IRF4 Expression Research Articles

The Transcription Factor SUB1 Is a Master Regulator of the Macrophage TLR Response in Atherosclerosis.

Toll‐like receptor 2 and 4 (TLR2, TLR4) signaling is implicated in atherosclerotic plaque formation. The two‐stage master regulator Virtual Inference of Protein‐activity by Enriched Regulon (VIPER) analysis of macrophage TLR2 and TLR4 signature genes integrated with coexpression network genes derived from 371 patient‐derived carotid specimens identifies activated RNA polymerase II transcriptional coactivator p15 (SUB1/Sub1, PC4) as a master regulon in the atherogenic TLR response. It is found that TLR2 and TLR4 signaling is proinflammatory and proatherosclerotic in chow‐fed apolipoprotein E‐deficient (ApoE −/−) mice. Through transgenic myeloid‐specific Sub1 knockout in ApoE −/− mice, it is discovered that these proatherosclerotic effects of TLR2 and TLR4 signaling are mediated by Sub1. Sub1 knockout in macrophages enhances anti‐inflammatory M2 macrophage polarization and cholesterol efflux. Irradiated low density lipoprotein receptor‐deficient (Ldlr −/−) mice transplanted with Sub1 −/− murine bone marrow display reduced atherosclerosis. Promoter analysis reveals Sub1‐dependent activation of interferon regulatory factor 1 (Irf1) transcription in a casein kinase 2 (Ck2)‐dependent manner, and Sub1‐knockout macrophages display decreased Irf1 expression. Artificial Irf1 overexpression in Sub1‐knockout macrophages enhances proinflammatory M1 skewing and lowers cholesterol clearance. In conclusion, the TLR master regulon Sub1, and its downstream effect on the transcription factor Irf1, promotes a proinflammatory M1 macrophage phenotype and enhances atherosclerotic burden in vivo.

Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity.

SUMMARYHumoral immunity relies on the efficient differentiation of memory B cells (MBCs) into antibody-secreting cells (ASCs). T helper (Th) signals upregulate B cell receptor (BCR) signaling by potentiating Src family kinases through increasing CD45 phosphatase activity (CD45 PA).In this study, we show that high CD45 PA in MBCs enhances BCR signaling and is essential for their effective ASC differentiation. Mechanistically, Th signals upregulate CD45 PA through intensifying the surface binding of a CD45 ligand, Galectin-1. CD45 PA works as a sensor of T cell help and defines high-affinity germinal center (GC) plasma cell (PC) precursors characterized by IRF4 expression in vivo. Increasing T cell help in vitro results in an incremental CD45 PA increase and enhances ASC differentiation by facilitating effective induction of the transcription factors IRF4 and BLIMP1. This study connects Th signals with BCR signaling through Galectin-1-dependent regulation of CD45 PA and provides a mechanism for efficient ASC differentiation of MBCs.

The Effects Of Exercise Preconditioning On Serum -free Mtdna And Mitochondrial Autoimmunity In Mice

PURPOSE: To explore the effects of exercise preconditioning on serum free mtDNA and mitochondria-associated immune proteins in skeletal muscles under acute centrifugation injury. METHODS: Eight-week-old C57BL male mice were randomly divided into three groups according to their body weight. Each group consisted of 6 mice (NC, RC, RE). Exercise pre-adaptation program: take a non-slope 20 m/min treadmill training for 40 min/d, training for 6 weeks. Acute Sports Injury Program: In the 6th week of exercise training in RE group, 3 days of 10 m/min and 5 minutes of adaptive training without gradients on the RC group were started in the RC group; all mice in the RC and RE groups took 16 m/min,-16 ° Downhill running, training 90 min. After 16 hours of injury training, the mice were sacrificed, and one side of the gastrocnemius muscle was fixed with paraformaldehyde and awaited for HE staining. The tissue samples were detected by PCR, RT-PCR, Western Blotting, and ELISA. RESULTS:1) Serum free mtDNA content in RC group was significantly higher than NC and RE group (P < 0.05) 2) IL6 levels in mouse serum were significantly lower in NC group than in RC and RE group (P < 0.05, P < 0.01); The group was significantly higher than the NC group and RE group (P < 0.05, P < 0.01); 3) The results of STING mRNA detection showed that NC group was higher than RE (P < 0.05) There was no significant difference between RC group and RC group, but RC group was significantly higher than that of RE group (P < 0.01). The results of mRNA expression tests of other molecules in STING pathway showed that only IRF3 expression was significantly lower in RE group than in NC group. (P < 0.05);4) The expression of PINK1 in skeletal muscle was highest in the RC group and significantly higher than that in the RE group (P < 0.01). Parkin was the highest in NC group in skeletal muscle, (P < 0.01). CONCLUSIONS: (1) Increase in serum free mtDNA content caused by acute centrifugation injury exercise is not a cause of inflammatory reaction to skeletal muscle injury, because acute centrifugation injury inhibits the transcription of mitochondria-associated immune proteins in skeletal muscle, and cGAS-STING-TBK1-IRF3 - The IFN-β signaling pathway is not activated. (2) Acute centrifugal injury exercise and exercise preconditioning did not affect PINK1/Parkin-mediated mitochondrial autophagy.

IRF4 and IRF8 expression are associated with clinical phenotype and clinico-hematological response to hydroxyurea in essential thrombocythemia.

The morbidity and mortality of myeloproliferative neoplasms (MPNs) are primarily caused by arterial and venous complications, progression to myelofibrosis, and transformation to acute leukemia. However, identifying molecular-based biomarkers for risk stratification of patients with MPNs remains a challenge. We have previously shown that interferon regulatory factor-8 (IRF8) and IRF4 serve as tumor suppressors in myeloid cells. In this study, we evaluated the expression of IRF4 and IRF8 and the JAK2V617F mutant allele burden in patients with MPNs. Patients with decreased IRF4 expression were correlated with a more developed MPN phenotype in myelofibrosis (MF) and secondary AML (sAML) transformed from MPNs versus essential thrombocythemia (ET). Negative correlations between the JAK2V617F allele burden and the expression of IRF8 (P < 0.05) and IRF4 (P < 0.001) and between white blood cell (WBC) count and IRF4 expression (P < 0.05) were found in ET patients. IRF8 expression was negatively correlated with the JAK2V617F allele burden (P < 0.05) in polycythemia vera patients. Complete response (CR), partial response (PR), and no response (NR) were observed in 67.5%,10%, and 22.5% of ET patients treated with hydroxyurea (HU), respectively, in 12 months. At 3 months, patients in the CR group showed high IRF4 and IRF8 expression compared with patients in the PR and NR groups. In the 12-month therapy period, low IRF4 and IRF8 expression were independently associated with the unfavorable response to HU and high WBC count. Our data indicate that the expression of IRF4 and IRF8 was associated with the MPN phenotype, which may serve as biomarkers for the response to HU in ET.

NFκB1 Dichotomously Regulates Pro-Inflammatory and Antiviral Responses in Asthma

Asthma exacerbations are commonly triggered by rhinovirus infections. Viruses can activate the NFκB pathway resulting in airway inflammation and increased Th2 cytokine expression. NFκB signaling is also involved in early activation of IFNβ, which is a central mediator of antiviral responses to rhinovirus infection. Using a mouse model, this study tests our hypothesis that NFκB signaling is involved in impaired IFNβ production at viral-induced asthma exacerbations. C57BL/6 wild-type and NFκB1^−/− mice were challenged with house dust mite for 3 weeks and were subsequently stimulated with the rhinoviral mimic poly(I:C). General lung inflammatory parameters and levels of the Th2 upstream cytokine IL-33 were measured after allergen challenge. At exacerbation, production of IFNβ and antiviral proteins as well as gene expression of pattern recognition receptors and IRF3/IRF7 was assessed. In the asthma exacerbation mouse model, lack of NFκB1 resulted in lower levels of IL-33 after allergen challenge alone and was associated with reduced eosinophilia. At exacerbation, mice deficient in NFκB1 exhibited enhanced expression of IFNβ and antiviral proteins. This was accompanied by increased IRF3/IRF7 expression and induction of pattern recognition receptor expression. In a human asthma dataset, a negative correlation between IRF3 and NFκB1 expression was observed. NFκB may impair antiviral responses at exacerbation, possibly by reducing expression of the transcription factors IRF3/IRF7. These findings suggest a therapeutic potential for targeting NFκB pathways at viral infection-induced exacerbations.

An autoimmune disease risk variant: A trans master regulatory effect mediated by IRF1 under immune stimulation?

Functional mechanisms remain unknown for most genetic loci associated to complex human traits and diseases. In this study, we first mapped trans-eQTLs in a data set of primary monocytes stimulated with LPS, and discovered that a risk variant for autoimmune disease, rs17622517 in an intron of C5ORF56, affects the expression of the transcription factor IRF1 20 kb away. The cis-regulatory effect specific to IRF1 is active under early immune stimulus, with a large number of trans-eQTL effects across the genome under late LPS response. Using CRISPRi silencing, we showed that perturbation of the SNP locus downregulates IRF1 and causes widespread transcriptional effects. Genome editing by CRISPR had suggestive recapitulation of the LPS-specific trans-eQTL signal and lent support for the rs17622517 site being functional. Our results suggest that this common genetic variant affects inter-individual response to immune stimuli via regulation of IRF1. For this autoimmune GWAS locus, our work provides evidence of the functional variant, demonstrates a condition-specific enhancer effect, identifies IRF1 as the likely causal gene in cis, and indicates that overactivation of the downstream immune-related pathway may be the cellular mechanism increasing disease risk. This work not only provides rare experimental validation of a master-regulatory trans-eQTL, but also demonstrates the power of eQTL mapping to build mechanistic hypotheses amenable for experimental follow-up using the CRISPR toolkit.

Defective interferon amplification and impaired host responses against influenza virus in obese mice.

Obesity is a major risk factor that increases morbidity and mortality upon infection. Although type I and type III interferon (IFN)-induced innate immune responses represent the first line of defense against viral infections, their functionality in the context of metabolic disorders remains largely obscure. This study aimed to investigate IFN responses upon respiratory viral infection in obese mice. The activation of IFNs as well as IFN regulatory factors (IRFs) upon H3N2 influenza infection in mice upon high-fat-diet feeding was investigated. Influenza infection of obese mice was characterized by higher mortalities. In-depth analysis revealed impaired induction of both type I and type III IFNs as well as markedly reduced IFN responses. Notably, it was found that IRF7 gene expression in obese animals was reduced in homeostasis, and its induction by the virus was strongly attenuated. The results suggest that the attenuated IRF7 expression and induction are responsible for the reduced expression levels of type I and III IFNs and, thus, for the higher susceptibility and severity of respiratory infections in obese mice.

Zebrafish Ism1 is a novel antiviral factor that positively regulates antiviral immune responses

Isthmin1 (Ism1), first identified as a secreted protein in Xenopus embryos in 2002, has been shown to perform multiple biological functions, but little is known currently regarding its role in immunity. Here we show that the expression of ism1 is inducible by challenge with Grass carp reovirus (GCRV) in zebrafish, suggesting involvement of Ism1 in antiviral response. We then demonstrate that recombinant Ism1 (rIsm1) reduces the cytopathic effect in the cells infected by GCRV, promotes the expression of type I IFN gene and IFN-inducible antiviral protein Mxa gene, and reduces the virus quantity in virus-infected cells and host. We also show that rIsm1 promotes the expression of tbk1, irf3 and irf7, suggesting it promotes the expression of type I IFN gene and Mxa gene via induction of Tbk1–Irf3–Ifn pathway. These data together indicate that Ism1 is a new immune-relevant factor functioning in antiviral immune response, and provides a target for controlling viral infection.

MiR-155 regulates Th9 differentiation in children with methicillin-resistant Staphylococcus aureus pneumonia by targeting SIRT1

Th9 is a subset of CD4+ T cells that mainly secrete IL-9. Th9/IL-9 participates in immune response during Staphylococcus aureus and methicillin-resistant Staphylococcus aureus pneumonia (MRSA) infection. Here, we collected bronchoalveolar lavage fluid (BALF) from 30 children with MRSA pneumonia (MRSA group) and 10 children with bronchial foreign bodies (Control group). RT-PCR, ELISA and flow cytometry were used to detect the expression of miR-155 and IL-9 in BALF and the number of Th9 cells. CD4+ T cells isolated from BALF of MRSA and Control group were transfected with miR-155 mimic or inhibitor, and then induced Th9 cell differentiation. The results showed that the expression of miR-155 and IL-9 were significantly increased in BALF and Th9 cell of MRSA group, as well as the number of Th9 cells. miR-155 mimic upregulated IL-9 mRNA expression, IL-9 secretion and increased number of Th9 cells. On the contrary, miR-155 inhibitor inhibited IL-9 mRNA expression, IL-9 secretion and decreased number of Th9 cells. The dual luciferase assays demonstrated miR-155 can target binding to SIRT1 3′UTR. Moreover, overexpression of SIRT1 could reverse the effect of miR-155 mimic on IL-9 expression level, Th9 cell number and transcription factors PU.1 and IRF4 expression. In conclusion, miR-155 regulates Th9 differentiation in children with MRSA by targeting SIRT1.

Grass carp (Ctenopharyngodon idella) interferon regulatory factor 8 down-regulates interferon1 expression via interaction with interferon regulatory factor 2 in vitro

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.

Exposure to the Gram-Negative Bacteria Pseudomonas aeruginosa Influences the Lung Dendritic Cell Population Signature by Interfering With CD103 Expression.

Lung dendritic cells (DCs) are divided into two major populations, which include CD103+XCR1+ cDC1s and CD11b+Sirpα+ cDC2s. The maintenance of their relative proportions is dynamic and lung inflammation, such as caused by exposure to lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, can have a significant impact on the local cDC signature. Alterations in the lung cDC signature could modify the capacity of the immune system to respond to various pathogens. We consequently aimed to assess the impact of the Gram-negative bacteria Pseudomonas aeruginosa on lung cDC1 and cDC2 populations, and to identify the mechanisms leading to alterations in cDC populations. We observed that exposure to P. aeruginosa decreased the proportions of CD103+XCR1+ cDC1s, while increasing that of CD11b+ DCs. We identified two potential mechanisms involved in this modulation of lung cDC populations. First, we observed an increase in bone marrow pre-DC IRF4 expression suggesting a higher propensity of pre-DCs to differentiate towards the cDC2 lineage. This observation was combined with a reduced capacity of lung XCR1+ DC1s to express CD103. In vitro, we demonstrated that GM-CSF-induced CD103 expression on cDCs depends on GM-CSF receptor internalization and RUNX1 activity. Furthermore, we observed that cDCs stimulation with LPS or P. aeruginosa reduced the proportions of intracellular GM-CSF receptor and decreased RUNX1 mRNA expression. Altogether, these results suggest that alterations in GM-CSF receptor intracellular localization and RUNX1 signaling could be involved in the reduced CD103 expression on cDC1 in response to P. aeruginosa. To verify whether the capacity of cDCs to express CD103 following P. aeruginosa exposure impacts the immune response, WT and Cd103-/- mice were exposed to P. aeruginosa. Lack of CD103 expression led to an increase in the number of neutrophils in the airways, suggesting that lack of CD103 expression on cDC1s could favor the innate immune response to this bacterium.

Effect of Xenon Treatment on Gene Expression in Brain Tissue after Traumatic Brain Injury in Rats.

The overactivation of inflammatory pathways and/or a deficiency of neuroplasticity may result in the delayed recovery of neural function in traumatic brain injury (TBI). A promising approach to protecting the brain tissue in TBI is xenon (Xe) treatment. However, xenon’s mechanisms of action remain poorly clarified. In this study, the early-onset expression of 91 target genes was investigated in the damaged and in the contralateral brain areas (sensorimotor cortex region) 6 and 24 h after injury in a TBI rat model. The expression of genes involved in inflammation, oxidation, antioxidation, neurogenesis and neuroplasticity, apoptosis, DNA repair, autophagy, and mitophagy was assessed. The animals inhaled a gas mixture containing xenon and oxygen (ϕXe = 70%; ϕO2 25–30% 60 min) 15–30 min after TBI. The data showed that, in the contralateral area, xenon treatment induced the expression of stress genes (Irf1, Hmox1, S100A8, and S100A9). In the damaged area, a trend towards lower expression of the inflammatory gene Irf1 was observed. Thus, our results suggest that xenon exerts a mild stressor effect in healthy brain tissue and has a tendency to decrease the inflammation following damage, which might contribute to reducing the damage and activating the early compensatory processes in the brain post-TBI.

ATF6-DGAT pathway is involved in TLR7-induced innate immune response in Ctenopharyngodon idellus kidney cells

DGAT1 and DGAT2 are two acyl-CoA:diacylglycerol O-acyltransferase (DGAT) enzymes that catalyze the final step in triglyceride (TG) synthesis. TGs are the primary constituents of lipid droplets (LDs). Although it has been demonstrated that LDs modulate immune and inflammatory responses in CIK cells, little is known about whether DGAT1 and DGAT2 involve in this process. Firstly, grass carp DGAT2 was isolated and characterized, encoding 361 amino acids, and all DGAT2 proteins in genomic structures are conserved in vertebrates. Then, using TLR7 agonist, we induced LDs accumulation in CIK cells. Only DGAT1b and DGAT2 were upregulated in forming TLR7 agonist induced-LDs. Next, we utilized small-molecule inhibitors of DGAT1 and DGAT2. The results indicated that DGAT1 inactivation attenuated TG content and the relative expressions of IFNα3, NF-κB, IL-1β, and TNFα genes, whereas DGAT2 inhibition decreased TG content and the relative expressions of MyD88, IRF7, IFNα3, NF-κB, IL-1β, and TNFα genes, implying that DGAT1-generated LDs and DGAT2-generated LDs contribute to TLR7-induced immune response via different signaling pathways. Finally, inhibiting ATF6 effectively decreased DGAT-generated LDs accumulation and the expression of TLR7 signaling-related genes induced by TLR7 agonist, implying that ATF6 UPR pathway may mediate the role of DGAT-generated LDs in TLR7 signaling. Overall, we demonstrate that DGAT1 and DGAT2-catalyzed TAG synthesis may generate different LDs to provide distinct signaling platforms for innate TLR7 signaling.

Development of a Novel Immune Infiltration-Related ceRNA Network and Prognostic Model for Sarcoma

Due to the rarity and heterogeneity, it is challenging to explore and develop new therapeutic targets for patients with sarcoma. Recently, immune cell infiltration in the tumor microenvironment (TME) was widely studied, which provided a novel potential approach for cancer treatment. The competing endogenous RNA (ceRNA) regulatory network has been reported as a critical molecular mechanism of tumor development. However, the role of the ceRNA regulatory network in the TME of sarcoma remains unclear. In this study, gene expression data and clinical information were obtained from The Cancer Genome Atlas (TCGA) sarcoma datasets, and an immune infiltration-related ceRNA network was constructed, which comprised 14 lncRNAs, 13 miRNAs, and 23 mRNAs. Afterward, we constructed an immune infiltration-related risk score model based on the expression of IRF1, MFNG, hsa-miR-940, and hsa-miR-378a-5p, presenting a promising performance in predicting the prognosis of patients with sarcoma.

High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

Abstract 2727: Heterodimeric IL-15 (hetIL-15) affects conventional dendritic cells and a distinct novel dendritic cell population in different mouse cancer models of breast and pancreatic cancer

Abstract Introduction: IL-15 is a cytokine inducing proliferation and cytotoxic function of lymphocytes, including CD8+ T and NKs cells. We have produced the native heterodimeric form of IL-15 (hetIL-15) which has advanced in clinical trials due to its anticancer activities in many different mouse cancer models. The objective of this study was to explore how hetIL-15 affects lymphoid and myeloid cell populations in the tumor microenvironment in different mouse cancer models of breast and pancreatic cancer. Study Design and Methods: We studied the therapeutic efficacy of intraperitoneal or locoregional administration of hetIL-15 in three different orthotopic mouse cancer models. We employed 4T1 and EO771 breast cancer models, which mimic human triple negative breast cancer and a KPC derived pancreatic cancer model. hetIL-15 effects on tumor infiltrating immune cells were evaluated by flow cytometry and immunohistochemistry. Results: hetIL-15 treatment showed a profound effect on the EO771 breast cancer model resulting in complete tumor regression in ~40% of the treated mice. Additionally, hetIL-15 caused tumor delay in 4T1 and KPC derived cancer models. Flow cytometry analysis revealed an increase of cytotoxic effector cells, and especially CD8+ T cells, with enhanced cytotoxic activity and proliferation in all cancer models tested. Immunohistochemistry verified the accumulation of cytotoxic effector cells intratumorally upon hetIL-15 treatment. Furthermore, flow analysis showed an increase of infiltrating conventional type 1 dendritic cells (cDC1s) which was found only in EO771 breast tumors. On the contrary, 4T1 breast and KPC derived pancreatic tumors, but not EO771 tumors, displayed an accumulation of infiltrating conventional type 2 dendritic cells (cDC2s). Importantly, the flow cytometry analysis revealed yet additional novel distinct dendritic cell population characterized by CD103intCD11b+ immunophenotype, which was mostly evident in tumor tissues treated with hetIL-15 in all three models. Phenotypic profiling of this novel DC population identified expression of several cDC1 specific markers, including CD103, IRF8 and XCR1. Both cDC1s and the novel DC population were inversely correlated with the tumor size in EO771 breast cancer model. Conclusions: hetIL-15 affects both T cells and conventional dendritic cells in syngeneic murine cancer models of breast and pancreatic cancer. We report that the treatment with hetIL-15 increases a novel distinct dendritic cell population (CD103intCD11b+) in all three models. Since we have previously reported that hetIL-15 induces long term immunological protection from tumor rechallenge, these findings suggest hetIL-15 as a promising therapeutic agent in treatment of triple negative breast and pancreatic cancer. Citation Format: Vasiliki Stravokefalou, Dimitris Stellas, Sevasti Karaliota, Bethany Nagy, Theresa Guerin, Serguei Kozlov, Barbara K. Felber, George N. Pavlakis. Heterodimeric IL-15 (hetIL-15) affects conventional dendritic cells and a distinct novel dendritic cell population in different mouse cancer models of breast and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2727.

Abstract 1613: A hypoxia-driven transcriptional program characterizes acquired resistance to immune checkpoint inhibitors in lung cancer

Abstract Immune checkpoint inhibitors (ICIs) that block negative regulators of T cell function can generate lasting responses in lung cancer patients, yet only a minority of tumors respond to therapy and ~50% of patients whose tumors initially respond eventually develop acquired resistance. Mechanisms of acquired resistance to ICIs in lung cancer are not fully understood and strategies to overcome resistance are lacking. To address this challenge, we developed a new mouse model of acquired resistance to ICIs by knocking out the DNA mismatch repair gene Msh2 in Kras mutant mouse lung adenocarcinoma cells, leading to a higher mutation burden in these cells. Unlike Msh2 WT controls, Msh2 KO tumors transplanted into syngeneic mice are sensitive to ICIs. Importantly, chronic treatment with αPD-1 or αPD-1 + αCTLA-4 led to Msh2 KO tumors that initially responded to therapy but then developed acquired resistance. Tumors remained resistant to ICIs upon transplantation to new hosts, suggesting that tumor-intrinsic programs drive resistance. Gene set enrichment analysis (GSEA) of single cell RNA sequencing data revealed that tumors with acquired resistance to ICIs downregulated antigen processing and presentation gene signatures compared to tumors that were regressing on therapy. Specifically, tumor cells with acquired resistance downregulated expression of MHC class II, which was confirmed by flow cytometry, and this was accompanied by a decrease in expression of Stat1, Irf1, and Ciita, which drive MHC-II expression in response to IFNγ. Depletion of CD4+ T cells during αPD-1 + αCTLA-4 treatment abrogated the response to therapy, suggesting a functional role for tumor cell MHC-II expression. GSEA also revealed that tumors with acquired resistance to ICIs upregulated hypoxia and glycolysis gene signatures compared to tumors responding to therapy. Consistent with these results, resistant tumor cells had increased hypoxia, detected by pimonidazole staining, compared to tumor cells responding to therapy. Tumor infiltrating lymphocytes have been shown to be excluded from hypoxic tumor regions, suggesting that hypoxia in the tumor microenvironment may contribute to acquired resistance to ICIs by excluding T cells from these regions. Moreover, we found that Msh2 KO tumor cells upregulated MHC-II in response to IFNγ under normoxic in vitro conditions, but had lower MHC-II expression under hypoxia. Our data suggest that tumor hypoxia may contribute to both tumor-cell intrinsic transcriptional programs, such as alterations in antigen processing and presentation machinery, as well as tumor-cell extrinsic effects, such as exclusion of T cells from the tumor microenvironment, which together may impact the development of acquired resistance to immune checkpoint blockade. Our results can inform strategies to overcome acquired resistance, addressing a critical unmet need in cancer therapy. Citation Format: Camila Robles-Oteiza, Katherine Hastings, Susan M. Kaech, Katerina Politi. A hypoxia-driven transcriptional program characterizes acquired resistance to immune checkpoint inhibitors in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1613.

Semaphorin 3E deficiency dysregulates dendritic cell functions: In vitro and in vivo evidence.

Regulation of dendritic cell functions is a complex process in which several mediators play diverse roles as a network in a context-dependent manner. The precise mechanisms underlying dendritic cell functions have remained to be addressed. Semaphorins play crucial roles in regulation of various cell functions. We previously revealed that Semaphorin 3E (Sema3E) contributes to regulation of allergen-induced airway pathology partly mediated by controlling recruitment of conventional dendritic cell subsets in vivo, though the underlying mechanism remained elusive. In this study, we investigate the potential regulatory role of Sema3E in dendritic cells. We demonstrated that bone marrow-derived dendritic cells differentiated from Sema3e-/- progenitors have an enhanced migration capacity both at the baseline and in response to CCL21. The enhanced migration ability of Sema3E dendritic cells was associated with an overexpression of the chemokine receptor (CCR7), elevated Rac1 GTPase activity and F-actin polymerization. Using a mouse model of allergic airway sensitization, we observed that genetic deletion of Sema3E leads to a time dependent upregulation of CCR7 on CD11b+ conventional dendritic cells in the lungs and mediastinal lymph nodes. Furthermore, aeroallergen sensitization of Sema3e-/- mice lead to an enhanced expression of PD-L2 and IRF-4 as well as enhanced allergen uptake in pulmonary CD11b+ DC, compared to wild type littermates. Collectively, these data suggest that Sema3E implicates in regulation of dendritic cell functions which could be considered a basis for novel immunotherapeutic strategies for the diseases associated with defective dendritic cells in the future.

FOXP1 Expression Is a Prognostic Biomarker in Follicular Lymphoma Treated with Immunochemotherapy and Is Associated with Distinct Molecular Features

Introduction: Follicular lymphoma (FL) is a clinically and molecularly highly heterogeneous disease, yet prognostication relies predominantly on clinical tools. We have recently demonstrated that integration of the mutation status of seven genes (including EZH2 and MEF2B) into a combined clinico-genetic risk model (m7-FLIPI) improves risk-stratification, and represents a promising approach to identify a subset of patients at highest risk of treatment failure. However, the mechanism underlying the association between mutations of the 7 genes included in the m7-FLIPI and outcome has thus far not been elucidated. We assessed whether downstream phenotypic commonalities of gene mutations predict outcome following immunochemotherapy and whether select genotypic and phenotypic alterations are associated with FOXP1 expression.Methods: We mined gene expression data to uncover genes that are differentially expressed in EZH2 - and MEF2B -mutated FLs. We focused on FOXP1 and assessed its protein expression by immunohistochemistry (IHC) in a total of 763 tissue biopsies. For outcome correlation, a population-based training cohort of 142 FL patients treated with R-CVP, and a clinical trial validation cohort comprising 395 patients treated with CHOP +/- rituximab were used. To differentiate cases with high vs. low FOXP1 expression, we chose the cut-off that maximized the log-rank test statistic for failure-free survival (FFS) in the training cohort. The effect of FOXP1 expression on FFS in the validation cohort was estimated using Cox regression analysis. In addition, for exploratory analyses, we used specimens from 350 BC Cancer Agency patients, 124 of whom overlapped with the above-mentioned training cohort.Results: We found FOXP1 to be significantly down-regulated in both EZH2 - and MEF2B -mutated cases. The cut-off value that best separated favorable from unfavorable FFS in the training cohort was determined to be >10% percent tumor cells expressing FOXP1 based on the log-rank test statistic. 76 specimens in the training cohort (54%) had high FOXP1 expression, which was associated with reduced 5-year failure-free FFS (55% vs. 70%, P=0.035). In the validation cohort, high FOXP1 expression status was observed in 248 patients (63%) and correlated with significantly shorter FFS in patients treated with R-CHOP (HR 1.95, P=0.017), but not in patients treated with CHOP (HR 1.15, P=0.44), demonstrating that FOXP1 is a predictive biomarker. In BCCA patients, high FOXP1 expression remained significantly associated with inferior FFS in a multivariate Cox regression model including rituximab maintenance (by intention to treat) and FLIPI high vs low/intermediate (HR 1.84, 95%-CI [1.10-3.08], P=0.021). Similarly, in the GLSG cohort, high FOXP1 expression was significantly associated with poor FFS in patients treated with R-CHOP, after adjusting for rituximab, FLIPI and the interaction of FOXP1 with rituximab (HR 1.84, 95%-CI [1.05-3.24], P=0.035). No significant association was observed in GLSG patients treated with CHOP (HR 1.06, 95%-CI [0.73-1.54], P=0.76). High FOXP1 expression was associated with distinct molecular features such as TP53 mutations, expression of IRF4, and gene expression signatures reminiscent of dark zone germinal centre or activated B cells. Conversely, low expression of FOXP1 was associated with - in addition to EZH2 and MEF2B mutations - GNA13 and TNFRSF14 mutations, as well as with expression of genes related to normal germinal centre light zone cells and germinal centre B cells. Taken together, these observations suggest that FOXP1 expression delineates FL cases with distinct genomic and phenotypic characteristics.Conclusion: In patients treated with immunochemotherapy, high FOXP1 expression identified patient populations with significantly shortened FFS. On the other hand, FOXP1 did not separate distinct outcome groups in patients treated without rituximab, suggesting that FOXP1 is a predictive biomarker. In multivariate analyses, high FOXP1 expression remained significantly associated with FFS in immunochemotherapy patients, after adjusting for the FLIPI. Moreover, FOXP1 high and low expression phenotypes are reflective of distinct sets of molecular alterations. Our results illustrate the value of understanding the intricate relationships between lymphoma biology and response to treatment. DisclosuresKlapper:Hoffmann La Roche: Other: Grant; Novartis: Other: Grant; Celgene: Other: Grant; Takeda Millenium: Other: Grant. Hiddemann:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Steidl:Affimed Therapeutics: Consultancy; Juno Therapeutics: Consultancy. Connors:Janssen: Research Funding; F Hoffmann-La Roche: Research Funding; Bayer Healthcare: Research Funding; Seattle Genetics: Research Funding; NanoString Technologies: Research Funding; Merck: Research Funding; Lilly: Research Funding; Genentech: Research Funding; Cephalon: Research Funding; Amgen: Research Funding; Bristol-Myers Squibb: Research Funding; NanoString Technologies, Amgen, Bayer, BMS, Cephalon, Roche, Genentech, Janssen, Lilly, Merck, Seattle Genetics, Takeda,: Research Funding; Takeda: Research Funding. Sehn:Roche/Genentech: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Hoster:Roche: Other: Travel support, Research Funding.

Higher Rates of Molecular Response to Peg-IFNa in JAK2V617F Vs. Calr Mutant MPN Patients Are Due to JAK1-Mediated STAT1 Activation and Autoregulation

Introduction: Pegylated interferon alpha (Peg-IFNa) treatment has been described to induce molecular remission in patients (pts) with myeloproliferative neoplasms (MPN), with partial molecular remission (PMR) being achieved in approximately 30-40% of pts. However, it is unclear whether pts with CALR mutations exhibit the same response as pts with JAK2V617F mutations. Also, the precise mechanisms accounting for the responses are still unknown.Methods: We report on hematological and molecular responses, according to ELN guidelines, in 52 MPN pts (JAK2V617F n=26 vs. CALR mutation n=26; ET n=25, prePMF n=8, PMF n=19), treated at Roskilde hospital with Peg-IFNa. The mechanism of IFNa response was assessed in stably transduced 32D murine cell lines and in a cohort of MPN patients within the Czech MPN registry MIND. All pts provided written informed consent, as approved by the local ethics committees. Cell proliferation was assessed by cell counting and/or MTT assay, signaling molecules were quantified using RT-PCR, flow cytometry and/or Western blotting.Results: Pts harboring JAK2V617F mutations were more likely to achieve PMR than those harboring CALR mutations (60% vs. 23%, p<0.05 by Chi square testing), while there was no significant difference in allelic burden before Peg-IFNa treatment. Accordingly, the median allelic burden decrease from baseline in the IFNa responding pts was 66% (range 15-99%) in JAK2V617F- vs. 29% (range 5-82%) in CALR-mutant pts. These differences were present independently of the fractions of ET, prePMF, or PMF pts in the two groups. There was no significant difference in the rates of hematological response. When examining primary cells from the Czech MPN cohort, cells from JAK2V617F positive pts showed higher expression of IFNa response-related genes such as Irf1 (p=0.0350) and Stat1 (trend at p=0.0874). Moreover, IFNa receptor expression was higher in JAK2V617F positive pts (p=0.0471) and the expression levels were associated with hemoglobin (p=0.0139) and hematocrit levels (p=0.0251). This was reflected in 32D cell lines, ectopically expressing both the MPL receptor and either JAK2V617F or CALR del 52. Here, the expression of Irf7, Irf-9, Ifi-27, and Stat2 was significantly higher in JAK2VF vs. CALR mutants (all p<0.001). In addition, JAK2V617F positive cells showed higher STAT1 phosphorylation and Stat1 mRNA expression levels (p=0.0499). In particular, STAT1 showed constitutive phosphorylation in JAK2V617F but not CALR mutant cells, indicating priming of these cells towards an IFNa response. Overexpression of STAT1 but not of a dominant-negative STAT1YF mutant was able to rescue cellular IFNa-induced growth arrest in CALR positive cells to the same level of JAK2V617F positive cells. In contrast, manipulation of STAT2 expression did not alter IFNa signaling. IFNa-induced growth arrest was counteracted by selective JAK1 inhibition (using itacitinib or filgotinib) but not selective JAK2 inhibition (using BMS-911543).Conclusions: MPN pts harboring JAK2V617F mutations show superior Peg-IFNa sensitivity against the malignant clone, as detected by molecular responses, compared to pts harboring CALR mutations. The higher IFNa sensitivity of JAK2V617F mutant cells was reflected in mutant-specific cell lines and is at least in part due to JAK1-mediated increases in STAT1 phosphorylation and STAT1 transcription. These results are of considerable importance for future clinical trials of Peg-IFNa as monotherapy or in combination with JAK inhibitors. DisclosuresBrümmendorf:Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding. Knudsen:Novartis: Other: Payed for ASH2016 registration fee, travel expenses and accommodation. Hasselbalch:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; AOR Orphan: Membership on an entity's Board of Directors or advisory committees. Koschmieder:Roche: Other: Clinical Trial participation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support.