derived suppressor cells (MDSCs). Here, we hypothesized that dead M. tuberculosis vaccines also may induce MDSCs that could impair the efficacy of vaccination. We found that repeated injections of M. tuberculosis vaccines (heat-killed M. tuberculosis in incomplete Freund’s adjuvant, such as Montanide) but not single or control vaccines without M. tuberculosis strongly expanded CD11b(+) myeloid cells in the spleen, leading to T cell suppression of proliferation and killing ex vivo. Dead M. tuberculosis vaccination induced the generation of CD11b(+)Ly6C(hi)CD115(+) iNOS/Nos2(+) monocytic MDSCs (M-MDSCs) upon application of inflammatory or microbial activation signals. In vivo these M-MDSCs were positioned strategically in the splenic bridging channels and then positioned in the white pulp areas. Notably, within 6–24 hours, in a Nos2-dependent fashion, they produced NO to rapidly kill conventional and plasmacytoid DCs while, surprisingly, sparing T cells in vivo. Thus, we demonstrate that M. tuberculosis vaccine induced M-MDSCs do not directly suppress effector T cells in vivo but, instead, indirectly by killing DCs. Collectively, we demonstrate that M. tuberculosis booster vaccines induce M-MDSCs in the spleen that can be activated to kill DCs. Our data suggest that formation of MDSCs by M. tuberculosis vaccines should be investigated also in clinical trials.

BackgroundAlveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a chronic zoonosis associated with significant modulation of the host immune response. A role of regulatory T-cells (Treg) in generating an immunosuppressive environment around the metacestode during chronic disease has been reported, but the molecular mechanisms of Treg induction by E. multilocularis remain elusive so far.Methodology/Principal findingsWe herein demonstrate that excretory/secretory (E/S) products of the E. multilocularis metacestode promote the formation of Foxp3+ Treg from CD4+ T-cells in vitro in a TGF-β-dependent manner. We also show that host T-cells secrete elevated levels of the immunosuppressive cytokine IL-10 in response to metacestode E/S products. Within the E/S fraction of the metacestode we identified an E. multilocularis activin A homolog (EmACT) that displays significant similarities to mammalian Transforming Growth Factor-β (TGF-β)/activin subfamily members. EmACT obtained from heterologous expression promoted host TGF-β-driven CD4+ Foxp3+ Treg conversion in vitro. Furthermore, like in the case of metacestode E/S products, EmACT-treated CD4+ T-cells secreted higher levels of IL-10. These observations suggest a contribution of EmACT in the in vitro expansion of Foxp3+ Treg by the E. multilocularis metacestode. Using infection experiments we show that intraperitoneally injected metacestode tissue expands host Foxp3+ Treg, confirming the expansion of this cell type in vivo during parasite establishment.Conclusions/SignificanceIn conclusion, we herein show that E. multilocularis larvae secrete a factor with clear structural and functional homologies to mammalian activin A. Like its mammalian homolog, this protein induces the secretion of IL-10 by T-cells and contributes to the expansion of TGF-β-driven Foxp3+ Treg, a cell type that has been reported crucial for generating a tolerogenic environment to support parasite establishment and proliferation.AUTHOR SUMMARYThe metacestode larval stage of the tapeworm E. multilocularis grows infiltratively, like a malignant tumor, within the organs of its human host, thus causing the lethal disease alveolar echinococcosis (AE). Immunosuppression plays an important role in both survival and proliferation of the metacestode, which mainly depends on factors that are released by the parasite. These parasite-derived molecules are potential targets for developing new anti-echinococcosis drugs and/or improving the effectiveness of current therapies. Additionally, an optimized use of such factors could help minimize pathologies resulting from over-reactive immune responses, like allergies and autoimmune diseases. The authors herein demonstrate that the E. multilocularis metacestode releases a protein, EmACT, with significant homology to activin A, a cytokine that might support host TGF-β in its ability to induce the generation of immunosuppressive regulatory T-cells (Treg) in mammals. Like its mammalian counterpart, EmACT was associated with the expansion of TGF-β-induced Treg and stimulated the release of elevated amounts of immunosuppressive IL-10 by CD4+ T-cells. The authors also demonstrate that Treg are locally expanded by the metacestode during an infection of mice. These data confirm an important role of Treg for parasite establishment and growth during AE and suggest a potential role of EmACT in the expansion of these immunosuppressive cells around the parasite.

Dendritic cells (DCs) are considered to play major roles during the induction of T cell immune responses as well as the maintenance of T cell tolerance. Naive CD4(+) T cells have been shown to respond with high plasticity to signals inducing their polarization into effector/helper or regulatory T cells. Data obtained from in vitro generated bone-marrow (BM)-derived DCs as well as genetic mouse models revealed an important but not exclusive role of DCs in shaping CD4(+) T cell responses. Besides the specialization of some conventional DC subsets for the induction of polarized immunity, also the maturation stage, activation of specialized transcription factors and the cytokine production of DCs have major impact on CD4(+) T cells. Since in vitro generated BM-DCs show a high diversity to shape CD4(+) T cells and their high similarity to monocyte-derived DCs in vivo, this review reports data mainly on BM-DCs in this process and only touches the roles of transcription factors or of DC subsets, which have been discussed elsewhere. Here, recent findings on 1) the conversion of naive into anergic and further into Foxp3(-) regulatory T cells (Treg) by immature DCs, 2) the role of RelB in steady state migratory DCs (ssmDCs) for conversion of naive T cells into Foxp3(+) Treg, 3) the DC maturation signature for polarized Th2 cell induction and 4) the DC source of IL-12 for Th1 induction are discussed.

CLN diseases are mostly fatal lysosomal storage diseases that lead to neurodegeneration in the CNS. We have previously shown that CD8+ T-lymphocytes contribute to axonal perturbation and neuron loss in the CNS of Ppt1(-/-) mice, a model of CLN1 disease. We now investigated the role of the inflammation-related cell adhesion molecule sialoadhesin (Sn) in Ppt1(-/-) and Cln3(-/-) mice, a model of the most frequent form, CLN3 disease. Microglia/macrophages in the CNS of both models showed an upregulation of Sn and markers for proinflammatory M1 polarization and antigen presentation. Sn+ microglia/macrophages associated with SMI32+ axonal spheroids and CD8+ T-lymphocytes. To analyze their pathogenic impact, we crossbred both models with Sn-deficient mice and scored axonal degeneration and neuronal integrity using immunohistochemistry, electron microscopy and optical coherence tomography. Degenerative alterations in the retinotectal pathway of Ppt1(-/-)Sn(-/-) and Cln3(-/-)Sn(-/-) mice were significantly reduced. Ppt1(-/-)Sn(-/-) mice also showed a substantially improved clinical phenotype and extended lifespan, attenuated numbers of M1-polarized microglia/macrophages and reduced expression levels of proinflammatory cytokines. This was accompanied by an increased frequency of CD8+CD122+ T-lymphocytes in the CNS of Ppt1(-/-)Sn(-/-) mice, the regulatory phenotype of which was demonstrated by impaired survival of CD8+CD122- effector T-lymphocytes in co-culture experiments. We show for the first time that increased Sn expression on microglia/macrophages contributes to neural perturbation in two distinct models of CLN disease. Our data also indicate that a rarely described CD8+CD122+ T-cell population can regulate the corresponding diseases. These studies provide insights into CLN pathogenesis and may guide in designing immuno-regulatory treatment strategies.

Aspergillus fumigatus is the main cause of invasive fungal infections occurring almost exclusively in immunocompromised patients. An improved understanding of the initial innate immune response is key to the development of better diagnostic tools and new treatment options. Mice are commonly used to study immune defense mechanisms during the infection of the mammalian host with A. fumigatus. However, little is known about functional differences between the human and murine immune response against this fungal pathogen. Thus, we performed a comparative functional analysis of human and murine dendritic cells (DCs), macrophages, and polymorphonuclear cells (PMNs) using standardized and reproducible working conditions, laboratory protocols, and readout assays. A. fumigatus did not provoke identical responses in murine and human immune cells but rather initiated relatively specific responses. While human DCs showed a significantly stronger upregulation of their maturation markers and major histocompatibility complex molecules and phagocytosed A. fumigatus more efficiently compared to their murine counterparts, murine PMNs and macrophages exhibited a significantly stronger release of reactive oxygen species after exposure to A. fumigatus. For all studied cell types, human and murine samples differed in their cytokine response to conidia or germ tubes of A. fumigatus. Furthermore, Dectin-1 showed inverse expression patterns on human and murine DCs after fungal stimulation. These specific differences should be carefully considered and highlight potential limitations in the transferability of murine host–pathogen interaction studies.

Influenza A virus (IAV) infection causes an acute respiratory disease characterized by a strong inflammatory immune response and severe immunopathology. Proinflammatory mechanisms are well described in the murine IAV infection model, but less is known about the mechanisms leading to the resolution of inflammation. Here, we analyzed the contribution of CD11b\(^{+}\)Ly6C\(^{++}\)Ly6G\(^{-}\) cells to this process. An accumulation of CD11b\(^{+}\)Ly6C\(^{++}\)Ly6G\(^{-}\) cells within the lungs was observed during the course of IAV infection. Phenotypic characterization of these CD11b\(^{+}\)Ly6C\(^{++}\)Ly6G\(^{-}\) cells by flow cytometry and RNA-Seq revealed an activated phenotype showing both pro- and anti-inflammatory features, including the expression of inducible nitric oxide synthase (iNOS) by a fraction of cells in an IFN-γ-dependent manner. Moreover, CD11b\(^{+}\)Ly6C\(^{++}\)Ly6G\(^{-}\) cells isolated from lungs of IAV-infected animals displayed suppressive activity when tested in vitro, and iNOS inhibitors could abrogate this suppressive activity. Collectively, our data suggest that during IAV infection, CD11b\(^{+}\)Ly6C\(^{++}\)Ly6G\(^{-}\) cells acquire immunoregulatory function, which might contribute to the prevention of pathology during this life-threatening disease.

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide-specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL-10(-) Foxp3(-) CTLA-4(+) CD25(low) Egr2(+) T cells could be converted into suppressive IL-10(+) Foxp3(-) CTLA-4(+) CD25(high) Egr2(+) cells resembling type-1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF-β during anergy induction favored Foxp3(+) Treg-cell induction, while TGF-β had little effect when added to the second stimulation. Expression of both CD28 and CTLA-4 molecules on anergic T cells was required to allow their conversion into Tr1-like cells. Suppressor activity was enabled via CD28-mediated CD25 upregulation, acting as an IL-2 sink, together with a CTLA-4-mediated inhibition of NFATc1/α activation to shut down IL-2-mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1-like cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls proliferation and survival of myeloid cells including monocytes. Here, we describe a time-dependent licensing process driven by GM-CSF in murine Ly6Chigh and human CD14+ monocytes that disables their inflammatory functions and promotes their conversion into suppressor cells. This 2-step licensing of monocytes requires activation of the AKT/mTOR/mTORC1 signaling cascade by GM-CSF followed by signaling through the interferon-γ receptor (IFN-γR)/interferon regulatory factor-1 (IRF-1) pathway. Only licensing-dependent adaptations in Toll-like receptor/inflammasome, IFN-γR, and phosphatidylinositol 3-kinase/AKT/mTOR signaling lead to stabilized expression of inducible nitric oxide synthase by mouse and indoleamine 2,3-dioxygenase (IDO) by human monocytes, which accounts for their suppressor activity. This study suggests various myeloid cells with characteristics similar to those described for monocytic myeloid-derived suppressor cells, Mreg, or suppressor macrophages may arise from licensed monocytes. Markers of GM-CSF-driven monocyte licensing, including p-Akt, p-mTOR, and p-S6, distinguish inflammatory monocytes from potentially suppressive monocytes in peripheral blood of patients with high-grade glioma.

Immature or semi-mature dendritic cells (DCs) represent tolerogenic maturation stages that can convert naive T cells into Foxp3\(^{+}\) induced regulatory T cells (iTreg). Here we found that murine bone marrow-derived DCs (BM-DCs) treated with cholera toxin (CT) matured by up-regulating MHC-II and costimulatory molecules using either high or low doses of CT (CT\(^{hi}\), CT\(^{lo}\)) or with cAMP, a known mediator CT signals. However, all three conditions also induced mRNA of both isoforms of the tolerogenic molecule cytotoxic T lymphocyte antigen 2 (CTLA-2α and CTLA-2β). Only DCs matured under CT\(^{hi}\) conditions secreted IL-1β, IL-6 and IL-23 leading to the instruction of Th17 cell polarization. In contrast, CT\(^{lo}\)- or cAMP-DCs resembled semi-mature DCs and enhanced TGF-β-dependent Foxp3\(^{+}\) iTreg conversion. iTreg conversion could be reduced using siRNA blocking of CTLA-2 and reversely, addition of recombinant CTLA-2α increased iTreg conversion in vitro. Injection of CT\(^{lo}\)- or cAMP-DCs exerted MOG peptide-specific protective effects in experimental autoimmune encephalomyelitis (EAE) by inducing Foxp3\(^{+}\) Tregs and reducing Th17 responses. Together, we identified CTLA-2 production by DCs as a novel tolerogenic mediator of TGF-β-mediated iTreg induction in vitro and in vivo. The CT-induced and cAMP-mediated up-regulation of CTLA-2 also may point to a novel immune evasion mechanism of Vibrio cholerae.

International audience; BACKGROUND:Using potential surrogate end-points for overall survival (OS) such as Disease-Free- (DFS) or Progression-Free Survival (PFS) is increasingly common in randomised controlled trials (RCTs). However, end-points are too often imprecisely defined which largely contributes to a lack of homogeneity across trials, hampering comparison between them. The aim of the DATECAN (Definition for the Assessment of Time-to-event End-points in CANcer trials)-Pancreas project is to provide guidelines for standardised definition of time-to-event end-points in RCTs for pancreatic cancer.METHODS:Time-to-event end-points currently used were identified from a literature review of pancreatic RCT trials (2006-2009). Academic research groups were contacted for participation in order to select clinicians and methodologists to participate in the pilot and scoring groups (>30 experts). A consensus was built after 2 rounds of the modified Delphi formal consensus approach with the Rand scoring methodology (range: 1-9).RESULTS:For pancreatic cancer, 14 time to event end-points and 25 distinct event types applied to two settings (detectable disease and/or no detectable disease) were considered relevant and included in the questionnaire sent to 52 selected experts. Thirty experts answered both scoring rounds. A total of 204 events distributed over the 14 end-points were scored. After the first round, consensus was reached for 25 items; after the second consensus was reached for 156 items; and after the face-to-face meeting for 203 items.CONCLUSION:The formal consensus approach reached the elaboration of guidelines for standardised definitions of time-to-event end-points allowing cross-comparison of RCTs in pancreatic cancer.

Removal of apoptotic cells, which appear during the steady state, is a pre-requisite to prevent generation of secondary necrotic cells that may lead to autoimmunity. The recognition of apoptotic material by dendritic cells (DCs) has been proposed to convert them into tolerogenic DCs equipped with specialized tolerogenic mechanisms on T cells. However, comparative studies to demonstrate functional alterations of DCs upon exposure to apoptotic cells have not been performed so far. Here we show that immature murine bone marrow-derived DCs generated with GM-CSF (GM-DCs) or Flt3L (FL-DCs) interact with live or apoptotic syngeneic thymocytes. As expected, GM-DCs phagocytose apoptotic but not live cells, FL-DCs only show trogocytosis of membrane parts. Interaction with live or apoptotic thymocytes did not lead to DC maturation. Both GM-DCs and FL-DCs present OVA as protein, peptide and membrane-associated antigens. Interestingly, only GM-DCs were able to induce T cell anergy or convert naïve T cells into FoxP3⁺ regulatory T cells (Tregs) but FL-DCs did not show either of these effects. Unexpectedly, exposure of immature GM-DCs to live or apoptotic thymocytes did not improve DC functions in both types of in vitro T cell tolerance induction assays. Together, our data suggest that these tolerogenic in vitro measures of immature BM-DCs are not further enhanced by exposure to apoptotic cells and may depend on the generating cytokine.

Mature dendritic cells (DC) secrete substantial amounts of vesicles that are primarily ingested by monocytes, leading to differentiation processes in these target cells towards monocyte-derived DC., During inflammation, murine and human monocytes can develop into dendritic cells (DC), but this process is not entirely understood. Here, we demonstrate that extracellular vesicles (EV) secreted by mature human DC (maDC) differentiate peripheral monocytes into immature DC, expressing a unique marker pattern, including 6-sulfo LacNAc (slan), Zbtb46, CD64, and CD14. While EV from both maDC and immature DC differentiated monocytes similar to GM-CSF/IL-4 stimulation, only maDC-EV produced precursors, which upon maturation stimulus developed into T-cell–activating and IL-12p70–secreting maDC. Mechanistically, maDC-EV induced cell signaling through GM-CSF, which was abundant in EV as were IL-4 and other cytokines and chemokines. When injected into the mouse skin, murine maDC-EV attracted immune cells including monocytes that developed activation markers typical for inflammatory cells. Skin-injected EV also reached lymph nodes, causing a similar immune cell infiltration. We conclude that DC-derived EV likely serve to perpetuate an immune reaction and may contribute to chronic inflammation.

Denileukin diftitox (DD), a diphtheria toxin fragment IL-2 fusion protein, is thought to target and kill CD25(+) cells. It is approved for the treatment of cutaneous T-cell lymphoma and is used experimentally for the depletion of regulatory T cells (Treg) in cancer trials. Curiously enough, clinical effects of DD did not strictly correlate with CD25 expression, and Treg depletion was not confirmed unambiguously. Here, we report that patients with melanoma receiving DD immediately before a dendritic cell (DC) vaccine failed to develop a tumor-antigen-specific CD4 and CD8 T-cell immune response even after repeated vaccinations. Analyzing the underlying mechanism, so far we found unknown effects of DD. First, DD modulated DCs toward tolerance by downregulating costimulatory receptors such as CD83 and CD25 while upregulating tolerance-associated proteins/pathways including Stat-3, β-catenin, and class II transactivator-dependent antigen presentation. Second, DD blocked Stat3 phosphorylation in maturing DCs. Third, only activated, but not resting, Treg internalized DD and were killed. Conversely, resting Treg showed increased survival because of DD-mediated antiapoptotic IL-2 signaling. We conclude that DD exerts functions beyond CD25(+) cell killing that may affect their clinical use and could be tested for novel indications.

Background Ovarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine on myeloid cells. Considering that tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) constitute up to 20 % of OvCA tissue, we investigated the effect of adenosine on myeloid cells and explored a possible contribution of myeloid cells to adenosine generation in vitro and ex vivo. Methods Monocytes were used as human blood-derived myeloid cells. After co-incubation with SK-OV-3 or OAW-42 OvCA cells, monocyte migration was determined in transwell assays. For conversion into M2-polarized “TAM-like” macrophages, monocytes were co-incubated with OAW-42 cells. Ex vivo TAMs were obtained from OvCA ascites. Macrophage phenotypes were investigated by intracellular staining for IL-10 and IL-12. CD39 and CD73 expression were assessed by FACS analysis both on in vitro-induced TAM-like macrophages and on ascites-derived ex situ-TAMs. Myeloid cells in solid tumor tissue were analyzed by immunohistochemistry. Generation of biologically active adenosine by TAM-like macrophages was measured in luciferase-based reporter assays. Functional effects of adenosine were investigated in proliferation-experiments with CD4+ T cells and specific inhibitors. Results When CD39 or CD73 activity on OvCA cells were blocked, the migration of monocytes towards OvCA cells was significantly decreased. In vivo, myeloid cells in solid ovarian cancer tissue were found to express CD39 whereas CD73 was mainly detected on stromal fibroblasts. Ex situ-TAMs and in vitro differentiated TAM-like cells, however, upregulated the expression of CD39 and CD73 compared to monocytes or M1 macrophages. Expression of ectonucleotidases also translated into increased levels of biologically active adenosine. Accordingly, co-incubation with these TAMs suppressed CD4+ T cell proliferation which could be rescued via blockade of CD39 or CD73. Conclusion Adenosine generated by OvCA cells likely contributes to the recruitment of TAMs which further amplify adenosine-dependent immunosuppression via additional ectonucleotidase activity. In solid ovarian cancer tissue, TAMs express CD39 while CD73 is found on stromal fibroblasts. Accordingly, small molecule inhibitors of CD39 or CD73 could improve immune responses in ovarian cancer. Electronic supplementary material The online version of this article (doi:10.1186/s40425-016-0154-9) contains supplementary material, which is available to authorized users.