A fluorescent reporter model for the visualization and characterization of TDC.

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T-cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecules (MHC)-II, and the T-cell receptor (TCR). Despite thorough characterization, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Here, we took advantage of Zbtb46-GFP reporter mice to explore the frequency and localization of TDC in different tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC sorted from Zbtb46-GFP mice have a gene signature that is distinct from conventional T cells and DC. In addition, this reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.

Zbtb46-dependent altered developmental program in embryonic stem cell-derived blood cell progenitors.

Zbtb46 is a recently identified dendritic cell (DC)-specific transcription factor with poorly defined biology. Although Zbtb46 is highly expressed in conventional DCs, evidence also points to its presence in erythroid progenitors and endothelial cells suggesting that this factor might influence the early hematopoietic development. Here, we probe the effect of this transcription factor in embryonic stem cell (ESC)-derived blood cell progenitors using chemically inducible mouse cell lines. Unexpectedly, forced expression of this protein elicited a broad repressive effect at the early stage of ESC differentiation. Ectopic expression of Zbtb46 interfered with the mesoderm formation and cell proliferation was also negatively impacted. More importantly, reduced number of CD11b+ myeloid blood cells were generated from ESC-derived Flk1+ mesoderm cells in the presence of Zbtb46. Consistent with this finding, our gene expression profiling revealed that numerous myeloid and immune response related genes, including Irf8, exhibited lower expression in the Zbtb46-primed cells. Despite these repressive effects, however, Zbtb46 overexpression was associated with enhanced formation of erythroid blood cell colonies and increased adult hemoglobin (Hbb-b1) expression at the early phase of ESC differentiation. Moreover, elevated percent of CD105 (Endoglin) positive cells were detected in the Zbtb46-primed samples. In summary, our results support that Zbtb46 suppresses the ESC-derived myeloid development and diverts mesoderm cells toward erythroid developmental pathway. Moreover, our transcriptomic data provide a resource for exploration of the Zbtb46 regulatory network in ESC-derived progenitors.

Low-dose 2-deoxy glucose stabilises tolerogenic dendritic cells and generates potent in vivo immunosuppressive effects.

Cell therapies for autoimmune diseases using tolerogenic dendritic cells (tolDC) have been promisingly explored. A major stumbling block has been generating stable tolDC, with low risk of converting to mature immunogenic DC (mDC), exacerbating disease. mDC induction involves a metabolic shift to lactate production from oxidative phosphorylation (OXPHOS) and ß-oxidation, the homeostatic energy source for resting DC. Inhibition of glycolysis through the administration of 2-deoxy glucose (2-DG) has been shown to prevent autoimmune disease experimentally but is not clinically feasible. We show here that treatment of mouse bone marrow-derived tolDC ex vivo with low-dose 2-DG (2.5 mM) (2-DGtolDC) induces a stable tolerogenic phenotype demonstrated by their failure to engage lactate production when challenged with mycobacterial antigen (Mtb). ~ 15% of 2-DGtolDC express low levels of MHC class II and 30% express CD86, while they are negative for CD40. 2-DGtolDC also express increased immune checkpoint molecules PDL-1 and SIRP-1α. Antigen-specific T cell proliferation is reduced in response to 2-DGtolDC in vitro. Mtb-stimulated 2-DGtolDC do not engage aerobic glycolysis but respond to challenge via increased OXPHOS. They also have decreased levels of p65 phosphorylation, with increased phosphorylation of the non-canonical p100 pathway. A stable tolDC phenotype is associated with sustained SIRP-1α phosphorylation and p85-AKT and PI3K signalling inhibition. Further, 2-DGtolDC preferentially secrete IL-10 rather than IL-12 upon Mtb-stimulation. Importantly, a single subcutaneous administration of 2-DGtolDC prevented experimental autoimmune uveoretinitis (EAU) in vivo. Inhibiting glycolysis of autologous tolDC prior to transfer may be a useful approach to providing stable tolDC therapy for autoimmune/immune-mediated diseases.

ZBTB46, SPDEF, and ETV6: Novel Potential Biomarkers and Therapeutic Targets in Castration-Resistant Prostate Cancer.

Prostate cancer (PCa) is the second most common killer among men in Western countries. Targeting androgen receptor (AR) signaling by androgen deprivation therapy (ADT) is the current therapeutic regime for patients newly diagnosed with metastatic PCa. However, most patients relapse and become resistant to ADT, leading to metastatic castration-resistant PCa (CRPC) and eventually death. Several proposed mechanisms have been proposed for CRPC; however, the exact mechanism through which CRPC develops is still unclear. One possible pathway is that the AR remains active in CRPC cases. Therefore, understanding AR signaling networks as primary PCa changes into metastatic CRPC is key to developing future biomarkers and therapeutic strategies for PCa and CRPC. In the current review, we focused on three novel biomarkers (ZBTB46, SPDEF, and ETV6) that were demonstrated to play critical roles in CRPC progression, epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) drug resistance, and the epithelial-to-mesenchymal transition (EMT) for patients treated with ADT or AR inhibition. In addition, we summarize how these potential biomarkers can be used in the clinic for diagnosis and as therapeutic targets of PCa.

Revisiting the specificity of the MHC class II transactivator CIITA in classical murine dendritic cells in vivo.

Ciita was discovered for its role in regulating transcription of major histocompatibility complex class II (MHCII) genes. Subsequently, CIITA was predicted to control many other genes based on reporter and ChIP-seq analysis but few such predictions have been verified in vivo using Ciita-/- mice. Testing these predictions for classical dendritic cells (cDCs) has been particularly difficult, since Ciita-/- mice lack MHCII expression required to identify cDCs. However, recent identification of the cDC-specific transcription factor Zbtb46 allows the identification of cDCs independently of MHCII expression. We crossed Zbtb46gfp mice onto the Ciita-/- background and found that all cDC lineages developed in vivo in the absence of Ciita. We then compared the complete transcriptional profile of wild-type and Ciita-/- cDCs to define the physiological footprint of CIITA for both immature and activated cDCs. We find that CIITA exerts a highly restricted control over only the MHCII, H2-DO and H2-DM genes, in DC1 and DC2 cDC subsets, but not over other proposed targets, including Ii. These findings emphasize the caveats needed in interpreting transcription factor binding sites identified by in-vitro reporter analysis, or by ChIP-seq, which may not necessarily indicate their functional activity in vivo.

The dual nature of TDC - bridging dendritic and T cells in immunity.

TDC are hematopoietic cells with unique features that provide intriguing insights into the interplay between innate and adaptive immunity. They express a combination of conventional dendritic cell (DC) and T-cell markers and are found in secondary lymphoid organs (SLOs), lungs and liver of naïve mice, as well as in human blood. When analyzed ex vivo, TDC can behave either as DCs or as T cells, depending on the provided stimuli. Notably, TDC numbers and activation significantly increase in SLOs following viral infection, suggesting a potential role for TDC in antiviral immune responses. In this review, we discuss the properties of these fascinating cells, which call for more investigation on their physiological role during immune responses to both pathogens and tumors.

Dendritic cells in liver injury and fibrosis: shortcomings and promises.

The phenotype and function of liver dendritic cells (LDCs) are poorly understood. This Snapshot summarizes our current knowledge on LDCs in the healthy and injured liver, and their role in fibrosis progression and reversal. It also draws attention to various pitfalls in the current experimental design and conclusions based on available data.

CircZBTB46 Protects Acute Myeloid Leukemia Cells from Ferroptotic Cell Death by Upregulating SCD.

Circular RNAs (circRNAs) have been shown to be closely linked to the tumorigenesis and treatment response of hematological malignancies. However, the biological functions and clinical implications of circRNAs in acute myeloid leukemia (AML) remain largely unknown. CircRNA microarray datasets were analyzed to screen differentially expressed circRNAs in AML patients. It was found that circZBTB46 was significantly upregulated in AML patients and AML cells. Moreover, the expression of circZBTB46 was associated with the stages of AML patients and showed high sensitivity and specificity for diagnosing AML. Silencing of circZBTB46 inhibited AML cell proliferation and induced cell cycle arrest. Importantly, the depletion of circZBTB46 notably increased ferroptosis and enhanced RSL3-induced ferroptosis in AML cells. Mechanistically, circZBTB46 upregulated the expression of stearoyl-CoA desaturase 1 (SCD) possibly by acting as a miRNA sponge. Finally, the circZBTB46 knockdown repressed the tumor growth of AML in vivo. In conclusion, circZBTB46 protects AML cells from ferroptosis and promotes the proliferation by upregulating SCD, thus suggesting that circZBTB46 may be a potential therapeutic target for AML.

Comparative transcriptomic analysis of Illumina and MGI next-generation sequencing platforms using RUNX3- and ZBTB46-instructed embryonic stem cells.

Introduction: We have previously observed phenotypic and developmental changes upon the ectopic expression of the RUNX3 or the ZBTB46 transcription factors in mouse embryonic stem cell (ESC) derived progenitors. In this study, we evaluated the gene expression profiles of the RUNX3- and the ZBTB46-instructed murine ESCs with RNA-seq testing two next-generation sequencing technologies. Methods: We compared the DNA nanoball-based DNBSEQ G400 sequencer (MGI) with the bridge-PCR-based NextSeq 500 instrument (Illumina) for RNA sequencing. Moreover, we also compared two types of MGI sequencing reagents (Standard versus Hot-massive parallel sequencing (MPS)) with the DNBSEQ G400. Results: We observed that both sequencing platforms showed comparable levels of quality, sequencing uniformity, and gene expression profiles. For example, highly overlapping RUNX3- and ZBTB46-regulated gene lists were obtained from both sequencing datasets. Moreover, we observed that the Standard and the Hot-MPS-derived RUNX3- and ZBTB46-regulated gene lists were also considerably overlapped. This transcriptome analysis also helped us to identify differently expressed genes in the presence of the transgenic RUNX3 or ZBTB46. For example, we found that Gzmb, Gzmd, Gzme, Gdf6, and Ccr7 genes were robustly upregulated upon the forced expression of Runx3; on the other hand, Gpx2, Tdpoz4, and Arg2 were induced alongside the ectopic expression of Zbtb46. Discussion: Similar gene expression profile and greatly overlapping RUNX3- and ZBTB46-regulated gene sets were detected with both DNA sequencing platforms. Our analyses demonstrate that both sequencing technologies are suitable for transcriptome profiling and target gene selection. These findings suggest that DNBSEQ G400 represents a cost-effective alternative sequencing platform for gene expression monitoring. Moreover, this analysis provides a resource for exploration of the RUNX3- and ZBTB46-dependent gene regulatory networks.

FLT3L-dependent dendritic cells control tumor immunity by modulating Treg and NK cell homeostasis.

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector T cells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.

Identification of Two Novel Candidate Genetic Variants Associated With the Responsiveness to Influenza Vaccination.

Background: Annual vaccination is the most effective prevention of influenza infection. Up to now, a series of studies have demonstrated the role of genetic variants in regulating the antibody response to influenza vaccine. However, among the Chinese population, the relationship between genetic factors and the responsiveness to influenza vaccination has not been clarified through genome-wide association study (GWAS). Method: A total of 1,968 healthy volunteers of Chinese descent were recruited and 1,582 of them were available for the subsequent two-stage analysis. In the discovery stage, according to our inclusion criteria, 123 of 1,582 subjects were selected as group 1 and received whole-genome sequencing to identify potential variants and genes. In the verification stage, 29 candidate variants identified by GWAS were selected for further validation in 481 subjects in group 2. Besides, we also analyzed nine variants from previously published reports in our study. Results: Multivariate logistic regression analysis showed that compared with the TT genotype of ZBTB46 rs2281929, the TC + CC genotype was associated with a lower risk of low responsiveness to influenza vaccination adjusted for gender and age (Group 2: P = 7.75E-05, OR = 0.466, 95%CI = 0.319-0.680; Combined group: P = 1.18E-06, OR = 0.423, 95%CI = 0.299-0.599). In the combined group, IQGAP2 rs2455230 GC + CC genotype was correlated with a lower risk of low responsiveness to influenza vaccination compared with the GG genotype (P = 8.90E-04, OR = 0.535, 95%CI = 0.370-0.774), but the difference was not statistically significant in group 2 (P = 0.008). The antibody fold rises of subjects with ZBTB46 rs2281929 TT genotype against H1N1, H3N2,and B were all significantly lower than that of subjects with TC + CC genotype (P < 0.001). Compared with IQGAP2 rs2455230 GC + CC carriers, GG carriers had lower antibody fold rises to H1N1 (P = 0.001) and B (P = 0.032). The GG genotype of rs2455230 tended to be correlated with lower antibody fold rises (P = 0.096) against H3N2, but the difference was not statistically significant. No correlation was found between nine SNPs from previously published reports and the serological response to influenza vaccine in our study. Conclusion: Our study identified two novel candidate missense variants, ZBTB46 rs2281929 and IQGAP2 rs2455230, were associated with the immune response to influenza vaccination among the Chinese population. Identifying these variants will provide more evidence for future research and improve the individualized influenza vaccination program.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Androgen deprivation-induced ZBTB46-PTGS1 signaling promotes neuroendocrine differentiation of prostate cancer.

Androgen receptor (AR) targeting is an important therapeutic strategy for treating prostate cancer. Most tumors progress to castration-resistant prostate cancer (CRPC) and develop the neuroendocrine (NE) phenotype under androgen deprivation therapy (ADT). The molecular basis for NE transdifferentiation after ADT remains incompletely understood. Herein, we show that an immunocyte expression protein, ZBTB46, induces inflammatory response gene expression and contributes to NE differentiation of prostate cancer cells. We demonstrated a molecular mechanism whereby ZBTB46 can be regulated by the androgen-responsive gene, SPDEF, and is associated with NE prostate cancer (NEPC) differentiation. In addition, ZBTB46 acts as a transcriptional coactivator that binds to the promoter of prostaglandin-endoperoxide synthase 1 (PTGS1) and transcriptionally regulated PTGS1 levels. Overexpression of ZBTB46 decreases the sensitivity of the combination of enzalutamide and a PTGS1 inhibitor; however, knockdown of ZBTB46 sensitizes the PTGS1 inhibitor and reduces tumor malignancy. ZBTB46 is inversely correlated with SPDEF and is increased in higher tumor grades and small-cell NE prostate cancer (SCNC) patients, which are positively associated with PTGS1. Our findings suggest that the induction of ZBTB46 results in increased PTGS1 expression, which is associated with NEPC progression and linked to the dysregulation of the AR-SPDEF pathway.

Transcriptional control of dendritic cell development and functions.

Dendritic cells (DCs) are major regulators of adaptive immunity, as they are not only capable to induce efficient immune responses, but are also crucial to maintain peripheral tolerance and thereby inhibit autoimmune reactions. DCs bridge the innate and the adaptive immune system by presenting peptides of self and foreign antigens as peptide MHC complexes to T cells. These properties render DCs as interesting target cells for immunomodulatory therapies in cancer, but also autoimmune diseases. Several subsets of DCs with special properties and functions have been described. Recent achievements in understanding transcriptional programs on single cell level, together with the generation of new murine models targeting specific DC subsets, advanced our current understanding of DC development and function. Thus, DCs arise from precursor cells in the bone marrow with distinct progenitor cell populations splitting the monocyte populations and macrophage populations from the DC lineage, which upon lineage commitment can be separated into conventional cDC1, cDC2, and plasmacytoid DCs (pDCs). The DC populations harbor intrinsic programs enabling them to react for specific pathogens in dependency on the DC subset, and thereby orchestrate T cell immune responses. Similarities, but also varieties, between human and murine DC subpopulations are challenging, and will require further investigation of human specimens under consideration of the influence of the tissue micromilieu and DC subset localization in the future.

Characterisation of ZBTB46 and DC-SCRIPT/ZNF366 in rainbow trout, transcription factors potentially involved in dendritic cell maturation and activation in fish.

ZBTB46 and DC-SCRIPT/ZNF366 are two zinc finger transcription factors that play important roles in regulating differentiation of dendritic cells in mammals. In this study, the ZBTB46 and DC-SCRIPT/ZNF366 homologues were identified in rainbow trout Oncorhynchus mykiss and their expression analysed in vivo and in vitro. As transcription factors, they are well conserved in sequence, genomic organisation and gene synteny. Their expression was differentially modulated by bacterial and viral PAMPs in the monocyte/macrophage-like cell line RTS-11, in primary head kidney (HK) macrophages, and in HK macrophages cultured with IL-4/13A. In the RTS-11 cells and primary HK macrophages, all the ZBTB46 and DC-SCRIPT/ZNF366 homologues were down-regulated by interferon gamma (type II IFN) but unaffected by IFN2 (type I IFN), administered as recombinant proteins to cell cultures. In fish gills, infection with amoebae (Paramoebae perurans) resulted in reduction of ZBTB46 and DC-SCRIPT/ZNF366 expression in Atlantic salmon Salmo salar, whilst infection with Yersinia ruckeri induced gene expression in rainbow trout.

COX-2 inhibitor prevents tumor induced down regulation of classical DC lineage specific transcription factor Zbtb46 resulting in immunocompetent DC and decreased tumor burden.

The interaction between the immune and tumor cells in the microenvironment is an important factor deciding the progression of cancer. Though many of the soluble mediators in the microenvironment that mediate immunosuppression are known, the mechanism by which the tumor affects the distal progenitors is not known. We report that the tumor derived prostanoids down regulated classical dendritic cells DC (cDC) lineage specific transcription factor Zbtb46 in the progenitor cells which affects its differentiation. Prostanoids also induced ERK/CREB/IL-10 signaling pathway in DC that is more important for maturation of DC. This was observed under in vitro as well as in vivo conditions leading to phenotypic and functional impairment of DC. siRNA mediated knockdown of Zbtb46 and not exogenous IL-10 mimicked the effects of tumor conditioned medium (TCM) on suppression of maturation markers. Treatment of tumor cells with COX-2 inhibitor NS-398 averted TCM induced phenotypic impairment of DC in vitro. Treatment of tumor bearing mice with NS-398 prevented tumor induced down regulation of Zbtb46 resulting in immunocompetent DC which in turn led to a decrease in tumor burden. The effects of NS-398 was indeed through immunomodulation was corroborated by no such response in SCID mice. Our study provides novel insight into the distal regulation of progenitor cells by tumor and the importance of Zbtb46 expression in anti-tumor immunity. These results identify Zbtb46 expression as an indicator of immunocompetent DC in tumor and also highlights that COX-2 inhibitors could be useful in cancer immunotherapy.

Microarray expression profile analysis of long non-coding RNAs in human breast cancer: a study of Chinese women.

Breast cancer (BC) is the most commonly diagnosed cancer and the second leading cause of cancer death among women. Long non-coding RNAs (lncRNAs) are key regulators of gene expression. Numerous lncRNAs have performed critical roles in cancer biology including breast cancer (BC). The expression levels of certain lncRNAs are associated with tumor development, recurrence, metastasis, and prognosis. However, the potential roles that lncRNAs regulate breast cancer tumorigenesis and tumor progression are still poorly understood. To investigate the potential roles of lncRNAs in the breast cancer, we constructed BC related lncRNA libraries by using microarray. Microarray expression profiling suggests 790 up-regulated and 637 down-regulated (log fold-change>2.3) lncRNAs were differently expressed between BC tissues and its paired adjacent tissues. Furthermore, we found differently expressed lncRNAs associated with immune regulation. RP4-583P15.10, an up-regulated lncRNA, was found to be located downstream of the natural antisense of the ZBTB46 gene, which may regulated breast cancer through influence immune system. In conclusion, our results for the first time indicate that distinct lncRNAs expression profiles of BC, which related to the immune network, may provide information for further research on immune regulation during the BC process.