Staphylococcus aureus-derived factors promote human Th9 cell polarization and enhance a transcriptional program associated with allergic inflammation.

T helper (Th) 9 cells, characterized by robust secretion of IL-9, have been increasingly associated with allergic diseases. However, whether and how Th9 cells are modulated by environmental stimuli remains poorly understood. In this study, we show that in vitro exposure of human PBMCs or isolated CD4 T-cells to Staphylococcus (S.) aureus-derived factors, including its toxins, potently enhances Th9 cell frequency and IL-9 secretion. Furthermore, as revealed by RNA sequencing analysis, S. aureus increases the expression of Th9-promoting factors at the transcriptional level, such as FOXO1, miR-155, and TNFRSF4. The addition of retinoic acid (RA) dampens the Th9 responses promoted by S. aureus and substantially changes the transcriptional program induced by this bacterium, while also altering the expression of genes associated with allergic inflammation. Together, our results demonstrate a strong influence of microbial and dietary factors on Th9 cell polarization, which may be important in the context of allergy development and treatment.

DNA methylation in infants with low and high body fatness.

BACKGROUND: Birth weight is determined by the interplay between infant genetics and the intrauterine environment and is associated with several health outcomes in later life. Many studies have reported an association between birth weight and DNA methylation in infants and suggest that altered epigenetics may underlie birthweight-associated health outcomes. However, birth weight is a relatively nonspecific measure of fetal growth and consists of fat mass and fat-free mass which may have different effects on health outcomes which motivates studies of infant body composition and DNA methylation. Here, we combined genome-wide DNA methylation profiling of buccal cells from 47 full-term one-week old infants with accurate measurements of infant fat mass and fat-free mass using air-displacement plethysmography. RESULTS: No significant association was found between DNA methylation in infant buccal cells and infant body composition. Moreover, no association between infant DNA methylation and parental body composition or indicators of maternal glucose metabolism were found. CONCLUSIONS: Despite accurate measures of body composition, we did not identify any associations between infant body fatness and DNA methylation. These results are consistent with recent studies that generally have identified only weak associations between DNA methylation and birthweight. Although our results should be confirmed by additional larger studies, our findings may suggest that differences in DNA methylation between individuals with low and high body fatness may be established later in childhood.

The transcriptional coregulator MAML1 affects DNA methylation and gene expression patterns in human embryonic kidney cells.

Mastermind-like 1 (MAML1) is a transcriptional coregulator that has been associated with early development of many systems such as neuronal, muscular and urogenital. The present study aimed to explore the genome wide effects of MAML1 on DNA methylation and RNA expression in human embryonic kidney cells. Infinium HumanMethylation450 BeadChip Illumina array, methylation-sensitive high-resolution melt technique, Chip Analysis Methylation Pipeline and RNA profiling approaches were used to study MAML1 effects on the epigenome. We found that 11802 CpG sites were differentially methylated in MAML1-expressing cells while only 225 genes were differentially expressed. MAML1 overexpression induced more global differential hypermethylation than hypomethylation changes. In addition, the differentially methylated regions were mapped predominantly to 3'untranslated regions, intragenic regions and gene bodies and to a lesser extent to gene regulatory sequences. Gene ontology analysis revealed that the differentially changed genes (including HOXC11, HTATIP2, SLFN12 and SOX11) are involved in the regulation of urogenital system development, cell adhesion and embryogenesis. This study is the first report that shows the global effect of a single coregulator on DNA methylation and gene expression. Our results stress and support the effects of transcriptional coregulators on the cell methylome.

Cystatin from the helminth Ascaris lumbricoides upregulates mevalonate and cholesterol biosynthesis pathways and immunomodulatory genes in human monocyte-derived dendritic cells.

Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclusion: Al-CPI modifies the transcriptome of moDC, increasing several transcripts encoding enzymes involved in cholesterol biosynthesis and SREBP signaling. Moreover, Al-CPI target several transcripts in the TNF-alpha signaling pathway influencing cytokine release by moDC. In addition, mRNA levels of genes encoding KLF10 and other members of the TGF beta and the IL-10 families were also modified by Al-CPI stimulation. The regulation of the mevalonate pathway and cholesterol biosynthesis suggests new mechanisms involved in DC responses to helminth immunomodulatory molecules.

Intravenous Infusion of High Dose Selenite in End-Stage Cancer Patients: Analysis of Systemic Exposure to Selenite and Seleno-Metabolites.

Cancer is one of the main causes of human death globally and novel chemotherapeutics are desperately required. As a simple selenium oxide, selenite is a very promising chemotherapeutic because of pronounced its dose-dependent tumor-specific cytotoxicity. We previously published a first-in-man systematic phase I clinical trial in patients with cancer (from IV to end-stage) (the SECAR trial) showing that selenite is safe and tolerable with an unexpectable high maximum tolerated dose (MTD) and short half-life. In the present study, we analyzed the selenium species in plasma samples, from the patients participating in the SECAR trial and from various time points and dose cohorts using LC-ICP-MS. In conclusion, selenite, selenosugars, and 1-2 unidentified peaks that did not correspond to any standard, herein denoted ui-selenium, were detected in the plasma. However, trimethylated selenium (trimethylselenonoium) was not detected. The unidentified ui-selenium was eluting close to the selenium-containing amino acids (selenomethionine and selenocysteine) but was not part of a protein fraction. Our data demonstrate that the major metabolite detected was selenosugar. Furthermore, the identification of selenite even long after the administration is remarkable and unexpected. The kinetic analysis did not support that dosing per the body surface area would reduce interindividual variability of the systemic exposure in terms of trough concentrations.

ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain.

Molecular diversity of microglia, the resident immune cells in the CNS, is reported. Whether microglial subsets characterized by the expression of specific proteins constitute subtypes with distinct functions has not been fully elucidated. Here we describe a microglial subtype expressing the enzyme arginase-1 (ARG1; that is, ARG1+ microglia) that is found predominantly in the basal forebrain and ventral striatum during early postnatal mouse development. ARG1+ microglia are enriched in phagocytic inclusions and exhibit a distinct molecular signature, including upregulation of genes such as Apoe, Clec7a, Igf1, Lgals3 and Mgl2, compared to ARG1- microglia. Microglial-specific knockdown of Arg1 results in deficient cholinergic innervation and impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, which in turn results in impaired long-term potentiation and cognitive behavioral deficiencies in female mice. Our results expand on microglia diversity and provide insights into microglia subtype-specific functions.

Effects of redox modulation by inhibition of thioredoxin reductase on radiosensitivity and gene expression.

The thioredoxin system is a promising target when aiming to overcome the problem of clinical radiation resistance. Altered cellular redox status and redox sensitive thiols contributing to induction of resistance strongly connect the ubiquitous redox enzyme thioredoxin reductase (TrxR) to the cellular response to ionizing radiation. To further investigate possible strategies in combating clinical radiation resistance, human radio-resistant lung cancer cells were subjected to a combination of single fractions of γ-radiation at clinically relevant doses and non-toxic levels of a well-characterized thioredoxin reductase inhibitor, the phosphine gold(I) compound [Au(SCN)(PEt(3))]. The combination of the TrxR-inhibitor and ionizing radiation reduced the surviving fractions and impaired the ability of the U1810 cells to repopulate by approximately 50%. In addition, inhibition of thioredoxin reductase caused changes in the cell cycle distribution, suggesting a disturbance of the mitotic process. Global gene expression analysis also revealed clustered genetic expression changes connected to several major cellular pathways such as cell cycle, cellular response to stress and DNA damage. Specific TrxR-inhibition as a factor behind the achieved results was confirmed by correlation of gene expression patterns between gold and siRNA treatment. These results clearly demonstrate TrxR as an important factor conferring resistance to irradiation and the use of [Au(SCN)(PEt(3))] as a promising radiosensitizing agent.

SWI/SNF associates with nascent pre-mRNPs and regulates alternative pre-mRNA processing.

The SWI/SNF chromatin remodeling complexes regulate the transcription of many genes by remodeling nucleosomes at promoter regions. In Drosophila, SWI/SNF plays an important role in ecdysone-dependent transcription regulation. Studies in human cells suggest that Brahma (Brm), the ATPase subunit of SWI/SNF, regulates alternative pre-mRNA splicing by modulating transcription elongation rates. We describe, here, experiments that study the association of Brm with transcribed genes in Chironomus tentans and Drosophila melanogaster, the purpose of which was to further elucidate the mechanisms by which Brm regulates pre-mRNA processing. We show that Brm becomes incorporated into nascent Balbiani ring pre-mRNPs co-transcriptionally and that the human Brm and Brg1 proteins are associated with RNPs. We have analyzed the expression profiles of D. melanogaster S2 cells in which the levels of individual SWI/SNF subunits have been reduced by RNA interference, and we show that depletion of SWI/SNF core subunits changes the relative abundance of alternative transcripts from a subset of genes. This observation, and the fact that a fraction of Brm is not associated with chromatin but with nascent pre-mRNPs, suggest that SWI/SNF affects pre-mRNA processing by acting at the RNA level. Ontology enrichment tests indicate that the genes that are regulated post-transcriptionally by SWI/SNF are mostly enzymes and transcription factors that regulate postembryonic developmental processes. In summary, the data suggest that SWI/SNF becomes incorporated into nascent pre-mRNPs and acts post-transcriptionally to regulate not only the amount of mRNA synthesized from a given promoter but also the type of alternative transcript produced.

Altered mRNA Expression Due to Rectal Perforation in a Porcine Model - A Pilot Study.

BACKGROUND: Anastomotic leakage is the most serious and unwelcome complication in rectal surgery. It has a great impact on postoperative morbidity and mortality. In this pilot study, changes of mRNA expression in blood were analyzed in an animal model designed to imitate anastomotic leakage. MATERIALS AND METHODS: Twelve pigs were randomized into two groups: A sham-operated control group and an experimental group in which iatrogenic rectal perforation was performed. The changes in the mRNA expression at 4 hours after creating the perforation were studied. Microarray analysis was performed using Gene Chip whole porcine genome array. mRNA expression of 19,124 genes was investigated. RESULTS: Significantly increased levels of genes with a fold change greater than 2 were found, including 276 coding for unknown proteins and 48 coding for known proteins. Eleven of those which coded for known proteins were up-regulated with a fold change >4. CONCLUSION: Eleven known genes were highly up-regulated after rectal perforation. These genes were mainly involved in inflammatory response, intracellular signaling and cell membrane regulation. Their corresponding proteins might potentially be clinical biomarkers of anastomotic leakage and should be evaluated in further clinical studies.

SWI/SNF regulates the alternative processing of a specific subset of pre-mRNAs in Drosophila melanogaster.

BACKGROUND: The SWI/SNF chromatin remodeling factors have the ability to remodel nucleosomes and play essential roles in key developmental processes. SWI/SNF complexes contain one subunit with ATPase activity, which in Drosophila melanogaster is called Brahma (Brm). The regulatory activities of SWI/SNF have been attributed to its influence on chromatin structure and transcription regulation, but recent observations have revealed that the levels of Brm affect the relative abundances of transcripts that are formed by alternative splicing and/or polyadenylation of the same pre-mRNA. RESULTS: We have investigated whether the function of Brm in pre-mRNA processing in Drosophila melanogaster is mediated by Brm alone or by the SWI/SNF complex. We have analyzed the effects of depleting individual SWI/SNF subunits on pre-mRNA processing throughout the genome, and we have identified a subset of transcripts that are affected by depletion of the SWI/SNF core subunits Brm, Snr1 or Mor. The fact that depletion of different subunits targets a subset of common transcripts suggests that the SWI/SNF complex is responsible for the effects observed on pre-mRNA processing when knocking down Brm. We have also depleted Brm in larvae and we have shown that the levels of SWI/SNF affect the pre-mRNA processing outcome in vivo. CONCLUSIONS: We have shown that SWI/SNF can modulate alternative pre-mRNA processing, not only in cultured cells but also in vivo. The effect is restricted to and specific for a subset of transcripts. Our results provide novel insights into the mechanisms by which SWI/SNF regulates transcript diversity and proteomic diversity in higher eukaryotes.

Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles.

BACKGROUND: Gcn5 is a transcriptional coactivator with histone acetyltransferase activity that is conserved with regard to structure as well as its histone substrates throughout the eukaryotes. Gene regulatory networks within cells are thought to be evolutionarily diverged. The use of evolutionarily divergent yeast species, such as S. cerevisiae and S. pombe, which can be studied under similar environmental conditions, provides an opportunity to examine the interface between conserved regulatory components and their cellular applications in different organisms. RESULTS: We show that Gcn5 is important for a common set of stress responses in evolutionarily diverged yeast species and that the activity of the conserved histone acetyltransferase domain is required. We define a group of KCl stress response genes in S. cerevisiae that are specifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent genes including Gcn5 repressed targets such as FLO8. Gcn5 regulates divergent sets of KCl responsive genes in S. cerevisiae and S. pombe. Genome-wide localization studies showed a tendency for redistribution of Gcn5 during KCl stress adaptation in S. cerevisiae from short genes to the transcribed regions of long genes. An analogous redistribution was not observed in S. pombe. CONCLUSIONS: Gcn5 is required for the regulation of divergent sets of KCl stress-response genes in S. cerevisiae and S. pombe even though it is required a common group of stress responses, including the response to KCl. Genes that are physically associated with Gcn5 require its activity for their repression or activation during stress adaptation, providing support for a role of Gcn5 as a corepressor as well as a coactivator. The tendency of Gcn5 to re-localise to the transcribed regions of long genes during KCl stress adaptation suggests that Gcn5 plays a specific role in the expression of long genes under adaptive conditions, perhaps by regulating transcriptional elongation as has been seen for Gcn5 in S. pombe. Interestingly an analogous redistribution of Gcn5 is not seen in S. pombe. The study thus provides important new insights in relation to why coregulators like Gcn5 are required for the correct expression of some genes but not others.

Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs.

Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59-RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation.

Accumulation of Progerin Affects the Symmetry of Cell Division and Is Associated with Impaired Wnt Signaling and the Mislocalization of Nuclear Envelope Proteins.

Hutchinson-Gilford progeria syndrome (HGPS) is the result of a defective form of the lamin A protein called progerin. While progerin is known to disrupt the properties of the nuclear lamina, the underlying mechanisms responsible for the pathophysiology of HGPS remain less clear. Previous studies in our laboratory have shown that progerin expression in murine epidermal basal cells results in impaired stratification and halted development of the skin. Stratification and differentiation of the epidermis is regulated by asymmetric stem cell division. Here, we show that expression of progerin impairs the ability of stem cells to maintain tissue homeostasis as a result of altered cell division. Quantification of basal skin cells showed an increase in symmetric cell division that correlated with progerin accumulation in HGPS mice. Investigation of the mechanisms underlying this phenomenon revealed a putative role of Wnt/ß-catenin signaling. Further analysis suggested an alteration in the nuclear translocation of ß-catenin involving the inner and outer nuclear membrane proteins, emerin and nesprin-2. Taken together, our results suggest a direct involvement of progerin in the transmission of Wnt signaling and normal stem cell division. These insights into the molecular mechanisms of progerin may help develop new treatment strategies for HGPS.

Effects of diazoxide on gene expression in rat pancreatic islets are largely linked to elevated glucose and potentially serve to enhance beta-cell sensitivity.

Diazoxide enhances glucose-induced insulin secretion from beta-cells through mechanisms that are not fully elucidated. Here, we used microarray analysis (Affymetrix) to investigate effects of diazoxide. Pancreatic islets were cultured overnight at 27, 11, or 5.5 mmol/l glucose with or without diazoxide. Inclusion of diazoxide upregulated altogether 211 genes (signal log(2) ratio > or =0.5) and downregulated 200 genes (signal log(2) ratio -0.5 or lower), and 92% of diazoxide's effects (up- and downregulation) were observed only after coculture with 11 or 27 mmol/l glucose. We found that 11 mmol/l diazoxide upregulated 97 genes and downregulated 21 genes. Increasing the glucose concentration to 27 mmol/l markedly shifted these proportions toward downregulation (101 genes upregulated and 160 genes downregulated). At 27 mmol/l glucose, most genes downregulated by diazoxide were oppositely affected by glucose (80%). Diazoxide influenced expression of several genes central to beta-cell metabolism. Diazoxide downregulated genes of fatty acid oxidation, upregulated genes of fatty acid synthesis, and downregulated uncoupling protein 2 and lactic acid dehydrogenase. Diazoxide upregulated certain genes known to support beta-cell functionality, such as NKX6.1 and PDX1. Long-term elevated glucose is permissive for most of diazoxide's effects on gene expression, the proportion of effects shifting to downregulation with increasing glucose concentration. Effects of diazoxide on gene expression could serve to enhance beta-cell functionality during continuous hyperglycemia.

Study on genetic stability in human urothelial cells in vitro.

Quality control studies addressing gene expression changes and genetic stability are of vital importance in regenerative medicine. In order to rule out that in vitro expansion gives rise to gene expression changes that could favour oncogenic events, this study applied a total human gene expression chip (Affymetrix®) and bioinformatics analysis using the Ingenuity web-based application in combination with an analysis of chromosomal copy number variations using array comparative genomic hybridization. Urothelial cells presented a general repression of genes required for cell cycle progression and upregulation of growth-inhibitory genes, as well as a decrease in deoxyribose nucleic acid replication after long-term culture. Molecules were identified with a potential to regulate human urothelial cell senescence, such as the micro-ribonucleic acid Let-7. Human urothelial cells did not acquire copy number variations after long-term culture and the cells had a normal expression of oncogenes and tumor suppressor genes. Altogether, both gene expression studies and array comparative genomic hybridization indicated a good quality of in vitro propagated cells. For tissue engineering purposes, these analyses could be used for quality control assessments before transplantation back to the patient. Copyright © 2016 John Wiley & Sons, Ltd.

Single-Stranded Nucleic Acids Regulate TLR3/4/7 Activation through Interference with Clathrin-Mediated Endocytosis.

Recognition of nucleic acids by endosomal Toll-like receptors (TLR) is essential to combat pathogens, but requires strict control to limit inflammatory responses. The mechanisms governing this tight regulation are unclear. We found that single-stranded oligonucleotides (ssON) inhibit endocytic pathways used by cargo destined for TLR3/4/7 signaling endosomes. Both ssDNA and ssRNA conferred the endocytic inhibition, it was concentration dependent, and required a certain ssON length. The ssON-mediated inhibition modulated signaling downstream of TLRs that localized within the affected endosomal pathway. We further show that injection of ssON dampens dsRNA-mediated inflammatory responses in the skin of non-human primates. These studies reveal a regulatory role for extracellular ssON in the endocytic uptake of TLR ligands and provide a mechanistic explanation of their immunomodulation. The identified ssON-mediated interference of endocytosis (SOMIE) is a regulatory process that temporarily dampens TLR3/4/7 signaling, thereby averting excessive immune responses.

Ameloblastoma RNA profiling uncovers a distinct non-coding RNA signature.

Ameloblastoma of the jaws remains the top difficult to treat odontogenic tumour and has a high recurrence rate. New evidence suggests that non-coding RNAs (ncRNAs) play a critical role in tumourgenesis and prognosis of cancer. However, ameloblastoma ncRNA expression data is lacking. Here we present the first report of ameloblastoma ncRNA signatures. A total of 95 ameloblastoma cases and a global array transcriptome technology covering > 285.000 full-length transcripts were used in this two-step analysis. The analysis first identified in a test cohort 31 upregulated ameloblastoma-associated ncRNAs accompanied by signalling pathways of cancer, spliceosome, mRNA surveillance and Wnt. Further validation in an independent cohort points out the long non-coding (lncRNAs) and small nucleolar RNA (snoRNAs): LINC340, SNORD116-25, SNORA11, SNORA21, SNORA47 and SNORA65 as a distinct ncRNA signature of ameloblastoma. Importantly, the presence of these ncRNAs was independent of BRAF-V600E and SMO-L412F mutations, histology type or tumour location, but was positively correlated with the tumour size. Taken together, this study shows a systematic investigation of ncRNA expression of ameloblastoma, and illuminates new diagnostic and therapeutic targets for this invasive odontogenic tumour.

Numerous Genes in Loci Associated With Body Fat Distribution Are Linked to Adipose Function.

Central fat accumulation is a strong risk factor for type 2 diabetes. Genome-wide association studies have identified numerous loci associated with body fat distribution. The objectives of the current study are to examine whether genes in genetic loci linked to fat distribution can be linked to fat cell size and number (morphology) and/or adipose tissue function. We show, in a cohort of 114 women, that almost half of the 96 genes in these loci are indeed associated with abdominal subcutaneous adipose tissue parameters. Thus, adipose mRNA expression of the genes is strongly related to adipose morphology, catecholamine-induced lipid mobilization (lipolysis), or insulin-stimulated lipid synthesis in adipocytes (lipogenesis). In conclusion, the genetic influence on body fat distribution could be mediated via several specific alterations in adipose tissue morphology and function, which in turn may influence the development of type 2 diabetes.

Regulation of myeloid cells by activated T cells determines the efficacy of PD-1 blockade.

Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor.