Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.

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 CD8CD107aIFN-γ 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.

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.

In a large cohort study of the German Neonatal Network (GNN) we aimed to evaluate whether less invasive surfactant administration (LISA) strategy is associated with complications of preterm birth. Within the observational period n = 7533 very-low-birth-weight infants (VLBWI) with gestational age 22 0/7 to 28 6/7 weeks were enrolled in GNN; n = 1214 VLBWI never received surfactant, n = 2624 VLBWI were treated according to LISA procedure, n = 3695 VLBWI had surfactant via endotracheal tube (ETT). LISA was associated with a reduced risk for adverse outcome measures including mortality [odds ratio (OR) 0.66 (95% CI: 0.51–0.84), p < 0.001] bronchopulmonary dysplasia [BPD; OR 0.55 (95% CI: 0.49–0.62), p < 0.001], intracerebral hemorrhage (ICH) grade II-IV [OR 0.55 (95% CI: 0.48–0.64), p < 0.001] and retinopathy of prematurity [ROP; OR 0.62 (95% CI: 0.45–0.85), p < 0.001]. Notably, LISA was associated with an increased risk for focal intestinal perforation [FIP; OR 1.49 (95% CI: 1.14–1.95), p = 0.002]. The differences in FIP rates were primarily observed in VLBWI born <26 weeks (LISA: 10.0 vs. ETT: 7.4%, p = 0.029). Our observational data confirm that LISA is associated with improved outcome. In infants <26 weeks we noted an increased risk for FIP. Future randomized controlled trials including LISA need to integrate safety analyses for this particular subgroup.

Key Points• Monoclonal antibody blockade of the common g chain attenuates acute and chronic GVHD.• Common g-chain cytokines increase granzyme B levels in CD8 T cells, which are reduced upon CD132 blockade in vivo.The common g chain (CD132) is a subunit of the interleukin (IL) receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Because levels of several of these cytokines were shown to be increased in the serum of patients developing acute and chronic graft-versus-host disease (GVHD), we reasoned that inhibition of CD132 could have a profound effect on GVHD. We observed that anti-CD132 monoclonal antibody (mAb) reduced acute GVHD potently with respect to survival, production of tumor necrosis factor, interferon-g, and IL-6, and GVHD histopathology. Anti-CD132 mAb afforded protection from GVHD partly via inhibition of granzyme B production in CD8 T cells, whereas exposure of CD8 T cells to IL-2, IL-7, IL-15, and IL-21 increased granzyme B production. Also, T cells exposed to anti-CD132 mAb displayed a more naive phenotype in microarray-based analyses and showed reduced Janus kinase 3 (JAK3) phosphorylation upon activation. Consistent with a role of JAK3 in GVHD, Jak3 2/2 T cells caused less severe GVHD. Additionally, anti-CD132 mAb treatment of established chronic GVHD reversed liver and lung fibrosis, and pulmonary dysfunction characteristic of bronchiolitis obliterans. We conclude that acute GVHD and chronic GVHD, caused by T cells activated by common g-chain cytokines, each represent therapeutic targets for anti-CD132 mAb immunomodulation. (Blood. 2015;125(3):570-580)

No abstract

Micro-RNAs (miR) are small non-coding RNAs that regulate gene expression, post-transcription, and manipulate immune responses in different types of cancer. 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 to 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 Interferon-γ (IFN-γ). Neutralization of IFN-γ in miR-146a-/- mice decreased survival and increased melanoma metastasis patterns to those of wild-type mice. In vitro, IFN-γ reduced melanoma cell migration, cell cycle activity, and basal metabolic rate. Conversely, IFN-γ 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/IFN-γ 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 anti-tumor immune response elicited by checkpoint therapy.

Acute graft-versus-host disease (GVHD) can affect the central nervous system (CNS). The role of microglia in CNS-GVHD remains undefined. In agreement with microglia activation, we found that profound morphological changes and MHC-II and CD80 upregulation occurred upon GVHD induction. RNA sequencing-based analysis of purified microglia obtained from mice with CNS-GVHD revealed TNF upregulation. Selective TNF gene deletion in microglia of Cx3cr1 creER Tnf fl/mice reduced MHC-II expression and decreased CNS T cell infiltrates and VCAM-1 + endothelial cells. GVHD increased microglia TGF-β-activated kinase-1 (TAK1) activation and NF-κB/p38 MAPK signaling. Selective Tak1 deletion in microglia using Cx3cr1 creER Tak1 fl/fl mice resulted in reduced TNF production and microglial MHC-II and improved neurocognitive activity. Pharmacological TAK1 inhibition reduced TNF production and MHC-II expression by microglia, Th1 and Th17 T cell infiltrates, and VCAM-1 + endothelial cells and improved neurocognitive activity, without blocking graft-versus-leukemia effects. Consistent with these findings in mice, we observed increased activation and TNF production of microglia in the CNS of GVHD patients. In summary, we prove a role for microglia in CNS-GVHD, identify the TAK1/TNF/MHC-II axis as a mediator of CNS-GVHD, and provide a TAK1 inhibitor-based approach against GVHD-induced neurotoxicity.