IRF4 Expression Research Articles

Preclinical and Early Clinical Results Indicate a Role for the Oral p300/CBP Inhibitor Inobrodib (CCS1477) in T-Cell Lymphoma

Inobrodib (CCS1477) is a potent, selective, and orally bioavailable inhibitor of the bromodomains of CBP and EP300, two homologous histone acetyltransferases with critical roles in cellular growth and differentiation. Lymphomas can harbor multiple putative predictive biomarkers for inobrodib, including deleterious mutations in CBP/EP300 that convey synthetic lethality and expression of the lineage transcription factors IRF4 and GATA3. The preclinical efficacy of inobrodib was assessed in a range of solid cancer and hematological cancer cell lines. Select models, including multiple myeloma, acute myeloid leukaemia, and lymphoma cell lines were sensitive to submicromolar concentrations of inobrodib. These in vitro results were extended to xenograft models in which inobrodib demonstrated dose-dependent efficacy. Based on these results, we initiated a clinical trial to test the safety and efficacy of inobrodib in advanced hematological malignancies (NCT04068597). Dose escalation was conducted in multiple myeloma patients and select B and T-cell lymphomas. The safety profile was similar to other hematological and solid tumour indications with readily manageable and reversible thrombocytopenia as the main toxicity. The recommended phase 2 dose was established at 35mg bidaily on a 4 days on/3 days off intermittent schedule. Initial data in peripheral T cell lymphoma - not otherwise specified (PTCL-NOS) shows a potential benefit with objective responses in 3 of 4 patients as of data cut-off date (20 Jul 2023). These included a complete metabolic response in a patient (with previously refractory disease) continuing on treatment for over 1.5 yrs and 2 patients with minor responses. Translational investigations indicate that responders all express IRF4 and GATA3, but do not harbour CBP/EP300 mutations. Together these data support the further development of inobrodib for the treatment of peripheral T cell lymphoma. Expansion continues with a focus on T-cell lymphomas, which may be driven by IRF4 and GATA3.

Genome Wide siRNA Screen Identifies CGAS-Sting Pathway As a Pharmacological Target That Promotes Survival of Hematopoietic Stem Cells Deficient in Fanconi Genes

Introduction Fanconi anemia (FA) is a homozygous recessive genetic disorder due to mutation in the Fanconi family of genes. The disease is characterized by early onset bone marrow failure and a predisposition to cancer. The FA genes are involved in DNA damage sensing and repair. Mutation in these genes leads to heightened DNA damage and extreme sensitivity to cross-linking agents. Hematopoietic stem cells of FA patients are defective in differentiation and self-renewal, leading to stem cell exhaustion and bone marrow failure early in life. The current treatment for bone marrow failure is allogeneic stem cell transplant from healthy donors. Due to difficulties in finding matching donors and the inherent problems associated with allogeneic transplants, pharmacological therapies that delay or prevent bone marrow failure are of clinical importance. Other proposed therapies including TGF-b, metformin, and resveratrol have met with limited clinical success. Methodology: To identify genes that promote survival of FA cells we performed a genome wide siRNA screen using isogenic pair of cell line with FANCD2 deletions and its corrected counterpart using transfection of siRNA libraries targeting 18,119 genes from Human Drug Targets, Human Druggable Genome, and Human Genome libraries at 2000 cells/well in 384-well plates followed by cell viability measurement after 72 hrs. We measured IRF3 phosphorylation and expression of immune related genes to analyze the CGAS-STING pathway. Using peripheral blood and bone marrow derived CD34+ cells transduced with lentivirus expressing shRNA against FANCD2 we tested the effect of CGAS inhibitor in promoting CFU-GM and CFU-E. Results: A genome wide siRNA screen identified IRF7 as one of the top 5 hits whose diminished expression resulted in increased viability of F-D2 mutant PD20 cells. IRF7 along with IRF3 serve as downstream transcription factors including various Toll-like receptors, CGAS-STING pathways, and other pattern recognition innate immune receptors. Using pIRF3 as an activation readout for CGAS-STING pathway we identified that both FANCD2 and FANCA mutant cells have elevated pIRF3 levels. The elevated pIRF3 levels were further increased upon addition of mitomycin C (MMC) in FA mutant cells as compared to gene corrected cells. The pIRF3 levels decreased upon both genetic KD and small molecule inhibition of both CGAS and STING. In line with increased pIRF3 FA mutant cells showed increased gene expression of various interferon-regulated genes, which were then also reduced by CGAS inhibitor. Overexpression of RNAseH1 partially reduced pIRF3 levels in FA mutant cells indicating that the RNA:DNA hybrids whose levels are elevated in FA mutant cells is one of the ligands of CGAS. Most importantly 10µM of CGAS inhibitor RU.51 was able to rescue FA-A mutant cell line 6914 cells from low dose MMC-induced cell death showing that the sensitivity of FA mutant cells to DNA damaging agents was at least partially driven by CGAS-STING pathway. We then tested if the protective effect of CGAS inhibitor extended to human CD34+ cells deficient in FA genes. 20µM of RU.51 was able to rescue the CFU-GM and CFU-E defects of CD34+ cells carrying lentiviral integrated shRNA against FANCD2 to a level to that in cells expressing non-silencing shRNAs. Interferon-regulated gene expression was also elevated in the FANCD2-deficient CD34+ cells, which was reversed by treatment with cGAS inhibitor. Conclusions: It has previously been shown that FA patients have elevated TNF-alpha, IFN-gamma levels, and NFkbactivation, and interferon alpha administration hastens the onset of bone marrow failure in FA patients. Our results show that FA mutant cells have elevated CGAS-STING signaling resulting in heightened interferon gene expression. Elevated R-loop levels in FA cells act as trigger for the CGAS-STING pathway. CGAS inhibitor is able to rescue low dose MMC-induced cell death in FA mutant fibroblasts and also restores the CFU-GM and CFU-E forming ability of CD34+ cells. These results indicate that CGAS-STING pathway is a pharmacologically tractable target to reverse the hematopoietic stem cell exhaustion seen in FA patients.

C5aR2 Regulates STING-Mediated Interferon Beta Production in Human Macrophages.

The complement system mediates diverse regulatory immunological functions. C5aR2, an enigmatic receptor for anaphylatoxin C5a, has been shown to modulate PRR-dependent pro-inflammatory cytokine secretion in human macrophages. However, the specific downstream targets and underlying molecular mechanisms are less clear. In this study, CRISPR-Cas9 was used to generate macrophage models lacking C5aR2, which were used to probe the role of C5aR2 in the context of PRR stimulation. cGAS and STING-induced IFN-β secretion was significantly increased in C5aR2 KO THP-1 cells and C5aR2-edited primary human monocyte-derived macrophages, and STING and IRF3 expression were increased, albeit not significantly, in C5aR2 KO cell lines implicating C5aR2 as a regulator of the IFN-β response to cGAS-STING pathway activation. Transcriptomic analysis by RNAseq revealed that nucleic acid sensing and antiviral signalling pathways were significantly up-regulated in C5aR2 KO THP-1 cells. Altogether, these data suggest a link between C5aR2 and nucleic acid sensing in human macrophages. With further characterisation, this relationship may yield therapeutic options in interferon-related pathologies.

Expression of constitutively active TβRI leads to attenuation of ovalbumin-induced allergic airway inflammation associated with augmented M2 polarization of alveolar macrophage

BackgroundTransforming growth factor-β (Tgf-β) plays an important role in the pathogenesis of asthma through the regulation of T cells and airway epithelium. Its functions in alveolar macrophage (AM) during allergic airway inflammation remain unknown. MethodsA murine asthma model was induced with ovalbumin (ova) in TβRICA/Fsp1-Cre transgenic mice expressing constitutively active Tgf-β receptor type I (TβRICA) under the control of Fsp1-Cre transgene. Cells in the bronchoalveolar lavage (BAL) were collected to study immune cell infiltration in the lungs. Cytokine levels in BAL fluid were measured by enzyme-linked immunoassay (ELISA). Lungs were sectioned and stained with hematoxylin and eosin, periodic acid-Schiff, and trichrome for histopathologic evaluation. AMs were assessed by flow cytometry and were sorted for quantitative polymerase chain reaction analysis. ResultsOur data indicated that TβRICA transcripts were induced in AMs of TβRICA/Fsp1-Cre mice. Following the ova challenges, TβRICA/Fsp1-Cre mice exhibited reduced cellular infiltration of the airway, reduced pulmonary fibrosis, and reduced bronchial mucus secretion as compared to ova-challenged wild-type mice. An alternatively activated macrophage (M2) polarization was significantly elevated in the lungs of ova-challenged TβRICA/Fsp1-Cre mice as reflected by increased numbers of AMs expressing M2 subtype marker, CD163, in the lungs and enhanced expression of CCR2 and CD206 in AMs. Moreover, TβRICA/Fsp1-Cre AMs showed augmented expression of transcription factors, Foxo1, and IRF4, which are known to be positive regulators for M2 polarization. ConclusionsExpression of TβRICA in AMs promoted M2 polarization and ameliorated allergic airway inflammation in an ova-induced asthma mouse model.

Role of macrophage colony stimulating factor and interferon regulatory factor 7 in modulating the immune profile of mouse testicular macrophages

Testicular macrophages (TM) are critical for the function of the testis by regulating homeostasis and inflammatory responses. However, the mechanisms by which TM fulfil these roles remain elusive. In this study, we explored the impact of two key testicular microenvironmental factors, namely 25-hydroxycholesterol (25HC), an oxysterol related to sex hormones and macrophage colony-stimulating factor (M-CSF), a factor crucial for macrophage survival and differentiation, on the regulation of the TM phenotype. Specifically, we examined their role in controlling the expression of the transcription factor interferon regulatory factor 7 (Irf7), a factor critical for maintaining the alternative macrophage phenotype. To achieve this, we used an in vitro bone marrow-derived macrophage (BMDM) model as a surrogate for TM to investigate the roles of 25HC and M-CSF in regulating the expression of Irf7 during the polarization of murine TM. M-CSF was identified as the main regulator of Irf7 expression, while 25HC production is a consequence of Irf7 activation in BMDM. In turn, 25HC plays a role in a negative feedback loop on the expression levels of Irf7 in BMDM. Using flow cytometry in Irf7-/- mouse testis the CD64loMHChi TM subpopulation was found to be decreased. Together with lower IL-10 protein levels in Irf7-/- TM this indicates a shift towards an M1-like macrophage profile. In summary, our data indicates that M-CSF could act as an inducer of high Irf7 expression levels in the mouse testis. However, the exact role of the high 25HC concentration in the testis in maintaining the local immune milieu still needs further study.

The Exposure to Lead (Pb) Exacerbates Immunological Abnormalities in BTBR T+ Itpr3tf/J Mice through the Regulation of Signaling Pathways Relevant to T Cells.

Autism spectrum disorder (ASD) is a common neurodevelopmental illness characterized by abnormal social interactions, communication difficulties, and repetitive and limited behaviors or interests. The BTBR T+ Itpr3tf/J (BTBR) mice have been used extensively to research the ASD-like phenotype. Lead (Pb) is a hazardous chemical linked to organ damage in the human body. It is regarded as one of the most common metal exposure sources and has been connected to the development of neurological abnormalities. We used flow cytometry to investigate the molecular mechanism behind the effect of Pb exposure on subsets of CD4+ T cells in the spleen expressing IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Furthermore, using RT-PCR, we studied the effect of Pb on the expression of numerous genes in brain tissue, including IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Pb exposure increased the population of CD4+IFN-γ+, CD4+T-bet+, CD4+STAT1+, CD4+STAT4+, CD4+IL-9+, CD4+IRF4+, CD4+IL-22+, and CD4+AhR+ cells in BTBR mice. In contrast, CD4+IL-10+ and CD4+Foxp3+ cells were downregulated in the spleen cells of Pb-exposed BTBR mice compared to those treated with vehicle. Furthermore, Pb exposure led to a significant increase in IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, and AhR mRNA expression in BTBR mice. In contrast, IL-10 and Foxp3 mRNA expression was significantly lower in those treated with the vehicle. Our data suggest that Pb exposure exacerbates immunological dysfunctions associated with ASD. These data imply that Pb exposure may increase the risk of ASD.

EZH2 Inhibition Overcomes Immunomodulatory Drug (IMiD) Resistance in Multiple Myeloma Cell Lines in a Cereblon Pathway Dependent Manner

Introduction Immunomodulatory agents (IMiDs) are effective therapies for multiple myeloma (MM), approved for use at all stages of disease. IMiDs function as molecular glues at cereblon (CRBN), part of the CRL4 CRBN E3 ubiquitin ligase complex, designating neo-substrates for proteasomal degradation. In myeloma cells the B cell transcription factors Ikaros and Aiolos are degraded, leading to downregulation of IRF4 which is critical for myeloma cell survival. Novel CRBN ligase modulators (CELMoDs) are currently in early phase clinical trials and have demonstrated activity in patients refractory to IMiDs, but resistance inevitably develops over time. EZH2 is a histone methyltransferase which acts at histone 3 lysine 27 (H3K27) leading to trimethylation (H3K27me3). The aberrant expression of EZH2 leads to abnormal gene repression, contributing to tumour development and progression. EZH2 inhibitors (EZH2i) have been shown to overcome drug resistance in several cancers and we hypothesized that combining EZH2i with IMiDs/CELMoDS would overcome resistance to these agents in MM cell lines. Methods MM cell lines KMS11, RPMI and MOLP8 were used as models of IMiD/CELMoD resistance and MM.1S as a control model of sensitivity. The isogenic pair of KMS11 with CRBN knockout (KMS11 CRBN KO) was established using CRISPR-Cas9. Following concentration/duration optimisation, cell lines were treated with EZH2i (Tazemetostat) 0.25-1µM or DMSO control for 5 days alone, and then in combination with IMiD (Lenalidomide, Len, 0-20 µM, Pomalidomide, Pom, 0-8 µM) or CELMoD (Iberdomide, CC-220, 0-2 µM and Mezigdomide, CC-92480, 0-0.1uM) for a further 5 days. Cell viability was measured using CellTiter Blue® and the level of synergy assessed using SynergyFinder (Bliss model). The effect on apoptosis and cell cycle was examined using flow cytometry after AnnexinV-PI and/or DAPI staining respectively. To elucidate the mechanism of synergy, the effect of the single agents and combinations on key CRBN pathway effectors (CRBN, Ikaros/Aiolos, IRF4) were examined using WB and qPCR. ChIP-PCR was employed to assess the changes in enrichment of H3K27me3 and key MM transcription factors (Ikaros/Aiolos) within the IRF4 promoter region upon EZH2i treatment. Results MM.1S cells were sensitive to IMiDs (Len/Pom) and CELMoDS (CC-220/CC-92480) with GI50s of 0.279 µM, 0.08 µM, 2 nM and 0.3 nM respectively. KMS11, MOLP8 and RPMI cell lines were resistant at more than 10x the GI50 in MM.1S. In these models of resistance, the cells express functional CRBN protein, demonstrated by the degradation of neo-substrates Ikaros/Aiolos. However, whilst their degradation results in reduced IRF4 expression and cell death in sensitive cells (e.g. MM.1S), this does not occur in the resistant models suggesting an uncoupling of Ikaros/Aiolos control of IRF4 expression in the resistant setting. Combining EZH2i with IMiDs/CELMoDs led to a reduction in cell viability (e.g. CC-220 shown in Fig.1A) and demonstrated synergy across resistant cell lines (KMS11, MOLP8 and RPMI). An increase in apoptosis was seen using AnnexinV/PI staining, with no demonstrable change in cell cycle. WB and qPCR results showed a significant reduction in IRF4 mRNA and protein with the combination treatment. The combination of EZH2i and IMiD/CELMoDs did not reduce cell viability or IRF4 expression in the KMS11 CRBN KO cell line ( Fig.1A), indicating that the observed synergistic effect is CRBN-dependent. Compared to DMSO control, EZH2i increased IRF4 mRNA, with a reduction of H3K27me3 enrichment observed within the IRF4 promoter region by ChIP-PCR in KMS11 ( Fig.1B). In addition, EZH2i also enhanced Ikaros and Aiolos enrichment within the IRF4 promoter region ( Fig.1B). Conclusions Our results suggest that combining EZH2i with IMiDs/CELMoDs can overcome resistance to these agents in MM cell line models, with synergy that is CRBN-dependent. By examining the key components of the CRBN pathway we identified that EZH2i reduced H3K27me3 and increased Ikaros and Aiolos association at the IRF4 promoter, suggesting a possible re-coupling of Ikaros/Aiolos to IRF4 expression, which may be responsible for reinstating IMiD/CELMoD activity, driving the synergistic effect seen.

SDC2 Expression Is Increased in Myeloma Cells in Response to Loss of Pro-Survival Surface Proteins, CD28 and CD86

Introduction: While recent advances in multiple myeloma (MM) have improved overall survival, most patients will succumb to late-stage and drug resistant disease. Plasma cell (PC) survival outside of the bone marrow is characteristic of late-stage myeloma and pivotal to disease progression. PCs are typically restricted to the bone marrow microenvironment, as they are dependent on stromal interactions for survival. One such interaction is the engagement of CD28 and its ligand CD86, proteins which are best understood for their roles in T-cell activation by antigen presenting cells. However, myeloma cells can express both CD28 and CD86 on the cell surface. Notably, high CD28 expression is seen in 59% of medullary recurrences, and over 90% of extramedullary relapses. Moreover, high CD86 expression is more common in malignant plasma cells as compared to precursor stages and has a reported role in driving expression of IRF4. We have previously shown that expression of CD28 and CD86 is associated with worse outcomes than that observed in patients with low expression in the MMRF CoMMpass study (NCT01454297). We have also shown that silencing of CD28 or CD86 results in cell death and loss of IRF4 protein expression in human myeloma cell lines (HMCL). We confirmed these studies using CRISPR/Cas9 editing and demonstrated loss of proliferation and induction of cell death in HMCLs. However, the mechanism(s) by which CD28 and CD86 contribute to MM cell survival is not fully understood. Methods: To shed light on the mechanistic basis of CD28 and CD86 in the survival of MM, we used CRISPR/Cas9 editing to disrupt expression from RPMI8226 myeloma cells. We then passaged transduced cells to select for populations that can survive loss of CD28 or CD86. These passaged cells were sorted into populations that either retained CD28 or CD86 expression as transduction controls (High), or populations that underwent CRISPR/Cas9 depletion (Low). Surface expression of CD28 High/Low and CD86 High/Low populations was confirmed via flow cytometry and were then processed for RNA isolation and cDNA library preparation. RNA-sequencing was performed using paired-end sequencing, and data were aligned using the STAR aligner. Differentially expressed genes were determined using edgeR with an FDR ≤ 0.05 and a fold-change ≥ 2.0. Validation of RNA-sequencing results was further assessed via western blot and flow cytometry. Results: Analysis of RNA-sequencing results indicated that 57 genes were differentially expressed between High and Low cells, with 23 genes common to both CD28 and CD86 High/Low comparisons. (FDR<0.05). Notably, our results reveal that loss of CD28 or CD86 is associated with an increase in mRNA expression of Syndecan-2 (SDC2). In addition, our preliminary experiments evaluating SDC2 surface expression via flow cytometry suggest that both CD28 Low and CD86 Low populations have increased expression of SDC2. Additionally, in the RNA-seq analysis we also observed that CD28 Low and CD86 Low cells were also able to sustain mRNA expression of IRF4. This result was later confirmed via western blot, demonstrating CD28 Low and CD86 Low cells sustain protein expression of IRF4. Conclusions: Our results demonstrate that RPMI8226 cells that lack CD28 or CD86 expression have increased mRNA and protein expression of SDC2, a member the heparan sulfate proteoglycan family. Since SDC2 expression has also been associated with a number of cancers including colorectal, lung, and pancreatic cancers, these results provide insight to potential alternative pathways that contribute to myeloma survival independent of CD28 and CD86 signaling. We also confirmed that CD28- and CD86-depleted populations were able to maintain IRF4 mRNA/protein levels. This indicates myeloma cells have mechanisms to sustain IRF4 expression in addition to CD28 and CD86 signaling. These results are also consistent with previous findings illustrating importance of IRF4 in myeloma. Finally, we have developed CRISPR/Cas9-depleted CD28 and CD86 populations in additional HMCLs. We are currently in the process of isolating CD28 High/Low and CD86 High/Low populations for additional analysis and will present these upcoming results.

Mechanisms of Cytokine-Induced NK Cell Therapy: IL-12/15/18 Induce a Unique Transcriptional, Epigenetic, and Functional Human NK Cell Memory Program

NK cells are innate lymphoid cells critical for mediating anti-tumor responses and are being advanced as a cancer immunotherapy. While conventional (c) NK cell-based therapies have been explored in clinical trials, challenges related to cancer target recognition, in vivo persistence, and functionality result in limited activity. To overcome these challenges, NK cell memory programs have been investigated. Cytokine-induced memory-like (CIML or ML) NK cells are generated after brief activation with IL-12, IL-15, and IL-18, resulting in enhanced functionality, cytotoxicity, and in vivo persistence. ML NK cells have been advanced in multiple clinical trials for leukemia with promising results. Despite this translational progress, the underlying cellular dynamics and molecular mechanisms of ML reprogramming remain unclear. Elucidating these molecular programs may optimize NK therapeutics and reveal new translational strategies. We hypothesized that transcriptional and epigenetic mechanisms contribute to ML NK cell heterogeneity arising after IL-12/15/18 activation. To test this, NK cells from healthy donors were activated with IL-12/15/18 (to become ML) or IL-15 (to maintain cNK) and differentiated in vitro for 7 days (Fig 1, n=3-40 donors), followed by the assessment of their transcriptome (CITE-seq) and epigenome (ATAC-seq). CITE-seq clustering revealed unexpected heterogeneity with ~50% clustering with cNK cells (effector, effcNK) and ~50% forming a unique enriched ML cluster (eML). eML clusters were significantly increased in frequency in the IL-12/15/18 condition, compared to cNK (Mean±SEM=53±10.5% for IL-12/15/18, 6.2±1.1% for IL-15, p<0.0001) and discovered to expressed ENTPD1 (CD39). Based on CITE-seq, a flow cytometry strategy was developed to distinguish eML NK cells from effector cNK and cNK cells. Next, we identified eML NK cells 3-4 weeks after adoptive transfer of IL-12/15/18 activated donor NK cells across three different clinical trials (mean=13.3±1.9%, p<0.001). eML NK cells produced significantly more IFNg compared to cNK cells and effector cNK cells when stimulated with leukemia targets (Fig. 2, p<0.0001), and had enhanced cytotoxicity compared to cNK cells (EC50 of eML=0.862, EC50 of cNK=2.223, p<0.0001). Bulk ATAC-seq on flow-sorted populations revealed that eML NK cells have a distinct global epigenetic profile with >4,500 significant differentially accessible regions (DARs) compared to effcNK, while effcNK and cNK cells were epigenetically similar. Further, we interrogated which transcription factors are enriched in DARs of eML NK cells revealing TBOX and IRF family motifs. Within CITE-seq, the eML cluster was comprised of two subsets (eML-1 and eML-2). eML1 had increased expression of the transcription factors TCF7 and TOX2. In contrast, eML-2 had increased expression of IRF4 and PRDM1, as well as terminal exhaustion genes, including LAG3 and TIGIT (p<0.05). To evaluate eML-1 and eML-2 function, we performed CITE-seq after stimulation of eML NK cells with leukemia targets. eML-1 NK cells were the most functional, with significantly increased IFNG compared to eML-2, while eML-2 was hypofunctional and marked by high TOX expression (p<0.05). These findings suggest that optimizing strategies that lead to eML-1 differentiation may enhance the therapeutic activity of IL-12/15/18-induced ML NK cells. In summary, we identify heterogeneity following IL-12/15/18 activation of NK cells, resulting in multiple cellular fates. Further, these data identify an eML NK cell population with a distinct single-cell transcriptional and epigenetic program that directly manifests as enhanced NK cell functional response to leukemia.

Expression of Irf3 and Irf7 in tumour cells drives pancreatic cancer development and progression

Expression of Irf3 and Irf7 in tumour cells drives pancreatic cancer development and progression

Tumor-Myeloid Cell Crosstalk Pathways Associated with Abscopal Responses in Breast Cancer

To identify predictive biomarkers for combined radiotherapy (RT) and immune checkpoint inhibitor (ICI) therapy, which can induce systemic anti-tumor immune responses (i.e., "abscopal" responses) in breast cancer. Four Trp53-/- Balb/c breast cancer syngeneic allograft models with low tumor mutational burden and resistance to dual ICI (anti-PD1 and anti-CTLA4, BioXcell) were used. Two lines exhibited abscopal responses to RT (8Gyx3) plus ICI combination therapy (i.e., "abscopal models"), whereas the other two did not (i.e., "non-abscopal models"). We performed spatial transcriptomics analysis (GeoMx whole transcriptome assay) of pan-cytokeratin positive (panCK+) tumor and tumor-adjacent CD45+ immune cell segments in orthotopically implanted tumors 10 days after initiating treatment with IgG control, RT alone, ICI alone, or RT/ICI combination (N>5 per group). Genes selectively induced by RT/ICI in panCK+ and CD45+ segments in abscopal models and were not induced in non-abscopal models were identified using two-tailed t-tests and FDR correction for multiple testing (FDR<5%). In vitro analyses of RT-induced secreted inflammatory signaling were conducted by exposing supernatant from irradiated (8Gy) tumor cells to a RAW264.7 macrophage cell line expressing an interferon-inducible luciferase reporter construct, followed by luciferase signal quantification (Invivogen). Hierarchical clustering of immune-related genes in CD45+ immune segments from breast cancer models with abscopal responses to RT/ICI revealed global differences in tumor-adjacent immune profiles at baseline and after RT/ICI treatment. Abscopal responsive breast cancer models at baseline showed an enrichment of myeloid cell subtypes expressing complement protein C1q, Ccl8, and Csf1r in tumor-adjacent CD45+ immune cells, and CD8+ T cells expressing Ccl5. 10 days after treatment with RT/ICI, tumor-adjacent CD45+ immune cells in abscopal models were enriched in Cxcl10, Irf7, and FcgRIV, whereas these genes were not induced in non-abscopal models. CyTOF analyses confirmed the induction of inflamed professional antigen presenting cells (CD11c+CD80+IA/IE+) by RT/ICI in abscopal models. Within panCK+ tumor segments, RT/ICI in abscopal models selectively induced Isg15 and Zbp1, both of which are involved in secreted inflammatory signaling. Consistently, RT-induced secreted inflammatory signaling from tumor cells to macrophages evaluated in vitro was significantly greater in abscopal models compared to non-abscopal models. Breast cancer models with abscopal responses to RT/ICI show increased Isg15- and Zbp1-dependent secreted inflammatory crosstalk that activates Cxcl10 and Irf7 expression in tumor-adjacent myeloid cells, which warrants further investigation as a potential predictive biomarker for combined RT/ICI therapy in breast cancer.

WEE1 Inhibition Promotes Radiotherapy-Induced Myeloid Suppressor Cells through cGAS/STING-STAT3-IRF3 Pathway

This study is the first to propose the WEE1 inhibitor combined with radiotherapy affect tumor microenvironment through cGAS/STING-STAT3-IRF3 pathway, and the effects of DNA damage caused by WEE1 inhibitor combined with radiotherapy on cGAS/STING-STAT3-IRF3 pathway and MDSC (myeloid-derived inhibitory cells) in tumor microenvironment were systematically expounded. MDA-MB-231 cells, A549 cells and HT29 cells were irradiated with 2GY, 12GY and 20GY X-ray respectively, and cell cycle tests were conducted. WEE1 inhibitor combined with radiotherapy was used to cells. EDU kit was used to detect the changes of DNA damage repair and proliferation of tumor cells after irradiation. Western-blot experiment was used to detect the expression changes of cGAS/STING-STAT3-IRF3 pathway related proteins TBK1, P-TBKI, Sting, P-Sting, IRF3, P-IRF3 and cGAS. Immunofluorescence double-label experiment was used to detect the expression changes of target molecules in tumor cell control group and experimental group. The tumor-bearing model of mice was established, and the survival time and tumor volume of mice were detected. The differential expression of CD11B, LY6C, CD45, IFN-γ, CD8a and other immune-related molecules was detected by flow cytometry. The results of cell cycle experiment show that WEE1 inhibitor combined with radiotherapy can effectively inhibit G2/M phase of cell cycle compared with the control group. Compared with the control group, the tumor volume of mice in WEE1 inhibitor combined with radiotherapy was significantly reduced and the survival time of mice was significantly prolonged. The infiltration of CD8T cells and the secretion level of IFN-γ in tumor tissues of mice were detected by flow cytometry. The WEE1 inhibitor combined with radiotherapy group was significantly higher than that of the control group. The results of immunofluorescence double-label experiment showed that WEE1 inhibitor combined with radiotherapy reduced the infiltration of MDSC in tumor microenvironment after radiotherapy compared with the control group. Western-blot results showed that inhibition of WEE1 could significantly down-regulate the expression of TBK1, P-TBKI, Sting, P-Sting, IRF3, P-IRF3, P-STAT3 and cGAS through STATE3, which indicated that the radiation resistance induced by WEE1 inhibitor combined with radiotherapy was related to cGAS/STING-STAT3-IRF3 pathway. WEE1 targeted therapy combined with radiotherapy can improve the microenvironment of inhibitory tumor and effectively alleviate radiotherapy resistance. The combination of WEE1 inhibitor and radiotherapy significantly inhibited the growth of tumor compared with single treatment, and the combined treatment achieved obvious inhibitory effect on tumor.

Epigenetic and transcriptional regulation of CCL17 production by glucocorticoids in arthritis

Glucocorticoids (GCs) are potent anti-inflammatory agents and are broadly used in treating rheumatoid arthritis (RA) patients, albeit with adverse side effects associated with long-term usage. The negative consequences of GC therapy provide an impetus for research into gaining insights into the molecular mechanisms of GC action. We have previously reported that granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced CCL17 has a non-redundant role in inflammatory arthritis. Here, we provide molecular evidence that GCs can suppress GM-CSF-mediated upregulation of IRF4 and CCL17 expression via downregulating JMJD3 expression and activity. In mouse models of inflammatory arthritis, GC treatment inhibited CCL17 expression and ameliorated arthritic pain-like behavior and disease. Significantly, GC treatment of RA patient peripheral blood mononuclear cells exvivo resulted in decreased CCL17 production. This delineated pathway potentially provides new therapeutic options for the treatment of many inflammatory conditions, where GCs are used as an anti-inflammatory drug but without the associated adverse side effects.

Molecular and functional characterization of viperin in golden pompano, Trachinotus ovatus

The radical S-adenosyl methionine domain-containing protein 2 (RSAD2), also known as viperin, plays a momentous and multifaceted role in antiviral immunity. However, the function of viperin is uninvestigated in golden pompano, Trachinotus ovatus. In the present study, a viperin homolog, named To-viperin, was cloned and characterized from golden pompano, and its role in response to grouper iridovirus (SGIV) and nervous necrosis virus (NNV) infection was investigated. The whole open reading frame (ORF) of To-viperin was composed of 1050 bp and encoded a polypeptide of 349 amino acids with 70.66%–83.51% identity with the known viperin homologs from other fish species. A variable N-terminal domain, a highly conserved C-terminal domain, and a conserved middle radical SAM domain (aa 61–271) with the three-cysteine motif CxxCxxC was found in To-viperin sequence. Expression analysis showed that To-viperin was constitutively expressed in all tested organs and was located mainly in the ER of golden pompano cells. Treatments with SGIV, poly I: C, or NNV could induce the up-regulation of viperin to varying degrees. The ectopic expression of To-viperin in vitro significantly reduced the viral titer of SGIV and NNV. Furthermore, To-viperin overexpression enhanced the expression of IFNc, IRF3, and ISG15 genes as well as, to a lesser extent, the IL-6 gene. In summary, our results suggested that the function of viperin is likely to be conserved in fish specise, as observed in other vertebrates, shedding light on the evolutionary conservation of viperin.

Fish Rhbdd3 positively regulates IFN response through RIG-I signaling pathway

Rhomboid domain-containing protein 3 (Rhbdd3) is a member of the rhomboid family, which can modulate the innate immune response in mammals. Nonetheless, the function and regulatory mechanism of fish Rhbdd3 during viral infection have not been characterized. In this study, Rhbdd3 was firstly cloned from common carp (Cyprinus carpio) and nominated as CcRhbdd3. Phylogenetically characterization showed that CcRhbdd3 shared a relatively long evolutionary distance with its mammalian homologs. In vivo experiment demonstrated that spring viraemia of carp virus (SVCV) infection promoted the expression of CcRhbdd3 in the liver, spleen, kidney and muscle tissues. Furthermore, overexpression of CcRhbdd3 significantly inhibited SVCV propagation, whereas knockdown of CcRhbdd3 markedly promoted SVCV replication in susceptible cells. RNA-seq and following validation showed that CcRhbdd3 overexpression upregulated the expression of several RIG-I signaling related genes, including TRIM25, TRAF2, MDA5, LGP2, IFN1, IFN3, RIG-I, IRF3 and ISG15. Moreover, CcRhbdd3 promoted the expression of NF-κB, a central immune regulator. Subcellular localization experiments showed that CcRhbdd3 was primarily distributed in the cytoplasm and co-localized with Rab5 in the early endosomes. Truncation experiments further demonstrated that the C-terminus containing the ubiquitin-binding associated domain, was crucial for both the subcellular localization and antiviral activity of CcRhbdd3. The findings in this study provide new insight into the host antiviral mechanism against aquatic RNA virus infection, and will facilitate the development of therapeutic strategies for the infection of SVCV.

BET proteins inhibitor JQ1 impairs GM-CSF-promoted peritoneal macrophage self-renewal and IL-4-induced alternative polarization

Peritoneal macrophages (PMs), which resided in peritoneal cavity, are crucial to maintain tissue homeostasis and immunity. Macrophage self-renewal and polarization states are critical for PM population homeostasis and function. However, the underlying molecular mechanism that regulates self-renewal and polarization of PMs is still unclear and needs to be explored. Here, we demonstrated that PMs self-renewal was stimulated by granulocyte macrophage colony-stimulating factor (GM-CSF), but not by macrophage colony-stimulating factor (M−CSF). Pharmacological inhibition of Bromodomain & Extraterminal (BET) Proteins by either JQ1 or ARV-825 significantly reduced GM-CSF-dependent peritoneal macrophage self-renewal by abrogating cell proliferation and decreasing self-renewal-related gene expression, such as MYC and Klf4, at transcriptional and protein levels. In addition, transcriptomic analysis showed that JQ1 blocked alternative PMs polarization by downregulating key transcriptional factor IRF4 expression, but not the activation of AKT or STAT6 in PMs. These findings illustrated that the significance of BET family proteins in GM-CSF-induced PMs self-renewal and IL-4-induced alternative polarization.

Inhibition of PI3K p110δ activity reduces IgE production in IL-4 and anti-CD40 stimulated human B cell cultures.

Phosphoinositide 3-kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL-4 and anti-CD40 antibodyto induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT-PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti-CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL-4 and anti-CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre-like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.

An in vitro model of human hematopoiesis identifies a regulatory role for the aryl hydrocarbon receptor

AbstractIn vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell–free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand–activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood–derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.

Higher pNRF2, SOCS3, IRF3, and RIG1 Tissue Protein Expression in NASH Patients versus NAFL Patients: pNRF2 Expression Is Concomitantly Associated with Elevated Fasting Glucose Levels.

Non-alcoholic fatty liver disease (NAFLD) embraces simple steatosis in non-alcoholic fatty liver (NAFL) to advanced non-alcoholic steatohepatitis (NASH) associated with inflammation, fibrosis, and cirrhosis. NAFLD patients often have metabolic syndrome and high risks of cardiovascular and liver-related mortality. Our aim was to clarify which proteins play a role in the progression of NAFL to NASH. The study investigates paraffin-embedded samples of 22 NAFL and 33 NASH patients. To detect potential candidates, samples were analyzed by immunohistochemistry for the proteins involved in innate immune regulation, autophagy, apoptosis, and antioxidant defense: IRF3, RIG-1, SOCS3, pSTAT3, STX17, SGLT2, Ki67, M30, Caspase 3, and pNRF2. The expression of pNRF2 immunopositive nuclei and SOCS3 cytoplasmic staining were higher in NASH than in NAFL (p = 0.001); pNRF2 was associated with elevated fasting glucose levels. SOCS3 immunopositivity correlated positively with RIG1 (r = 0.765; p = 0.001). Further, in NASH bile ducts showed stronger IRF3 immunostaining than in NAFL (p = 0.002); immunopositive RIG1 tissue was higher in NASH than in NAFL (p = 0.01). Our results indicate that pNRF2, SOCS3, IRF3, and RIG1 are involved in hepatic lipid metabolism. We suggest that they may be suitable for further studies to assess their potential as therapeutics.

Proteomic analysis of EPC cell replication effect of Hsp70 on SVCV infection at different temperatures

Spring Viremia of Carp Virus (SVCV) has a high fatality rate and is highly contagious. Once aquaculture is infected, significant economic losses will ensue. In this study, the influence of five temperatures on the activity and proteomic alterations of SVCV-infected carp epithelial cell line (EPC) was investigated. MTT results showed that at 26 °C, the activity of both uninfected and infected cells with the SVCV virus peaked, while virus titers peaked at 31 °C. For further research, 26 °C and 31 °C infected for 24 h were chosen. Flow cytometry revealed that the percentage of apoptosis increased at 26 °C and 31 °C. At 31 °C, qPCR results revealed that the expression of the SVCV-G gene was significantly different from that of the control group (p < 0.05). According to the results of proteomics, 1665 distinct proteins were identified. TLR3, IRF3, IFN-β, and SVCV-G expression was significantly upregulated following HSP70 knockdown. Through screening and validation, we explored the influence of Hsp70 replication with promising results in order to gain a deeper understanding of the pathogenesis of SVCV and to develop new treatment and prevention strategies for SVCV.