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The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma. Implications: MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned.

The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma.Implications:MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned.

Supplementary Figures Legends and Methods

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic RAS mutations, causing constitutive activation of multiple tumor-promoting pathways, are frequently found in human cancer. So far in hematologic malignancies, RAS mutations have only been reported to occur in hematopoietic cells. In this study, we investigated the effect of oncogenic Kras expression in the BMME in a chimeric mouse model. We observed that an activating mutation of Kras in the nonhematopoietic system leads to a phenotype resembling myelodysplastic syndrome (MDS) characterized by peripheral cytopenia, marked dysplasia within the myeloid lineage as well as impaired proliferation and differentiation capacity of hematopoietic stem and progenitor cells. The phenotypic changes could be reverted when the BM was re-isolated and transferred into healthy recipients, indicating that the KrasG12D-activation in the nonhematopoietic BMME was essential for the MDS phenotype. Gene expression analysis of sorted nonhematopoietic BM niche cells from KrasG12D mice revealed upregulation of multiple inflammation-related genes including IL1-superfamily members (Il1α, Il1β, Il1f9) and the NLPR3 inflammasome. Thus, pro-inflammatory IL1-signaling in the BMME may contribute to MDS development. Our findings show that a single genetic change in the nonhematopoietic BMME can cause an MDS phenotype. Oncogenic Kras activation leads to pro-inflammatory signaling in the BMME which impairs HSPCs function.Implications:These findings may help to identify new therapeutic targets for MDS.

S1. KrasG12D recipient mice develop a myelodysplastic syndrome within the transferred WT hematopoietic system. S2. Gating strategy used for the isolation of bone marrow (BM) niche cells. S3. The frequency of BM niche cells is not affected in KrasG12D recipient mice. S4. Differential pathway analysis in MSPCs and endothelial cells of KrasG12D versus WT mice. S5. Differential pathways analysis in CaR and osteoblast cells of KrasG12D versus WT recipient mice. S6. Expression of phosphorylated H2AX (γ-H2AX) in BM of WT or KrasG12D recipient mice. S7. Analysis of patient-derived BM for KRAS mutations by digital droplet polymerase chain reaction (ddPCR).

(S1) Supplemental Figure S1 shows the flow cytometry based analysis of B16 cells transformed with a lentiviral vector with Luc-GFP-neo (S2-S7) Supplemental Figures S2-S7 show complete western blot images (S8) Supplemental Figure S8 shows the impact of IFN gamma on melanoma cells with respect to gene expression, OCR and ROS production (S9) Supplemental Figure S9 shows the effect of prolonging combination therapy regimen in melanoma bearing mice (S10) Supplemental Figure S10 shows the combined effects of IFN-γ and miR-146a inhibition on melanoma cells in vitro (S11) Supplemental Figure S11 shows the proposed mechanism of action.

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.

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic RAS mutations, causing constitutive activation of multiple tumor-promoting pathways, are frequently found in human cancer. So far in hematologic malignancies, RAS mutations have only been reported to occur in hematopoietic cells. In this study, we investigated the effect of oncogenic Kras expression in the BMME in a chimeric mouse model. We observed that an activating mutation of Kras in the nonhematopoietic system leads to a phenotype resembling myelodysplastic syndrome (MDS) characterized by peripheral cytopenia, marked dysplasia within the myeloid lineage as well as impaired proliferation and differentiation capacity of hematopoietic stem and progenitor cells. The phenotypic changes could be reverted when the BM was re-isolated and transferred into healthy recipients, indicating that the KrasG12D-activation in the nonhematopoietic BMME was essential for the MDS phenotype. Gene expression analysis of sorted nonhematopoietic BM niche cells from KrasG12D mice revealed upregulation of multiple inflammation-related genes including IL1-superfamily members (Il1α, Il1β, Il1f9) and the NLPR3 inflammasome. Thus, pro-inflammatory IL1-signaling in the BMME may contribute to MDS development. Our findings show that a single genetic change in the nonhematopoietic BMME can cause an MDS phenotype. Oncogenic Kras activation leads to pro-inflammatory signaling in the BMME which impairs HSPCs function. Implications: These findings may help to identify new therapeutic targets for MDS.

Vaccination against the ongoing COVID-19 is the key point in fight against the pandemic. The Spike (S) glycoprotein of SARS-CoV-2 is the major target of the neutralizing humoral response. We evaluated analytical and clinical performances of a surrogate virus neutralization test (sVNT) (iFlash-2019-nCoV Nab assay, Ylho, China) compared to the conventional neutralization tests (cVNT) and anti-S eCLIA assays (Roche Diagnostics, Switzerland) in recovered and/or vaccinated health care workers. Our results indicate that sVNT displayed high specificity and no cross reactivity. Both Roche and iFlash immunoassays were good in identifying cVNT serum dilution > 1:16. Optimal thresholds in identifying cVNT titers ≥ 1:16, were 74.5 U/ml and 49.4 IU/ml for anti-S eCLIA and sVNT, respectively. Our data show that Nab neutralizing antibodies titers depend on individuals and may abate over time. Specific assays such as sVNT may be a reliable complementary tool to routine anti-S serology assays.

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysM(cre) Mcl1(fl/fl) mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.

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.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies.

Acute myeloid leukemia (AML) relapse after allogeneic hematopoietic cell transplantation (allo-HCT) has a dismal prognosis. We found that T cells of patients relapsing with AML after allo-HCT exhibited reduced glycolysis and interferon-γ production. Functional studies in multiple mouse models of leukemia showed that leukemia-derived lactic acid (LA) interfered with T cell glycolysis and proliferation. Mechanistically, LA reduced intracellular pH in T cells, led to lower transcription of glycolysis-related enzymes, and decreased activity of essential metabolic pathways. Metabolic reprogramming by sodium bicarbonate (NaBi) reversed the LA-induced low intracellular pH, restored metabolite concentrations, led to incorporation of LA into the tricarboxylic acid cycle as an additional energy source, and enhanced graft-versus-leukemia activity of murine and human T cells. NaBi treatment of post–allo-HCT patients with relapsed AML improved metabolic fitness and interferon-γ production in T cells. Overall, we show that metabolic reprogramming of donor T cells is a pharmacological strategy for patients with relapsed AML after allo-HCT.

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic

Immune checkpoint inhibitor (ICI) therapy has shown a significant benefit in the treatment of a variety of cancer entities. However, immune-related adverse events (irAEs) occur frequently and can lead to ICI treatment termination. MicroRNA-146a (miR-146a) has regulatory functions in immune cells. We observed that mice lacking miR-146a developed markedly more severe irAEs compared with WT mice in several irAE target organs in 2 different murine models. miR-146a(–/–) mice exhibited increased T cell activation and effector function upon ICI treatment. Moreover, neutrophil numbers in the spleen and the inflamed intestine were highly increased in ICI-treated miR-146a(–/–) mice. Therapeutic administration of a miR-146a mimic reduced irAE severity. To validate our preclinical findings in patients, we analyzed the effect of a SNP in the MIR146A gene on irAE severity in 167 patients treated with ICIs. We found that the SNP rs2910164 leading to reduced miR-146a expression was associated with an increased risk of developing severe irAEs, reduced progression-free survival, and increased neutrophil counts both at baseline and during ICI therapy. In conclusion, we characterized miR-146a as a molecular target for preventing ICI-mediated autoimmune dysregulation. Furthermore, we identified the MIR146A SNP rs2910164 as a biomarker to predict severe irAE development in ICI-treated patients.

Conditioning-induced damage of the intestinal tract plays a critical role during the onset of acute graft-versus-host disease (GVHD). Therapeutic interference with these early events of GVHD is difficult, and currently used immunosuppressive drugs mainly target donor T cells. However, not donor T cells but neutrophils reach the sites of tissue injury first, and therefore could be a potential target for GVHD prevention. A detailed analysis of neutrophil fate during acute GVHD and the effect on T cells is difficult because of the short lifespan of this cell type. By using a novel photoconverter reporter system, we show that neutrophils that had been photoconverted in the ileum postconditioning later migrated to mesenteric lymph nodes (mLN). This neutrophil migration was dependent on the intestinal microflora. In the mLN, neutrophils colocalized with T cells and presented antigen on major histocompatibility complex (MHC)-II, thereby affecting T cell expansion. Pharmacological JAK1/JAK2 inhibition reduced neutrophil influx into the mLN and MHC-II expression, thereby interfering with an early event in acute GVHD pathogenesis. In agreement with this finding, neutrophil depletion reduced acute GVHD. We conclude that neutrophils are attracted to the ileum, where the intestinal barrier is disrupted, and then migrate to the mLN, where they participate in alloantigen presentation. JAK1/JAK2-inhibition can interfere with this process, which provides a potential therapeutic strategy to prevent early events of tissue damage-related innate immune cell activation and, ultimately, GVHD.

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysM(cre) Mcl1(fl/fl) mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.

Oncogenic Ras mutations occur in various leukemias. It was unclear if, besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation-related effect of KRAS contributes to the disease. Here, we identify a functional link between oncogenic KrasG12D and NLRP3 inflammasome activation in murine and human cells. Mice expressing active KrasG12D in the hematopoietic system developed myeloproliferation and cytopenia, which is reversed in KrasG12D mice lacking NLRP3 in the hematopoietic system. Therapeutic IL-1-receptor blockade or NLRP3-inhibition reduces myeloproliferation and improves hematopoiesis. Mechanistically, KrasG12D-RAC1 activation induces reactive oxygen species (ROS) production causing NLRP3 inflammasome-activation. In agreement with our observations in mice, patient-derived myeloid leukemia cells exhibit KRAS/RAC1/ROS/NLRP3/IL-1β axis activity. Our findings indicate that oncogenic KRAS not only act via its canonical oncogenic driver function, but also enhances the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis. This paves the way for a therapeutic approach based on immune modulation via NLRP3 blockade in KRAS-mutant myeloid malignancies., Oncogenic Ras mutations are common drivers in myeloid leukemia. Here, the authors show in patient cells and in mice that oncogenic K-Ras activates NLRP3 inflammasome to drive myeloproliferation, which can be reversed by genetic or pharmacologic NLRP3 blockade.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F–myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient–derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1–mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Contains fulltext : 190745.pdf (Publisher’s version ) (Closed access) Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD(+) leukemia cells. This synergized with the allogeneic CD8(+) T cell response, leading to long-term survival in six mouse models of FLT3-ITD(+) AML. Sorafenib-related IL-15 production caused an increase in CD8(+)CD107a(+)IFN-gamma(+) T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD(+) AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8(+) T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive PD-L1 expression may help in developing new therapeutic strategies. Here, we show that oncogenic JAK2 activity caused STAT3 and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2(V617F)-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2(V617F)-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2(V617F)-myeloproliferative neoplasms (MPN) compared to healthy individuals and declined upon JAK2 inhibition. JAK2(V617F) mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2(V617F)-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2(V617F)-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for different malignant and non-malignant hematologic diseases. Major life-threatening complications after allo-HCT are acute graft-versus-host disease (aGVHD) and severe infections. The incidence of GVHD after allo-HCT remains high, despite prophylactic immunosuppressive medication. According to the CIBMTR database, 60% of patients undergoing allo-HCT develop at least grade II-IV aGVHD. Microbial invasion into the intestinal mucosa after allo-HCT triggers the activation of neutrophil granulocytes (neutrophils) and requires antibiotic treatment to prevent sepsis. However, antibiotics lead to a loss of microbial diversity, which is connected to a higher incidence of aGVHD. Anti-microbial therapies which eliminate invading bacteria and diminish neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution for this could be the use of anti-microbial antibodies that target and mark invading pathogens resulting in their elimination by innate immune cells. Therefore we investigated the potency of both active and passive immunization against the conserved microbial surface polysaccharide Poly-N-acetylglucosamine (PNAG) which is expressed on various pathogens in a murine aGVHD model. We could detect reduced aGVHD related mortality in mice which underwent treatment with an antibody against PNAG (anti-PNAG) or were actively immunized against PNAG. Sequencing analysis of the microbiota before and after allo-HCT showed that anti-PNAG treatment did not have any effect luminal microbial diversity. Mechanistically, we could show that anti-PNAG antibody treatment caused more abundant neutrophil recruitment to the intestinal submucosa. This supports the concept that neutrophils are able to effectively eliminate any opsonized bacteria in this tissue, leading to reduced local inflammation. In agreement with this we observed lower intestinal inflammation and reduced myeloperoxidase activity as well as proliferation of neutrophils in the small intestine of mice treated with anti-PNAG antibody. We demonstrate the potency of targeting PNAG as a novel antimicrobial treatment option in aGVHD by reducing uncontrolled neutrophil activation and thereby interfering with aGVHD without affecting commensal intestinal microbial diversity. Anti-PNAG-antibodies enhance anti-microbial immunity, while reducing the activation of neutrophil-mediated downstream immunopathology which is a novel approach to reduce the severity of GVHD. This could be translated into a clinical application given the modest toxicity profile of vaccination and the availability of fully human anti-PNAG antibodies, both of which have been tested in phase 1 trials in humans (ClinicalTrials.gov Identifier: NCT02853617). Disclosures Cywes-Bentley: OneBiopharma, Inc.: Honoraria. Koenecke:Novartis: Other: none. Pier:OneBiopharma, Inc.: Equity Ownership, Honoraria; Alopexx Vaccine, LLC: Equity Ownership, Honoraria.

During development, proliferation must be tightly controlled for organs to reach their appropriate size. While the Hippo signaling pathway plays a major role in organ growth control, how it senses and responds to increased cell density is still unclear. In this study, we use the zebrafish lateral line primordium (LLP), a group of migrating epithelial cells that form sensory organs, to understand how tissue growth is controlled during organ formation. Loss of the cell junction-associated Motin protein Amotl2a leads to overproliferation and bigger LLP, affecting the final pattern of sensory organs. Amotl2a function in the LLP is mediated together by the Hippo pathway effector Yap1 and the Wnt/β-catenin effector Lef1. Our results implicate for the first time the Hippo pathway in size regulation in the LL system. We further provide evidence that the Hippo/Motin interaction is essential to limit tissue size during development. DOI: http://dx.doi.org/10.7554/eLife.08201.001, eLife digest How do organs and tissues know when to stop growing? A cell communication pathway known as Hippo signaling plays a central role as it can tell cells to stop dividing. It is activated when cells in developing tissues come into contact with each other and causes a protein called Yap1 to be modified, which prevents it from entering the cell nucleus to activate genes that are involved in cell division. In a zebrafish embryo, an organ called the lateral line forms from a cluster of cells that migrate along the embryo's length. At regular intervals, the cluster deposits small bunches of cells from its trailing end. The resulting loss of cells from the cluster is balanced by cell division at the front of the cluster, which is triggered by another signaling pathway called Wnt signaling. A protein of the ‘Motin’ family called Amotl2a is present in this migrating cluster. Motin proteins form junctions between cells and inhibit the activity of Yap1, but it is not known whether they are involved in regulating the size of organs. Here, Agarwala et al. used the lateral line as a model to study the control of organ size in zebrafish embryos. The experiments show that when Amotl2a is absent, the migrating cell cluster becomes larger, with the highest levels of cell division occurring at its trailing end. Yap1 and a protein involved in Wnt signaling called Lef1 are also present in the cluster and are required for it to be normal in size. In zebrafish that lack Amotl2a, the additional loss of Yap1 prevents this cluster from becoming too large. From these and other results, it appears that Amotl2a regulates the size of the lateral line cell cluster by restricting the ability of Yap1 and Lef1 to promote cell division. Agarwala et al.'s findings demonstrate a role for Amotl2a in controlling the size of organs. A future challenge is to understand the details of how it restricts the activities of Yap1 and Lef1. DOI: http://dx.doi.org/10.7554/eLife.08201.002