Search

MicroRNAs (miR) are small noncoding RNAs that regulate gene expression, posttranscription, and manipulate immune responses in different types of cancers. In this study, we identify miR-146a as a negative regulator of immune activation, comparable to immune-checkpoint molecules. miR-146a levels were increased in melanoma microenvironmental tissue, and miR-146a−/− mice survived longer and developed less metastases in comparison with wild-type melanoma-bearing mice. T cells isolated from miR-146a−/− mice revealed higher expression levels of the miR-146a target gene Stat1 and the Stat1-regulated cytokine IFNγ. Neutralization of IFNγ in miR-146a−/− mice decreased survival and increased melanoma metastasis patterns to those of wild-type mice. In vitro, IFNγ reduced melanoma cell migration, cell-cycle activity, and basal metabolic rate. Conversely, IFNγ also increased PD-L1 levels on the melanoma cells, which may counterbalance some of the beneficial effects increasing immune escape in vivo. Combined treatment with a miR-146a antagomiR and anti–PD-1 resulted in improved survival over isotype control or anti–PD-1 treatment alone. In summary, these data show that miR-146a plays a central role within the STAT1/IFNγ axis in the melanoma microenvironment, affecting melanoma migration, proliferation, and mitochondrial fitness as well as PD-L1 levels. Additionally, combined inhibition of PD-1 and miR-146a could be a novel strategy to enhance antitumor immune response elicited by checkpoint therapy. Significance: These findings identify a microRNA–based mechanism by which melanoma cells escape the immune system, providing a new therapeutic strategy to improve the current management of patients with melanoma.

Introduction Complete Freund's Adjuvant (CFA) emulsified with an antigen is a widely used method to induce autoimmune disease in animal models, yet the contribution of CFA to the immune response is not well understood. We compared the effectiveness of CFA with Incomplete Freund's Adjuvant (IFA) or TiterMax Gold Adjuvant (TMax) in experimental autoimmune myocarditis (EAM) in male mice. Methods EAM was induced in A/J, BALB/c, and IL6KO BALB/c male mice by injection of the myocarditogenic peptide in CFA, IFA, or TMax on days 0 and 7. EAM severity was analyzed by histology on day 21. In addition, specific flow cytometry outcomes were evaluated on day 21. Results Only mice immunized with CFA and myocarditogenic peptide on both days 0 and 7 developed substantial myocarditis as measured by histology. We observed a significantly increased level of IL6 in the spleen 3 days after CFA immunization. In the spleen and heart on day 21, there was an expansion of myeloid cells in CFA-immunized mice, as compared to IFA or TMax-immunized animals. Recombinant IL-6 at the time of IFA immunization partially restored susceptibility of the mice to EAM. We also treated EAM-resistant IL-6 knockout mice with recombinant IL-6 around the time of the first immunization, on days −1 to 2, completely restoring disease susceptibility, showing that the requirement for IL-6 coincides with primary immunization. Examining APC populations in the lymph node draining the immunization site evidenced the contribution of IL-6 to the CFA-dependence of EAM was through controlling local dendritic cell (DC) trafficking. Conclusions CFA used with myocarditogenic peptide twice is required to induce EAM in both A/J and Balb/c mice. Although IFA and TiterMax induce antibody responses, only CFA preferentially induced autoantigen-specific responses. CFA expands monocytes in the heart and in the spleen. IL-6 signaling is required during short window around primary immunization to induce EAM. In addition, IL-6 deficient mice resistance to EAM could be reversed by injecting IL-6 around first immunization. IL-6 expands dendritic cell and monocytic populations and ultimately leads to a robust T-cell driven immune response in CFA immunized mice.

Acute Graft-versus-host disease (GVHD) is a major immunological complication after allogeneic hematopoietic cell transplantation and a better understanding of the molecular regulation of the disease could help to develop novel targeted therapies. Here we found that a G/C polymorphism within the human microRNA-146a (miR-146a) gene of transplant recipients, which causes reduced miR-146a levels, was strongly associated with the risk of developing severe acute GVHD (n=289). In mice, deficiency of miR-146a in the hematopoietic system or transfer of recipient-type miR-146a-/- dendritic cells (DCs) enhanced GVHD, while miR-146a mimic-transfected DCs ameliorated disease. Mechanistically, lack of miR-146a enhanced JAK2-STAT1 pathway activity, which led to higher expression of class II-transactivator (CIITA) and consecutively increased MHCII-levels on DCs. Inhibition of JAK1/2 or CIITA knockdown in DCs prevented miR-146a-/- DC-induced GVHD exacerbation. Consistent with our findings in mice, patients with the miR-146a polymorphism rs2910164 in hematopoietic cells displayed higher MHCII levels on monocytes, which could be targeted by JAK1/2 inhibition. Our findings indicate that the miR-146a polymorphism rs2910164 identifies patients at high risk for GVHD before allo-HCT. Functionally we show that miR-146a acts as a central regulator of recipient-type DC activation during GVHD by dampening the pro-inflammatory JAK-STAT/CIITA/MHCII axis, which provides a scientific rationale for early JAK1/2 inhibition in selected patients.

Acute graft-versus-host disease (GVHD) limits the success of allogeneic hematopoietic cell transplantation (allo-HCT); therefore, a better understanding of its biology may improve therapeutic options. We observed miR-146a up-regulation in T cells of mice developing acute GVHD compared with untreated mice. Transplanting miR-146a(-/-) T cells caused increased GVHD severity, elevated tumor necrosis factor (TNF) serum levels, and reduced survival. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was up-regulated in miR-146a(-/-) T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased nuclear factor-κB activity and TNF production in miR-146a(-/-) T cells. Conversely, the detrimental effect of miR-146a deficiency in T cells was antagonized by TNF blockade, whereas phytochemical induction of miR-146a or its overexpression using a miR-146a mimic reduced GVHD severity. In humans, the minor genotype of the single nucleotide polymorphism rs2910164 in HCT donors, which reduces expression of miR-146a, was associated with severe acute GVHD (grade III/IV). We show that miR-146a functions as a negative regulator of donor T cells in GVHD by targeting TRAF6, leading to reduced TNF transcription. Because miR-146a expression can be exogenously enhanced, our results provide a novel targeted molecular approach to mitigate GVHD.

Podocyte loss is a major determinant of progressive CKD. Although recent studies showed that a subset of parietal epithelial cells can serve as podocyte progenitors, the role of podocyte turnover and regeneration in repair, aging, and nephron loss remains unclear. Here, we combined genetic fate mapping with highly efficient podocyte isolation protocols to precisely quantify podocyte turnover and regeneration. We demonstrate that parietal epithelial cells can give rise to fully differentiated visceral epithelial cells indistinguishable from resident podocytes and that limited podocyte renewal occurs in a diphtheria toxin model of acute podocyte ablation. In contrast, the compensatory programs initiated in response to nephron loss evoke glomerular hypertrophy, but not de novo podocyte generation. In addition, no turnover of podocytes could be detected in aging mice under physiologic conditions. In the absence of podocyte replacement, characteristic features of aging mouse kidneys included progressive accumulation of oxidized proteins, deposits of protein aggregates, loss of podocytes, and glomerulosclerosis. In summary, quantitative investigation of podocyte regeneration in vivo provides novel insights into the mechanism and capacity of podocyte turnover and regeneration in mice. Our data reveal that podocyte generation is mainly confined to glomerular development and may occur after acute glomerular injury, but it fails to regenerate podocytes in aging kidneys or in response to nephron loss.

CD4+ T cells play a central role in inflammatory heart disease, implicating a cytokine product associated with Th cell effector function as a necessary mediator of this pathophysiology. IFN-γ–deficient mice developed severe experimental autoimmune myocarditis (EAM), in which mice are immunized with cardiac myosin peptide, whereas IL-17A–deficient mice were protected from progression to dilated cardiomyopathy. We generated IFN-γ−/−IL-17A−/− mice to assess whether IL-17 signaling was responsible for the severe EAM of IFN-γ−/− mice. Surprisingly, IFN-γ−/−IL-17A−/− mice developed a rapidly fatal EAM. Eosinophils constituted a third of infiltrating leukocytes, qualifying this disease as eosinophilic myocarditis. We found increased cardiac production of CCL11/eotaxin, as well as Th2 deviation, among heart-infiltrating CD4+ cells. Ablation of eosinophil development improved survival of IFN-γ−/−IL-17A−/− mice, demonstrating the necessity of eosinophils in fatal heart failure. The severe and rapidly fatal autoimmune inflammation that developed in the combined absence of IFN-γ and IL-17A constitutes a novel model of eosinophilic heart disease in humans. This is also, to our knowledge, the first demonstration that eosinophils have the capacity to act as necessary mediators of morbidity in an autoimmune process.

Direct contact with stromal cells protects chronic lymphocytic leukaemia (CLL) B cells from chemotherapy-induced apoptosis in vitro. Blockade of CXCR4 signalling antagonizes stroma-mediated interactions and restores CLL chemosensitivity. In vivo, administration of CXCR4 antagonists effectively mobilizes haematopoietic progenitor cells. Therefore, combinations of CXCR4 blockade and cytoreductive treatment with selective activity on CLL cells may avoid potential haematotoxicity. Hence, we tested CXCR4 antagonists in the context of passive and active immunotherapeutic approaches. We evaluated how efficiently rituximab, alemtuzumab and cytotoxic T cells killed CLL cells cocultured with stromal cells in the presence and absence of a CXCR4 antagonist. Stromal cell contact attenuated rituximab- and alemtuzumab-induced complement-dependent cytotoxicity of CLL cells. Addition of CXCR4 antagonists abrogated the protective effect of stroma. In contrast, stromal cells did not impair antibody-dependent cell-mediated cytotoxicity and cytotoxicity induced by activated T cells. Destruction of microtubules in CLL target cells restored the protective effect of stroma coculture for CLL cells during Natural Killer cell attack by preventing mitochondrial relocalization towards the immunological synapse. Our data identify the combination of CXCR4 antagonists with passive - but not active - immunotherapy as a promising potential treatment concept in CLL.

Conditioning therapies before transplantation induce the release of uric acid, which triggers the NLRP3 inflammasome and IL-1β production contributing to graft-versus-host disease., The success of allogeneic hematopoietic cell transplantation is limited by acute graft-versus-host disease (GvHD), a severe complication accompanied by high mortality rates. Yet, the molecular mechanisms initiating this disease remain poorly defined. In this study, we show that, after conditioning therapy, intestinal commensal bacteria and the damage-associated molecular pattern uric acid contribute to Nlrp3 inflammasome–mediated IL-1β production and that gastrointestinal decontamination and uric acid depletion reduced GvHD severity. Early blockade of IL-1β or genetic deficiency of the IL-1 receptor in dendritic cells (DCs) and T cells improved survival. The Nlrp3 inflammasome components Nlrp3 and Asc, which are required for pro–IL-1β cleavage, were critical for the full manifestation of GvHD. In transplanted mice, IL-1β originated from multiple intestinal cell compartments and exerted its effects on DCs and T cells, the latter being preferentially skewed toward Th17. Compatible with these mouse data, increased levels of active caspase-1 and IL-1β were found in circulating leukocytes and intestinal GvHD lesions of patients. Thus, the identification of a crucial role for the Nlrp3 inflammasome sheds new light on the pathogenesis of GvHD and opens a potential new avenue for the targeted therapy of this severe complication.

Acute Graft-versus-host disease (GvHD) is a major immunological complication following allogeneic hematopoietic cell transplantation (allo-HCT) occurring in 30-40% of patients. Although immunosuppression with corticosteroids serves as standard first-line therapy in acute GvHD, sustained responses are achieved in less than 50% of patients. Therefore, the identification of risk factors and a better understanding of the molecular mechanisms underlying the disease are indispensable to develop novel therapeutic strategies. MicroRNAs control various key biological processes, including innate and adaptive immune responses. In particular, microRNA-146a (miR-146a) is emerging as a central regulator of innate immune cell signaling. Here we found that a G/C single nucleotide polymorphism (SNP) within the pre-miR-146a gene, which reduces the amount of miR-146a produced from the C allele, was strongly associated with the risk to develop severe acute GvHD. Patients with the rs2910164 CC genotype had a significantly higher risk to develop acute GvHD grade III-IV than all other allo-HCT recipients (p=0.002) as well as an overall increased histopathological GvHD severity. In order to functionally dissect the role of miR-146a in recipient cells during GvHD, we used gene targeted (miR-146a-/-) mice and a major histocompatibility complex (MHC)-mismatched experimental model for acute GvHD. MiR-146a deficiency of the recipient mice resulted in increased GvHD severity, elevated serum levels of pro-inflammatory cytokines and accelerated mortality compared to wild type (WT) recipients after allo-HCT. Chimeric mice with a lack of miR-146a specifically in the hematopoietic system showed an even more distinct disease exacerbation, whereas miR-146a deficiency only in non-hematopoietic recipient cells did not affect GvHD severity. Moreover, co-transplantation of recipient type miR-146a-/-dendritic cells (DCs) elicited a more aggressive course of GvHD, while overexpression of miR-146a in DCs using a specific miR-146a mimic ameliorated disease. Phenotypically, miR-146a-/- DCs displayed a more activated phenotype with increased MHC class II, CD80 and CD86 surface expression levels after endotoxin stimulation when compared to WT DCs. Global gene expression analysis revealed upregulation of the JAK-STAT signaling pathway in miR-146a-/- DCs, which we confirmed at the protein and phosphoprotein level using Western blot and flow cytometry. Importantly, inhibition of JAK-STAT signaling in DCs using the JAK1/2 inhibitor ruxolitinib prevented miR-146a-/- DC-induced GvHD exacerbation. In conclusion, our findings indicate that miR-146a acts as a central regulator of recipient type DCs during GvHD, by dampening cytokine receptor signaling via the JAK-STAT pathway. We detected the SNP rs2910164 as a marker to identify patients at high risk for GvHD before allo-HCT, facilitating the opportunity to apply pre-emptive interventions. Disclosures No relevant conflicts of interest to declare.

Introduction: Acute graft-versus-host disease (GvHD) arises from the attack of recipient tissues by donor allogeneic T cells and represents one of the major limitations of allogeneic hematopoietic cell transplantation (allo-HCT). In spite of many clinical trials, the standard immunosuppressive regimens for prevention of acute GvHD have improved little in the last two decades. Hence, a better understanding of the biology of acute GvHD may improve therapeutic options. MicroRNA-146a (miR-146a) was found to be increased in the sera of patients with GvHD. Therefore, we aimed to decipher the role of miR-146a in allogeneic donor T cells during GvHD by functional studies and in patients undergoing allo-HCT by single nucleotide polymorphism (SNP) analysis. Methods: We used two different murine major MHC mismatch models for acute GvHD. Recipient mice were conditioned with irradiation before transplantation of bone marrow and either wildtype or miR-146a deficient T cells from allogeneic donor mice. Furthermore, genomic DNA from 289 patients that underwent allo-HCT and their respective hematopoietic stem cell donors was isolated in order to determine their miR-146a rs2910164genotype. Results: We observed miR-146a upregulation in T cells of mice developing acute GvHD compared to untreated mice in a major MHC and a minor histocompatibility antigen mismatch model. Transfer of miR-146a deficient T cells caused increased GvHD severity, elevated TNF serum levels and reduced survival. Conversely, the phytochemical induction of miR-146a or its overexpression in donor T cells using a specific miR-146a mimic reduced GvHD severity. TNF receptor-associated factor 6 (TRAF6), a verified target of miR-146a, was upregulated in miR-146a-/- T cells following alloantigen stimulation. Higher TRAF6 levels translated into increased NF-κB activity and TNF production in miR-146a-/- T cells, while other pro-inflammatory cytokine levels were unaffected. The detrimental effect of miR-146a deficiency in T cells could be antagonized by TNF blockade in vivo. Moreover, in contrast to WT T cells, over expression of miR-146a in Tnf deficient T cells had no effect on their alloreactivity. In the human system, the minor genotype of the SNP rs2910164, which causes reduced miR-146a expression, was more frequent in patients developing acute GvHD grade III/IV compared to all other allo-HCT recipients (n=289). Conclusions: Taken together we show that miR-146a functions as a negative regulator of the TRAF6/TNF-axis in allogeneic donor T cells during GvHD, leading to reduced TNF transcription. Given our observation on the predictive role of the SNP leading to decreased miR-146a expression in acute GvHD in patients and the possibility to exogenously enhance miR-146a expression, we provide a novel and targeted molecular approach to mitigate GvHD. Disclosures No relevant conflicts of interest to declare.

Abstract 800 Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. The highly variable prognosis of the disease may be predicted using a number of biomarkers, including the expression level of CD38. Human CD38 is a surface glycoprotein with enzymatic activity and the ability to mediate cell-cell interactions by binding the non-substrate ligand CD31. CD31/CD38 interactions drive CLL proliferation and chemotaxis, and increase CXCL-12-mediated signals and homing of CLL cells towards lymphoid organs. We and others have previously identified SYK, a key component of the BCR signalling pathway, as a candidate for targeted therapy in CLL due to its enhanced expression and activity and the apoptotic effects of pharmacological SYK inhibition even in the context of the microenvironment. In this study, we demonstrate direct involvement of SYK in the CD38 signaling pathway and that SYK inhibition may prevent CD38−mediated CLL cell proliferation and migration. CD38 stimulation of primary CLL cells by its ligand CD31 resulted in SYK activation as indicated by phospho-protein analysis by flow cytometry (n=18, p=0.0002) and immunoblotting. Analysis of CD38 expression on CLL cells by flow cytometry with SYK inhibition using the pharmacological SYK inhibitor R406 (Sellek) revealed a significant decrease of CD38 by 31% (n=25, p Taken together, our data demonstrate direct involvement of SYK in the CD38 signaling pathway. Beyond the known effects of SYK inhibition on chemokine- and integrin mediated CLL-stroma interactions, these data establish interference with the CD31/CD38 axis as novel therapeutic mode of action of SYK inhibition in CLL. Since CD38 expression identifies CLL cells with high proliferative and migratory function and clinically associates with poor responsiveness to chemotherapy SYK inhibition may be a particularly promising therapeutic option in CLL patients expressing CD38. Disclosures: No relevant conflicts of interest to declare.