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Rationale: Breast cancer preferentially develops osteolytic bone metastasis, which makes patients suffer from pain, fractures and spinal cord compression. Accumulating evidences have shown that exosomes play an irreplaceable role in pre-metastatic niche formation as a communication messenger. However, the function of exosomes secreted by breast cancer cells remains incompletely understood in bone metastasis of breast cancer. Methods: Mouse xenograft models and intravenous injection of exosomes were applied for analyzing the role of breast cancer cell-derived exosomes in vivo. Effects of exosomes secreted by the mildly metastatic MDA231 and its subline SCP28 with highly metastatic ability on osteoclasts formation were confirmed by TRAP staining, ELISA, microcomputed tomography, histomorphometric analyses, and pit formation assay. The candidate exosomal miRNAs for promoting osteoclastogenesis were globally screened by RNA-seq. qRT-PCR, western blot, confocal microscopy, and RNA interfering were performed to validate the function of exosomal miRNA. Results: Implantation of SCP28 tumor cells in situ leads to increased osteoclast activity and reduced bone density, which contributes to the formation of pre-metastatic niche for tumor cells. We found SCP28 cells-secreted exosomes are critical factors in promoting osteoclast differentiation and activation, which consequently accelerates bone lesion to reconstruct microenvironment for bone metastasis. Mechanistically, exosomal miR-21 derived from SCP28 cells facilitates osteoclastogenesis through regulating PDCD4 protein levels. Moreover, miR-21 level in serum exosomes of breast cancer patients with bone metastasis is significantly higher than that in other subpopulations. Conclusion: Our results indicate that breast cancer cell-derived exosomes play an important role in promoting breast cancer bone metastasis, which is associated with the formation of pre-metastatic niche via transferring miR-21 to osteoclasts. The data from patient samples further reflect the significance of miR-21 as a potential target for clinical diagnosis and treatment of breast cancer bone metastasis.

Tcf-1 (encoded by Tcf7) not only plays critical roles in promoting T cell development and differentiation but also has been identified as a tumor suppressor involved in preventing T cell malignancy. However, the comprehensive mechanisms of Tcf-1 involved in T cell transformation remain poorly understood. In this study, Tcf7(fl/fl) mice were crossed with Vav-cre, Lck-cre, or Cd4-cre mice to delete Tcf-1 conditionally at the beginning of the HSC, DN2–DN3, or DP stage, respectively. The defective T cell development phenotypes became gradually less severe as the deletion stage became more advanced in distinct mouse models. Interestingly, consistent with Tcf7(−/−) mice, Tcf7(fl/fl)Vav-cre mice developed aggressive T cell lymphoma within 45 weeks, but no tumors were generated in Tcf7(fl/fl)Lck-cre or Tcf7(fl/fl)Cd4-cre mice. Single-cell RNA-seq (ScRNA-seq) indicated that ablation of Tcf-1 at distinct phases can subdivide DN1 cells into three clusters (C1, C2, and C3) and DN2–DN3 cells into three clusters (C4, C5, and C6). Moreover, Tcf-1 deficiency redirects bifurcation among divergent cell fates, and clusters C1 and C4 exhibit high potential for leukemic transformation. Mechanistically, we found that Tcf-1 directly binds and mediates chromatin accessibility for both typical T cell regulators and proto-oncogenes, including Myb, Mycn, Runx1, and Lyl1 in the DN1 phase and Lef1, Id2, Dtx1, Fyn, Bcl11b, and Zfp36l2 in the DN2–DN3 phase. The aberrant expression of these genes due to Tcf-1 deficiency in very early T cells contributes to subsequent tumorigenesis. Thus, we demonstrated that Tcf-1 plays stage-specific roles in regulating early thymocyte development and transformation, providing new insights and evidence for clinical trials on T-ALL leukemia.

N6-methyladenosine (m6A) is the most prevalent internal RNA modification in mammals that regulates homeostasis and function of modified RNA transcripts. Here, we aimed to investigate the role of YTH m6A RNA-binding protein 1 (YTHDF1), a key regulator of m6A methylation in gastric cancer tumorigenesis. Multiple bioinformatic analyses of different human cancer databases identified key m6A-associated genetic mutations that regulated gastric tumorigenesis. YTHDF1 was mutated in about 7% of patients with gastric cancer, and high expression of YTHDF1 was associated with more aggressive tumor progression and poor overall survival. Inhibition of YTHDF1 attenuated gastric cancer cell proliferation and tumorigenesis in vitro and in vivo. Mechanistically, YTHDF1 promoted the translation of a key Wnt receptor frizzled7 (FZD7) in an m6A-dependent manner, and mutated YTHDF1 enhanced expression of FZD7, leading to hyperactivation of the Wnt/β-catenin pathway and promotion of gastric carcinogenesis. Our results demonstrate the oncogenic role of YTHDF1 and its m6A-mediated regulation of Wnt/β-catenin signaling in gastric cancer, providing a novel approach of targeting such epigenetic regulators in this disease. Significance: This study provides a rationale for controlling translation of key oncogenic drivers in cancer by manipulating epigenetic regulators, representing a novel and efficient strategy for anticancer treatment.

Hematopoietic stem cells (HSCs) have the capacity for self-renewal to maintain the HSCs' pool and the ability for multilineage differentiation, which are responsible for sustained production of multiple blood lineages. The regulation of HSC development is controlled precisely by complex signal networks and hematopoietic microenvironment, which has been termed the HSCs' niche. The Wnt signaling pathway is one of a variety of signaling pathways that have been involved in HSC self-renewal and maintenance. Previous studies are indeterminant on the regulation of adult HSCs upon canonical Wnt signaling pathways because of the different experimental systems and models used. In this study, we generated the conditional knockout Wnt coreceptor low-density lipoprotein receptor-related protein 5 (Lrp5) and low-density lipoprotein receptor-related protein 6 (Lrp6) mice in adult hematopoiesis via Vav-Cre Loxp system. Inactivation of Lrp5 and -6 in a hematopoietic system diminished the pool of HSCs, but there were no obvious defects in mature immune cells. Lrp5 and -6 double deficiency HSCs showed intrinsic defects in self-renewal and differentiation due to reduced proliferation and increased quiescence of the cell cycle. Analysis of HSC gene expression suggested that the quiescence regulators were significantly up-regulated, such as Egr1, Cdkn1a, Nr4a1, Gata2, Junb and Btg2, and the positive cell cycle regulators were correspondingly down-regulated, such as Ccna2 and Ranbp1. Taken together, we investigated the roles of Lrp5 and -6 in HSCs by functional and bioinformatic assays, and we demonstrated that Lrp5 and -6 are required for the self-renewal and differentiation of adult HSCs. The canonical Wnt pathway may contribute to maintaining the HSC pool and regulate the differentiation of adult HSCs by controlling cell cycle gene regulatory module.-Liu, J., Cui, Z., Wang, F., Yao, Y., Yu, G., Liu, J., Cao, D., Niu, S., You, M., Sun, Z., Lian, D., Zhao, T., Kang, Y., Zhao, Y., Xue, H.-H., Yu, S. Lrp5 and Lrp6 are required for maintaining self-renewal and differentiation of hematopoietic stem cells.

RNA binding protein SRSF1 controls the development of late thymocytes via posttranscriptional regulation., The underlying mechanisms of thymocyte maturation remain largely unknown. Here, we report that serine/arginine-rich splicing factor 1 (SRSF1) intrinsically regulates the late stage of thymocyte development. Conditional deletion of SRSF1 resulted in severe defects in maintenance of late thymocyte survival and a blockade of the transition of TCRβhiCD24+CD69+ immature to TCRβhiCD24−CD69− mature thymocytes, corresponding to a notable reduction of recent thymic emigrants and diminished periphery T cell pool. Mechanistically, SRSF1 regulates the gene networks involved in thymocyte differentiation, proliferation, apoptosis, and type I interferon signaling pathway to safeguard T cell intrathymic maturation. In particular, SRSF1 directly binds and regulates Irf7 and Il27ra expression via alternative splicing in response to type I interferon signaling. Moreover, forced expression of interferon regulatory factor 7 rectifies the defects in SRSF1-deficient thymocyte maturation via restoring expression of type I interferon–related genes. Thus, our work provides new insight on SRSF1-mediated posttranscriptional regulatory mechanism of thymocyte development.

T follicular helper (TFH) cells are specialized effector CD4+ T cells critical to humoral immunity. Whether post-transcriptional regulation has a function in TFH cells is unknown. Here, we show conditional deletion of METTL3 (a methyltransferase catalyzing mRNA N6-methyladenosine (m6A) modification) in CD4+ T cells impairs TFH differentiation and germinal center responses in a cell-intrinsic manner in mice. METTL3 is necessary for expression of important TFH signature genes, including Tcf7, Bcl6, Icos and Cxcr5 and these effects depend on intact methyltransferase activity. m6A-miCLIP-seq shows the 3′ UTR of Tcf7 mRNA is subjected to METTL3-dependent m6A modification. Loss of METTL3 or mutation of the Tcf7 3′ UTR m6A site results in accelerated decay of Tcf7 transcripts. Importantly, ectopic expression of TCF-1 (encoded by Tcf7) rectifies TFH defects owing to METTL3 deficiency. Our findings indicate that METTL3 stabilizes Tcf7 transcripts via m6A modification to ensure activation of a TFH transcriptional program, indicating a pivotal function of post-transcriptional regulation in promoting TFH cell differentiation.

The kinase PDK1 is a crucial regulator for immune cell development by connecting PI3K to downstream AKT signaling. However, the roles of PDK1 in CD4+ T cell differentiation, especially in T follicular helper (Tfh) cell, remain obscure. Here we reported PDK1 intrinsically promotes the Tfh cell differentiation and germinal center responses upon acute infection by using conditional knockout mice. PDK1 deficiency in T cells caused severe defects in both early differentiation and late maintenance of Tfh cells. The expression of key Tfh regulators was remarkably downregulated in PDK1-deficient Tfh cells, including Tcf7, Bcl6, Icos, and Cxcr5. Mechanistically, ablation of PDK1 led to impaired phosphorylation of AKT and defective activation of mTORC1, resulting in substantially reduced expression of Hif1α and p-STAT3. Meanwhile, decreased p-AKT also suppresses mTORC2-associated GSK3β activity in PDK1-deficient Tfh cells. These integrated effects contributed to the dramatical reduced expression of TCF1 and ultimately impaired the Tfh cell differentiation.

Glaucoma is the leading cause of irreversible blindness. For glaucoma screening, the cup to disc ratio (CDR) is a significant indicator, whose calculation relies on the segmentation of optic disc(OD) and optic cup(OC) in color fundus images. This study proposes a residual multi-scale convolutional neural network with a context semantic extraction module to jointly segment the OD and OC. The proposed method uses a W-shaped backbone network, including image pyramid multi-scale input with the side output layer as an early classifier to generate local prediction output. The proposed method includes a context extraction module that extracts contextual semantic information from multiple level receptive field sizes and adaptively recalibrates channel-wise feature responses. It can effectively extract global information and reduce the semantic gaps in the fusion of deep and shallow semantic information. We validated the proposed method on four datasets, including DRISHTI-GS1, REFUGE, RIM-ONE r3, and a private dataset. The overlap errors are 0.0540, 0.0684, 0.0492, 0.0511 in OC segmentation and 0.2332, 0.1777, 0.2372, 0.2547 in OD segmentation, respectively. Experimental results indicate that the proposed method can estimate the CDR for a large-scale glaucoma screening.

Long noncoding RNAs (lncRNAs) are emerging as critical mediators of various biological processes in the immune system. The current data showed that the lncRNA Malat1 is highly expressed in T cell subsets, but the function of Malat1 in T cell remains unclear. In this study, we detected the T cell development and both CD8(+) and CD4(+) T cell response to LCMV infection using Malat1(−/-) mice model. To our surprise, there were no significant defects in thymocytes at different developmental stages and the peripheral T cell pool with ablation of Malat1. During LCMV infection, Malat1(−/-) mice exhibited normal effector and memory CD8(+) T cells as well as T(FH) cells differentiation. Our results indicated that Malat1 is not essential for T cell development and T cell-mediated antiviral response though it expresses at very high level in different T cell populations.

Activation-induced cytidine deaminase (AID) mediates class switching by binding to a small fraction of single-stranded DNA (ssDNA) to diversify the antibody repertoire. The precise mechanism for highly selective AID targeting in the genome has remained elusive. Here, we report an RNA-binding protein, ROD1 (also known as PTBP3), that is both required and sufficient to define AID-binding sites genome-wide in activated B cells. ROD1 interacts with AID via an ultraconserved loop, which proves to be critical for the recruitment of AID to ssDNA using bi-directionally transcribed nascent RNAs as stepping stones. Strikingly, AID-specific mutations identified in human patients with hyper-IgM syndrome type 2 (HIGM2) completely disrupt the AID interacting surface with ROD1, thereby abolishing the recruitment of AID to immunoglobulin (Ig) loci. Together, our results suggest that bi-directionally transcribed RNA traps the RNA-binding protein ROD1, which serves as a guiding system for AID to load onto specific genomic loci to induce DNA rearrangement during immune responses.

Xing et al demonstrate the requirements for Tle transcriptional corepressors in CD8+ T cell development. Tle proteins are differentially partitioned to the Runx and Tcf/Lef complexes to promote CD8+ lineage choice and establish CD8+ T cell identity, respectively., Tle/Groucho proteins are transcriptional corepressors interacting with Tcf/Lef and Runx transcription factors, but their physiological roles in T cell development remain unknown. Conditional targeting of Tle1, Tle3 and Tle4 revealed gene dose–dependent requirements for Tle proteins in CD8+ lineage cells. Upon ablating all three Tle proteins, generation of CD8+ T cells was greatly diminished, largely owing to redirection of MHC-I–selected thymocytes to CD4+ lineage; the remaining CD8-positive T cells showed aberrant up-regulation of CD4+ lineage-associated genes including Cd4, Thpok, St8sia6, and Foxp3. Mechanistically, Tle3 bound to Runx-occupied Thpok silencer, in post-selection double-positive thymocytes to prevent excessive ThPOK induction and in mature CD8+ T cells to silence Thpok expression. Tle3 also bound to Tcf1-occupied sites in a few CD4+ lineage-associated genes, including Cd4 silencer and St8sia6 introns, to repress their expression in mature CD8+ T cells. These findings indicate that Tle corepressors are differentially partitioned to Runx and Tcf/Lef complexes to instruct CD8+ lineage choice and cooperatively establish CD8+ T cell identity, respectively., Graphical Abstract

In order to study the thallus changes on microscopic morphology and mechanical properties ofCandida albicansantagonized byStreptococcus sanguinisbacteriocin, the adhesion ability and Young’s modulus of thalli and hypha ofCandida albicanswere measured by the relative measurement method using atomic force microscope’s (AFM) tapping model. The results showed that the average adhesion ability and Young’s modulus of thalli were7.35±0.77 nN and7.33±1.29 Mpa, respectively; the average adhesion ability and Young’s modulus of hypha were9.82±0.39 nN and4.04±0.76 Mpa, respectively. After being antagonized byStreptococcus sanguinisbacteriocin, the adhesion ability was decreased along with the increasing of deformation in reaction region and Young’s modulus followed the same changes. It could be concluded that the adhesion ability of hypha was greater than thalli, Young’s modulus of hypha was less than thalli, and adhesion ability and Young’s modulus ofCandida albicanswere decreased significantly after being antagonized byStreptococcus sanguinisbacteriocin.

Spaceflight leads to health risks including bone demineralization, skeletal muscle atrophy, cardiovascular dysfunction, and disorders of almost all physiologic systems. However, the impacts of microgravity on blood lineage cells and hematopoietic stem cells (HSCs) in vivo are largely unknown. In this study, we analyzed peripheral blood samples from 6 astronauts who had participated in spaceflight missions and found significant changes in several cell populations at different time points. These dynamic alterations of lineage cells and the role of HSCs were further studied in a mouse model, using hindlimb unloading (HU) to simulate microgravity. Large reductions in the frequency of NK cells, B cells, and erythrocyte precursors in the bone marrow of the HU mice were observed, together with an increased frequency of T cells, neutrophils, and HSCs. T cell levels recovered faster than those of B cells and erythrocyte precursors, whereas the recovery rates of NK cells and granulocytes were slow. In addition, competitive reconstitution experiments demonstrated the impaired function of HSCs, although these changes were reversible. Deep sequencing showed changes in the expression of regulatory molecules important for the differentiation of HSCs. This study provides the first determination of altered HSC function under simulated microgravity in vivo. The impairment of HSC function and differentiation provides an explanation for the immune disorders that occur under simulated microgravity. Thus, our findings demonstrated that spaceflight and simulated microgravity disrupt the homeostasis of immune system and cause dynamic alterations on both HSCs and lineage cells.-Cao, D., Song, J., Ling, S., Niu, S., Lu, L., Cui, Z., Li, Y., Hao, S., Zhong, G., Qi, Z., Sun, W., Yuan, X., Li, H., Zhao, D., Jin, X., Liu, C., Wu, X., Kan, G., Cao, H., Kang, Y., Yu, S., Li, Y. Hematopoietic stem cells and lineage cells undergo dynamic alterations under microgravity and recovery conditions.

Differentiation of effector and memory CD8+ T cells is accompanied by extensive changes in the transcriptome and histone modifications at gene promoters; however, the enhancer repertoire and associated gene regulatory networks are poorly defined. Using histone mark chromatin immunoprecipitation coupled with deep sequencing, we mapped the enhancer and super-enhancer landscapes in antigen-specific naive, differentiated effector, and central memory CD8+ T cells during LCMV infection. Epigenomics-based annotation revealed a highly dynamic repertoire of enhancers, which were inherited, de novo activated, decommissioned and re-activated during CD8+ T cell responses. We employed a computational algorithm to pair enhancers with target gene promoters. On average, each enhancer targeted three promoters and each promoter was regulated by two enhancers. By identifying enriched transcription factor motifs in enhancers, we defined transcriptional regulatory circuitry at each CD8+ T cell response stage. These multi-dimensional datasets provide a blueprint for delineating molecular mechanisms underlying functional differentiation of CD8+ T cells.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

The transcription factors TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4(+)CD8(+) double-positive (DP) stage are unknown. By specific ablation of these factors in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished the output of CD4(+) T cells and redirected CD4(+) T cells to a CD8(+) T cell fate. The role of TCF-1 and LEF-1 in the CD4-versus-CD8 lineage 'choice' was mediated in part by direct positive regulation of the transcription factor Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused derepression of CD4 expression in T cells committed to the CD8(+) lineage without affecting the expression of Runx transcription factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence Cd4. Thus, TCF-1 and LEF-1 adopted distinct genetic 'wiring' to promote the CD4(+) T cell fate and establish CD8(+) T cell identity.

GA binding protein (GABP) is a ubiquitously expressed transcription factor that regulates the development of multiple cell types, including osteoblast, hematopoietic stem cells, B cells and T cells. However, so little is known about its biological function in the development of central nervous system. In this report, we show that GABP is highly expressed in neural stem/progenitor cells (NSPCs) and down-regulated in neurons, and that GABPβ1 is required for the proper proliferation of NSPCs. Knockdown of GABPα resulted in an elevated expression level of GABPβ1, and GABPβ1 down-regulation significantly decreased the proliferation of NSPCs, whereas GABPβ2 knockdown did not result in any changes in the proliferation of NSPCs. We observed that there was nearly a 21-fold increase of the GABPβ1S mRNA level in GABPβ1L KO NSPCs compared to WT cells, and knocking down of GABPβ1S in GABPβ1L KO NSPCs could further reduce their proliferation potential. We also found that knockdown of GABPβ1 promoted neuronal and astrocytic differentiation of NSPCs. Finally, we identified dozens of downstream target genes of GABPβ1, which are closely associated with the cell proliferation and differentiation. Collectively, our results suggest that both GABPβ1L and GABPβ1S play an essential role in regulating the proper proliferation and differentiation of NSPCs. Keywords: GABP, Neural stem cell, GABPβ1L, GABPβ1S, Cell proliferation, Neurogenesis

Hematopoietic and leukemic stem cells (HSCs and LSCs) have self-renewal ability to maintain normal hematopoiesis and leukemia propagation, respectively. Tcf1 and Lef1 transcription factors are expressed in HSCs, and targeting both factors modestly expanded the size of the HSC pool due to diminished HSC quiescence. Functional defects of Tcf1/Lef1-deficient HSCs in multi-lineage blood reconstitution was only evident under competitive conditions or when subjected to repeated regenerative stress. These are mechanistically due to direct positive regulation of Egr and Tcf3 by Tcf1 and Lef1, and significantly, forced expression of Egr1 in Tcf1/Lef1-deficient HSCs restored HSC quiescence. In a preclinical CML model, loss of Tcf1/Lef1 did not show strong impact on leukemia initiation and progression. However, when transplanted into secondary recipients, Tcf1/Lef1-deficient LSCs failed to propagate CML. By induced deletion of Tcf1 and Lef1 in pre-established CML, we further demonstrated an intrinsic requirement for these factors in LSC self-renewal. When combined with imatinib therapy, genetic targeting of Tcf1 and Lef1 potently diminished LSCs and conferred better protection to the CML recipients. LSCs are therefore more sensitive to loss of Tcf1 and Lef1 than HSCs in their self-renewal capacity. The differential requirements in HSCs and LSCs thus identify Tcf1 and Lef1 transcription factors as novel therapeutic targets in treating hematological malignancies, and inhibition of Tcf1/Lef1-regulated transcriptional programs may thus provide a therapeutic window to eliminate LSCs with minimal side effect on normal HSC functions.

Although evolutionarily just as ancient as IgM, it has been thought for many years that IgD is not present in birds. Based on the recently sequenced genomes of 48 bird species as well as high-throughput transcriptome sequencing of immune-related tissues, we demonstrate in this work that the ostrich (Struthio camelus) possesses a functional δ gene that encodes a membrane-bound IgD H chain with seven CH domains. Furthermore, δ sequences were clearly identified in many other bird species, demonstrating that the δ gene is widely distributed among birds and is only absent in certain bird species. We also show that the ostrich possesses two μ genes (μ1, μ2) and two υ genes (υ1, υ2), in addition to the δ and α genes. Phylogenetic analyses suggest that subclass diversification of both the μ and υ genes occurred during the early stages of bird evolution, after their divergence from nonavian reptiles. Although the positions of the two υ genes are unknown, physical mapping showed that the remaining genes are organized in the order μ1-δ-α-μ2, with the α gene being inverted relative to the others. Together with previous studies, our data suggest that birds and nonavian reptile species most likely shared a common ancestral IgH gene locus containing a δ gene and an inverted α gene. The δ gene was then evolutionarily lost in selected birds, whereas the α gene lost in selected nonavian reptiles. The data obtained in this study provide significant insights into the understanding of IgH gene evolution in tetrapods.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens in the swine industry worldwide. Due to the lack of robust cell lines and small animal models, the pathogenesis of PRRSV infection and mechanism for protective vaccination are still not yet well understood. To obtain useful cell lines, several groups have attempted to construct different transgenic cell lines with three PRRSV receptors: CD163, CD169, and CD151. The results showed that CD163 is essential for PRRSV entry into target cells and replication, and both CD169 and CD151 play key roles during PRRSV infection. However, their interplay and combined effect remains unclear. In this study, we generated transgenic BHK-21 derived cell lines co-expressing different combinations of the three receptors, which were transfected with CD163 alone, or the combination of CD163 and CD169, or the combination of CD163 and CD151, or the combination of CD163, CD169, and CD151 using the PiggyBac transposon system. Our results showed that the synergistic interaction among the three receptors was important to improve the susceptibility of cells during PRRSV infection. Through a series of comparable analyses, we confirmed that the cell line co-expressing triple receptors sustained viral infection and replication, and was superior to the current cell platform used for the PRRSV study, MARC-145 cells. Moreover, we found that PRRSV infection of the transgenic cell lines could trigger IFN-stimulated gene responses similar to those of porcine alveolar macrophages and MARC-145 cells. In summary, we developed a stable transgenic cell line susceptible to PRRSV, which may not only provide a useful tool for virus propagation, vaccine development, and pathogenesis studies, but also establish the foundation for small animal model development.

Summary Effective treatment of chronic myelogenous leukemia (CML) largely depends on the eradication of CML leukemic stem cells (LSCs). We recently showed that CML LSCs depend on Tcf1 and Lef1 factors for self-renewal. Using a connectivity map, we identified prostaglandin E1 (PGE1) as a small molecule that partly elicited the gene expression changes in LSCs caused by Tcf1/Lef1 deficiency. Although it has little impact on normal hematopoiesis, we found that PGE1 treatment impaired the persistence and activity of LSCs in a pre-clinical murine CML model and a xenograft model of transplanted CML patient CD34 + stem/progenitor cells. Mechanistically, PGE1 acted on the EP4 receptor and repressed Fosb and Fos AP-1 factors in a β-catenin-independent manner. Misoprostol, an FDA-approved EP4 agonist, conferred similar protection against CML. These findings suggest that activation of this PGE1-EP4 pathway specifically targets CML LSCs and that the combination of PGE1/misoprostol with conventional tyrosine-kinase inhibitors could provide effective therapy for CML.

T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Rα and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized β-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized β-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.

Summary Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) are both capable of self-renewal, with HSCs sustaining multiple blood lineage differentiation and LSCs indefinitely propagating leukemia. The GABP complex, consisting of DNA binding GABPα subunit and transactivation GABPβ subunit, critically regulates HSC multipotency and self-renewal via controlling an essential gene regulatory module. Two GABPβ isoforms, GABPβ1L and GABPβ2, contribute to assembly of GABPα 2 β 2 tetramer. We demonstrate that GABPβ1L/β2 deficiency specifically impairs HSC quiescence and survival, with little impact on cell cycle or apoptosis in differentiated blood cells. The HSC-specific effect is mechanistically ascribed to perturbed integrity of the GABP-controlled gene regulatory module in HSCs. Targeting GABPβ1L/β2 also impairs LSC self-renewal in p210 BCR-ABL - induced chronic myelogenous leukemia (CML) and exhibits synergistic effects with tyrosine kinase inhibitor imatinib therapy in inhibiting CML propagation. These findings identify the tetramer-forming GABPβ isoforms as specific HSC regulators and potential therapeutic targets in treating LSC-based hematological malignancy.