T-cell Transcription Factor Research Articles

Deregulated expression of circular RNAs in acute myeloid leukemia

Circular RNAs (circRNAs) are dynamically regulated during differentiation and show cell type–specific expression, which is altered in cancer and can have a direct impact on its various hallmarks. We hypothesized that circRNA expression is deregulated in acute myeloid leukemia (AML) and that circRNA candidates might contribute to the pathogenesis of the disease. To identify leukemia-associated and differentiation-independent changes in circRNA expression, we determined the circular RNAome of 61 AML patients and 16 healthy hematopoietic stem and progenitor cell (HSPC) samples using ribosomal RNA–depleted RNA sequencing. We found hundreds of circRNAs that were differentially expressed between AML and healthy HSPCs. Gene set analysis found that many of these circRNAs were transcribed from genes implicated in leukemia biology. We discovered a circRNA derived from the T-cell transcription factor gene B cell CLL/lymphoma 11B, circBCL11B, which was exclusively expressed in AML patients, but not detected in healthy HSPCs, and associated with a T-cell–like gene expression signature. We were able to validate this finding in an independent cohort of 332 AML patients. Knockdown of circBCL11B had a negative effect on leukemic cell proliferation and resulted in increased cell death of leukemic cells, thereby suggesting circBCL11B as a novel functionally relevant candidate in AML pathogenesis. In summary, our study enables comprehensive insights into circRNA expression changes upon leukemic transformation and provides valuable information on the biology of leukemic cells and potential novel pathway dependencies that are relevant for AML therapy.

Localization of T-cell factor 4 positive fibroblasts and CD206-positive macrophages during skeletal muscle regeneration in mice

Skeletal muscle regeneration is initiated by the activation of the transcription factor paired box 7 (Pax7), which is expressed in the satellite cells. The nuclear transcription factor T-cell factor 4 (Tcf4) is expressed in the fibroblasts and is involved in muscle tissue repair, while M2-like macrophages play an important role in skeletal muscle regeneration. However, the localization of M2-like macrophages and the expression of Tcf4 over a period of time during skeletal muscle regeneration remain unknown. Therefore, the murine masseter muscle was immunofluorescence investigated for the surface protein CD206 of M2-like macrophages and Tcf4 of fibroblasts during skeletal muscle regeneration to understand the changes in the CD206 and Tcf4 expression over time. We observed that CD206 entered the cytoplasm of some regenerating muscle fibers 5–7 days after the experimental muscle damage, that is, in the early stage of maturation of the regenerating muscle fibers with central nuclei. In addition, Tcf4 was expressed in the nuclei of the fibroblasts around the regenerating muscle fibers and in the central nuclei of the regenerating muscle fibers. Furthermore, the expression of laminin adjacent to Tcf4-positive cells was observed to partially disappear, and the shape of this missing part was observed to be identical to that of the nuclei of Tcf4-positive cells adjacent to the laminin. Clathrin was also expressed in these sites, demonstrating endocytosis. Thus, these results suggest that in the early stage of maturation of the regenerating muscle fibers, M2-like macrophages and Tcf4-positive fibroblasts enter the cytoplasm of the regenerating muscle fibers, thereby regulating the expression of various maturation factors.

Retinoic acid–responsive CD8 effector T cells are selectively increased in IL-23–rich tissue in gastrointestinal GVHD

AbstractGastrointestinal (GI) graft-versus-host disease (GVHD) is a major barrier in allogeneic hematopoietic stem cell transplantation (allo-HSCT). The metabolite retinoic acid (RA) potentiates GI-GVHD in mice via alloreactive T cells expressing the RA receptor-α (RARα), but the role of RA-responsive cells in human GI-GVHD remains undefined. Therefore, we used conventional and novel sequential immunostaining and flow cytometry to scrutinize RA-responsive T cells in tissues and blood of patients who had received allo-HSCT and to characterize the impact of RA on human T-cell alloresponses. Expression of RARα by human mononuclear cells was increased after exposure to RA. RARαhi mononuclear cells were increased in GI-GVHD tissue, contained more cellular RA-binding proteins, localized with tissue damage, and correlated with GVHD severity and mortality. By using a targeted candidate protein approach, we predicted the phenotype of RA-responsive T cells in the context of increased microenvironmental interleukin-23 (IL-23). Sequential immunostaining confirmed the presence of a population of RARαhi CD8 T cells with the predicted phenotype that coexpressed the effector T-cell transcription factor T-bet and the IL-23–specific receptor (IL-23R). These cells were increased in GI- but not skin-GVHD tissues and were also selectively expanded in the blood of patients with GI-GVHD. Finally, functional approaches demonstrated that RA predominantly increased alloreactive GI-tropic RARαhi CD8 effector T cells, including cells with the phenotype identified in vivo. IL-23–rich conditions potentiated this effect by selectively increasing β7 integrin expression on CD8 effector T cells and reducing CD4 T cells with a regulatory cell phenotype. In summary, we have identified a population of RA-responsive effector T cells with a distinctive phenotype that is selectively expanded in human GI-GVHD and that represents a potential new therapeutic target.

Abstract 2627: Opposite functions of Claudin-2 involving Wnt signaling in liver cancer cells

Abstract Background: We previously identified a unique Wnt target gene CLAUDIN-2, whose expression level was regulated by isoforms of the Wnt signal transcription factor T-cell factor-4 (Exp Cell Res 2011, Liver Int 2013). CLAUDIN-2 (CLDN-2) was highly expressed in combined hepatocellular-cholangiocarcinoma cell lines, and the molecule contributed to high cellular proliferation, sphere-forming ability, and tumorigenicity of the cells (Koga et al., The Liver Meeting 2018). However, such tumor-promoting action of CLDN-2 was not demonstrated in all liver cancer cell lines tested. The opposite functions of CLDN-2 included extremely low proliferative activity and no tumorigenicity, that were, at least, associated with the tumor suppressor LKB1-mediated AMPK activation. Thus, the Aim of this study was to further investigate molecular basis for the two-faced actions of CLDN-2 in liver cancer cells. Methods: The human liver cancer cell lines Hep3B and HLF were used in this study. Plasmid-mediated CLAUDIN-2 cDNA (Origene) and lentivirus-mediated delivery of siRNA for CLAUDIN-2 (Santa Cruz) were utilized to generate stable overexpressing and knock-down (KD) cells, respectively. A cDNA microarray analysis and qPCR were employed for exploring genetic determinants that controlled the two-faced actions of CLDN-2. Results: CLDN-2-overexpressing HLF (CLDN-2-OE-HLF) cells exhibited clear G1 cell-cycle arrest and no tumorigenicity, although CLDN-2-OE-Hep3B cells did not. The cDNA microarray analysis comparing CLDN-2-OE-HLF cells with CLDN-2-OE-Hep3B cells and the validation qPCR revealed 7.9-fold increase of LRP4 and 1.6-fold increase of APC in the CLDN-2-OE-HLF cells, while 0.4-fold and 0.7-fold in the latter, respectively. Of interest, in western blot analysis, the CLDN-2-OE-HLF cells exhibited increased expression of the stem cell marker Nanog, in concert with those of phosphorylated (p-)LKB1 and p-AMPKalpha1, that together resulted in upregulation of p53 and p21 in both mRNA and protein levels. Conclusion: LRP4 has been reported to have suppressive function on the Wnt signaling pathway interplaying with Frizzled at cell membrane, and APC is a strong tumor suppressor to inhibit beta-catenin-mediated Wnt signaling. Therefore, the findings obtained from this study suggested that the Wnt target gene CLDN-2 differentially regulated feedforward and feedback signal relays in the Wnt signal pathway in a context-dependent manner in human liver cancer cells. Citation Format: Hironori Koga, Yasuko Imamura, Toru Nakamura, Hideki Iwamoto, Takahiko Sakaue, Atsutaka Masuda, Toshimitsu Tanaka, Dan Nakano, Hiroyuki Suzuki, Hirohisa Yano, Takuji Torimura. Opposite functions of Claudin-2 involving Wnt signaling in liver cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2627.

Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.

Light-Mediated Control over TRPC3-Mediated NFAT Signaling.

Canonical transient receptor potential (TRPC) channels were identified as key players in maladaptive remodeling, with nuclear factor of activated T-cells (NFAT) transcription factors serving as downstream targets of TRPC-triggered Ca2+ entry in these pathological processes. Strikingly, the reconstitution of TRPC-NFAT signaling by heterologous expression yielded controversial results. Specifically, nuclear translocation of NFAT1 was found barely responsive to recombinant TRPC3, presumably based on the requirement of certain spatiotemporal signaling features. Here, we report efficient control of NFAT1 nuclear translocation in human embryonic kidney 293 (HEK293) cells by light, using a new photochromic TRPC benzimidazole activator (OptoBI-1) and a TRPC3 mutant with modified activator sensitivity. NFAT1 nuclear translocation was measured along with an all-optical protocol to record local and global Ca2+ pattern generated during light-mediated activation/deactivation cycling of TRPC3. Our results unveil the ability of wild-type TRPC3 to produce constitutive NFAT nuclear translocation. Moreover, we demonstrate that TRPC3 mutant that lacks basal activity enables spatiotemporally precise control over NFAT1 activity by photopharmacology. Our results suggest tight linkage between TRPC3 activity and NFAT1 nuclear translocation based on global cellular Ca2+ signals.

MiR-7 Functions as a Tumor Suppressor by Targeting the Oncogenes TAL1 in T-Cell Acute Lymphoblastic Leukemia.

Background:T-cell acute lymphoblastic leukemia is a hematologic malignancy characterized by T-cell proliferation, and in many cases, the ectopic expression of the oncogenic transcription factor T-cell acute lymphocytic leukemia protein 1 (TAL1). MicroRNA-7 has been shown to play a critical role in proliferation, migration, and treatment sensitivity in a diverse array of cancers. In this study, we sought to establish a novel link between microRNA-7 and T-cell acute lymphoblastic leukemia oncogenesis.Material and Method:To do so, we characterized gene expression of microRNA-7 as well as TAL1 in both T-cell acute lymphoblastic leukemia patient-derived tissue and cell lines, as well as performing functional luciferase assays to assess microRNA-7 binding to the TAL1 3′-untranslated region. We also performed growth, apoptosis, and migration experiments using 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide, Annexin V, and transwell assays in the context of microRNA-7 overexpression.Results:We found that microRNA-7 expression is attenuated and inversely correlated with TAL1 expression in TAL1 + T-cell acute lymphoblastic leukemia cells. Additionally, microRNA-7 directly targets and suppresses TAL1 levels. Finally, microRNA-7 overexpression reduces growth, motility, and migration while inducing apoptosis in T-cell acute lymphoblastic leukemia cells, phenotypes that can be rescued by concomitant overexpression of TAL1.Conclusions:These results indicate that microRNA-7 functions as a potent tumor suppressor by inhibiting the oncogene TAL1 and suggest microRNA-7 could function as a prognostic biomarker and possible therapeutic in the clinical management of T-cell acute lymphoblastic leukemia.

Gene variants of TCF7L2 are histopathologically important in colorectal cancers but do not have direct association with MYC expression

Rapidly accumulating preclinical and clinical studies have helped us to unveil underlying mechanisms of colorectal cancer development and progression. Deregulated signaling pathways play instrumental role in carcinogenesis, drug resistance and metastasis. Wnt signaling cascade has attracted considerable attention in colorectal cancer as many ground-breaking researches have highlighted central role of Wnt pathway in pathogenesis of colorectal cancer. T-Cell Transcription Factors (TCFs) have been shown to work synchronously with β-catenin to fuel colorectal cancer development and progression. Chromatin immuno-precipitation coupled with high-throughput sequencing (ChIP-Seq) data sets has deepened our knowledge about critical role of risk-associated SNPs. Increasingly it is being reported that many risk-associated SNPs are located within binding sites for transcription factors and consequently risk status of these SNPs may modify binding pattern of transcriptional factors and thus rewire the transcriptional regulation. DNA was extracted from peripheral blood samples of 117 colorectal cancer patients and 127 healthy subjects. TCF7L2 variants (rs6983267, rs7903146) were examined by the PCR-RFLP method. Tumor and the surrounding tissues were dissected from 37 CRC patients and RNA isolation was performed. The gene expression of c-myc was determined by RT-PCR. T allele carriage of rs6983267 variant was found to be associated with CRC (p=0.042). TT genotype of rs7903146 was associated with late tumor stage (T3+T4) (p=0.037) and presence of mucinous component (p=0.031). TTCT haplotype was found to be statistically higher in CRC compared to the control group (p=0.007). There was no statistically significant difference in c-myc gene expression. TCF7L2 gene variants may play an important role in histopathologic aspects associated with CRC and it is independent of c-myc gene expression.

PD-1 Blockade Reinvigorates Bone Marrow CD8+ T Cells from Patients with Multiple Myeloma in the Presence of TGF-β Inhibitors

Programmed cell death (PD)-1/PD-Ligand 1(PD-L1) blockade that reinvigorates exhausted T cells has been approved for the treatment of various solid tumors and hematological malignancies. However, in a clinical trial of multiple myeloma (MM) patients, anti-PD-1 monotherapy did not result in a clinical response. Furthermore, clinical trials of combining PD-1 blockade with immunomodulatory drugs or anti-CD38 monoclonal antibody failed to demonstrate clinical benefits in MM patients. To overcome the limitation of anti-PD-1 therapy in MM, the phenotype and differentiation of CD8+ T cells need to be characterized in the bone marrow (BM) of MM patients, particularly by analyzing myeloma antigen-specific CD8+ T cells. In addition, the role of immunosuppressive factors abundant in the MM microenvironment should be considered, including TGF-β. First, we confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of MM patients. PD-1-expressing CD8+ T cells from the BM of MM patients co-expressed other checkpoint inhibitory receptors including Tim-3, LAG-3, and TIGIT. We also investigated the expression of T-cell transcription factors, such as T-bet, and EOMES, which are related to T-cell differentiation. In BM from MM patients, PD-1+CD8+ T cells had a higher percentage of EomeshiT-betlo cells than PD-1-CD8+ T cells. These data demonstrate that PD-1-expressing CD8+ T cells from the BM of MM patients exhibit a terminally differentiated phenotype with co-expression of multiple immune checkpoint inhibitory receptors. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. Next, we investigated proliferation and cytokine production of BM CD8+ T cells from MM patients. BM CD8+ T cells from MM patients exhibited reduced proliferation and cytokine production upon T cell receptor (TCR) stimulation, compared to BM CD8+ T cells from other control group such as of undetermined significance. However, both anti-PD-1 alone and combined blockade of PD-1 with other immune checkpoint receptors, such as Tim-3, Lag-3, or TIGIT, did not increase the proliferation of BM CD8+ T cells from MM patients. Likewise, anti-PD-1 treatment failed to induce reinvigoration of BM CD8+ T cells stimulated with HLA-A*0201-restricted myeloma antigen peptides, including NY-ESO-1157-165 and HM1.2422-30 peptides. These data demonstrate that blocking PD-1 is not sufficient to restore the function of BM CD8+ T cells from MM patients. It has been known that TGF-β, which is actively secreted by malignant plasma cells and BM stromal cells, can inhibit T-cell responses. We confirmed that the major source of TGF- β1 is plasma cells including myeloma cells among BMMCs from MM patients, and the number of TGF- β1-producing plasma cells, including myeloma cells, is increased in the BM of MM patients. We investigated whether blocking TGF-β signaling enhances reinvigoration of BM CD8+ T cells from MM patients. The combined blockade of PD-1 and TGF- β significantly increased the proliferation of BM CD8+ T cells from MM patients in the presence of TCR stimulation. The production of IFN-γ and TNF by BM CD8+ T cells was also rescued by combined blockade of PD-1 and TGF-β. Moreover, combination of anti-PD-1 antibody and TGF-β inhibitors increased proliferative responses of BM CD8+ T cells from HLA-A2+ MM patients stimulated with a mixture of HLA-A*0201-restricted myeloma antigen peptides (NY-ESO-1157-165 and HM1.2422-30 peptides). Thus, PD-1 blockade reinvigorates BM CD8+ T cells from MM patients in the presence of TGF-β inhibitors. Taken together, BM CD8+ T cells and myeloma antigen-specific CD8+ T cells express increased levels of PD-1 and have a terminally exhausted phenotype in MM patients. Under TGF-β inhibition, anti-PD-1 reinvigorates BM CD8+ T cells from MM patients, but PD-1 blockade alone does not restore the function of BM CD8+ T cells. Blocking both TGF-β and PD-1 can be a promising therapeutic strategy for the treatment of MM. Disclosures No relevant conflicts of interest to declare.

Expression of immune regulatory genes correlate with the abundance of specific Clostridiales and Verrucomicrobia species in the equine ileum and cecum

Billions of bacteria inhabit the gastrointestinal tract. Immune-microbial cross talk is responsible for immunological homeostasis, and symbiotic microbial species induce regulatory immunity, which helps to control the inflammation levels. In this study we aimed to identify species within the equine intestinal microbiota with the potential to induce regulatory immunity. These could be future targets for preventing or treating low-grade chronic inflammation occurring as a result of intestinal microbial changes and disruption of the homeostasis. 16S rRNA gene amplicon sequencing was performed on samples of intestinal microbial content from ileum, cecum, and colon of 24 healthy horses obtained from an abattoir. Expression of genes coding for IL-6, IL-10, IL-12, IL-17, 18 s, TNFα, TGFβ, and Foxp3 in the ileum and mesenteric lymph nodes was measured by qPCR. Intestinal microbiota composition was significantly different in the cecum and colon compared to the ileum, which contains large abundances of Proteobacteria. Especially members of the Clostridiales order correlated positively with the regulatory T-cell transcription factor Foxp3 and so did the phylum Verrucomicrobia. We conclude that Clostridiales and Verrucomicrobia have the potential to induce regulatory immunity and are possible targets for intestinal microbial interventions aiming at regulatory immunity improvement.

Genome organization and definition of the Penaeus monodon viral responsive protein 15 (PmVRP15) promoter

The viral responsive protein 15 from the black tiger shrimp Penaeus monodon (PmVRP15) is a highly responsive gene upon white spot syndrome virus (WSSV) challenge. It is identified from hemocyte and important for WSSV trafficking and assembly. However, the knowledge of PmVRP15 gene regulation is limited. In the present study, the genome organization and 5′upstream promoter sequences of PmVRP15 gene were investigated. The PmVRP15 gene was found to contain 4 exons interrupted by 3 introns and the start codon was located in the exon 2. The transcription start site and TATA box were also determined from the 5′ upstream sequence. By using the narrow down experiment, the 5′ upstream promoter active region was determined to be at the nucleotide positions -525 to +612. Mutagenesis of the putative transcription factor (TF) binding sites revealed that the binding site of interferon regulatory factor (IRF) (-495/-479) was a repressor-binding site whereas those of the octamer transcription factor 1 (Oct-1) (-275/-268) and the nuclear factor of activated T-cells transcription factor (NFAT) (-228/-223) were activator-binding sites. This is the first report on the transcription factors that might play essential roles in modulating the PmVRP15 gene expression. Nevertheless, the underlying regulation mechanism of PmVRP15 gene expression needs further investigation.

Inhibition of transcription factor T-cell factor 3 (TCF3) using the oligodeoxynucleotide strategy increases embryonic stem cell stemness: possible application in regenerative medicine.

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches.

Immune Checkpoint Inhibitor-induced Reinvigoration of Tumor-infiltrating CD8+ T Cells is Determined by Their Differentiation Status in Glioblastoma.

Immune checkpoint inhibitors (ICI) are used for the treatment of various cancers, but clinical trials of anti-programmed cell death protein 1 (PD-1) with patients with recurrent glioblastoma (GBM) have failed to show clinical benefits. In this study, we examined the differentiation status of CD8+ tumor-infiltrating lymphocytes (TIL) from patients with primary GBM and their reinvigoration by ICIs to understand the nature of T-cell exhaustion in GBM. We isolated TILs from 98 patients with newly diagnosed GBM and examined the expression of immune checkpoint receptors and T-cell transcription factors using flow cytometry. TILs were ex vivo stimulated with anti-CD3 in the presence of anti-PD-1 and/or anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) and their proliferation assessed. CD8+ TILs had significantly increased expression of immune checkpoint receptors, including PD-1 and CTLA-4, compared with peripheral blood CD8+ T cells. Among CD8+ TILs, PD-1+ cells exhibited more terminally differentiated phenotypes (i.e., EomeshiT-betlo) than PD-1- cells. These data were confirmed by analyzing NY-ESO-1157-specific CD8+ TILs. Evaluating the proliferation of CD8+ TILs after ex vivo stimulation with anti-CD3 and anti-PD-1, we found that proliferation inversely correlated with the percentage of EomeshiT-betlo cells among PD-1+CD8+ TILs. When anti-CTLA-4 was used in combination with anti-PD-1, an additional increase in CD8+ TIL proliferation was observed in patients with low percentages of EomeshiT-betlo CD8+ TILs, who responded well to anti-PD-1 in ex vivo assays, but not in patients with high percentages of EomeshiT-betlo CD8+ TILs, who did not respond to anti-PD-1. In primary GBM, the differentiation status of CD8+ TILs determines their reinvigoration ability upon ICI treatment.

Downregulation of T-Cell Transcription Factors in Adult Latent Autoimmune Diabetes with High-Titer Glutamic Acid Decaroxylase Antibody.

IntroductionLatent autoimmune diabetes in adults (LADA) shows a heterogeneous clinical profile that is dependent on the glutamic acid decaroxylase antibody (GADA) titer. We speculated that LADA patients with a high or low GADA titer may have distinct T-lymphocyte subset profiles and distinct expression patterns of transcription factors involved in T-cell immunomodulation.MethodsPatients with LADA (n = 40) and type 2 diabetes (T2DM; n = 14) were recruited to the study, and peripheral blood mononuclear cells were isolated. The proportions of T-lymphocyte subsets (Th1 [T helper type 1], Th2 [T helper type 2], Treg [regulatory T], and Th17 [T helper type 17] cells) were determined by flow cytometry. Real-time polymerase chain reaction (PCR) was performed to estimate mRNA expression levels of the T-cell subtype-enriched transcription factors T-bet (Th1), GATA3 (Th2), transcription factor forkhead box protein 3 (FOXP3) (Treg), and RORC (Th17).ResultsThe frequency of Th1 (as a percentage of total CD4+T cells) was greater in the LADA patients with high-titer GADA than in the LADA patients with low-titer GADA (11.06 ± 1.62 vs. 7.05 ± 0.86, P = 0.030). Compared to the T2DM group, in the low-titer GADA group the frequency of Th1 was significantly reduced (7.05 ± 0.86 vs. 16.75 ± 3.73, P = 0.017) and the frequency of Th17 frequency was signficantly increased (1.11 ± 0.09 vs. 0.74 ± 0.16, P = 0.017). Compared to T2DM patients, in the high-titer GADA group there was a significantly reduced expression of FOXP3 (0.35 ± 0.13 vs. 1.75 ± 0.54, P = 0.002), RORC (0.53 ± 0.19 vs. 2.00 ± 0.77, P = 0.046), and GATA3 (0.74 ± 0.17 vs. 2.31 ± 0.91, P = 0.046). Similarly, the high-titer GADA group expressed reduced levels of FOXP3 and RORC compared to the low-titer GADA group (0.35 ± 0.13 vs. 1.50 ± 0.41, P = 0.027; 0.53 ± 0.19 vs. 1.35 ± 0.21, P = 0.027, respectively). There was a negative correlation between FOXP3 expression level and GADA titer for the entire cohort (r = − 0.0433, P = 0.015) and a stronger negative correlation in LADA patients (r = − 0.606, P = 0.008).ConclusionLADA patients with high-titer GADA express lower levels of T-cell transcription factors, including the Treg transcription factor FOXP3, which may contribute to differences in the clinical profile compared to LADA patients with low-titer GADA.Trial RegistrationClinicalTrials.gov identifier, NCT01159847.

GATA3 Immunohistochemical Staining in Hodgkin Lymphoma: Diagnostic Utility in Differentiating Classic Hodgkin Lymphoma From Nodular Lymphocyte Predominant Hodgkin Lymphoma and Other Mimicking Entities.

Classic Hodgkin lymphoma (CHL) and nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) are clinically distinct entities, with different prognostic and treatment implications. In addition, several large B-cell lymphomas and some T-cell lymphomas can mimic CHL. Differentiating these entities from CHL is crucial for ensuring appropriate therapy. GATA3 is a T-cell transcription factor involved in T-cell maturation and has been previously shown to be overexpressed in CHL cells via gene expression profiling. We investigated the utility of GATA3 immunostain in differentiating CHL from NLPHL and other mimicking entities. We accrued 17 NLPHLs, 49 CHLs [23 nodular sclerosis (NS), 3 syncytial variants, 3 lymphocyte rich and 13 mixed cellularity types], 4 primary mediastinal large B-cell lymphomas (PMBLs), 2 Epstein-Barr virus (EBV) positive diffuse large B-cell lymphomas (DLBCLs) (EBV+LBCLs), 2 T-cell/histiocyte-rich large B-cell lymphomas (TCHRBCLs), 1 gray zone lymphoma, and 2 tissue microarrays consisting of 72 DLBCLs. One slide from each was stained with GATA3 and percent positive tumor cells and intensity of nuclear expression was semiquantitatively graded independently by 2 board certified hematopathologists. GATA3 was positive in 80% of CHLs. Both percent positivity and intensity of staining varied greatly. Syncytial variant of NS subtype showed the highest positivity rate (3/3; 100%), followed by NS (20/23; 87%), mixed cellularity (9/13; 70%), and lymphocyte rich (2/3; 67%). GATA3 was negative in all NLPHLs, EBV+LBCLs, TCRBCLs, and DLBCLs stained. The single gray zone lymphoma and 3/4 PMBLs were positive. Nuclear expression of GATA3 can be used to delineate CHL from NLPHL. GATA3 positivity effectively excludes NLPHL with 100% negative predictive value. However, as 20% of CHL can be negative for GATA3, CHL cannot be ruled out with negative GATA3. Additional findings include GATA3 positivity among PMBLs, whereas all 72 DLBCLs were negative for GATA3. This finding further highlights similarities between CHL and PMBL.

MicroRNA-124 represses wound healing by targeting SERP1 and inhibiting the Wnt/β-catenin pathway.

Wound healing is a complex process which restores cellular structures and tissue layers after their destruction. Accumulating evidence has proven that microRNAs (miRs) are involved in wound healing. The aim of the study was to research the role of miR-124 in wound healing. Keratinocytes were respectively transfected with miR-124 mimic, scrambled miRNA (a negative control of miR-124 mimic: mimic NC), antisense oligonucleotides against miR-124 (ASOmiR124), or a negative control of ASO-miR-124 (ASO-NC), and then cell viability, colony formation, cell cycle, expression of cell cycle-associated proteins, and collagen content were all evaluated. The target gene of miR-124 was predicted using TargetScan and verified with luciferase assay. Subsequently, the effects of target gene overexpression on cell viability, colony formation and collagen synthesis were all evaluated. Finally, the expression levels of key kinases in the Wnt/β-catenin pathway were detected using western blot analysis. Cell viability, colony formation, expression levels of cell cycle-associated proteins, and collagen content were all significantly reduced by miR-124 overexpression. As predicted using bioinformatics and validated with luciferase assay, stress-associated endoplasmic reticulum protein 1 (SERP1) is a target gene of miR-124. Meanwhile, the miR-124 mimic-induced decrease in cell viability, colony formation and collagen synthesis was reversed by SERP1 overexpression. Furthermore, the miR-124 mimic obviously upregulated glycogen synthase kinase 3β (GSK-3β) while downregulating β-catenin, T cell transcription factor 4 (TCF-4) and leukemia enhancer factor 1 (LEF-1). Additionally, all the effects of ASO-miR-124 were the opposite of those of the miR-124 mimic. We found that miR-124 inhibited keratinocyte proliferation, collagen biosynthesis and activation of Wnt/β-catenin by targeting SERP1.

21st Century FOX: Toward Gene Therapy for the Regulatory T Cell Deficiency Syndrome IPEX.

IPEX is a fatal X-linked autoimmune disorder that results from mutations in the canonical regulatory Tcell transcription factor FOXP3. In this issue of Cell Stem Cell, Masiuk etal. (2019) demonstrate an effective and potentially durable approach to eliminate the IPEX phenotype in murine models.

Simulation of P2X-mediated calcium signalling in microglia.

A computational model of P2X channel activation in microglia was developed that includes downfield Ca2+ -dependent signalling pathways. This model provides quantitative insights into how diverse signalling pathways in microglia converge to control microglial function. Microglia function is orchestrated through highly coupled signalling pathways that depend on calcium (Ca2+ ). In response to extracellular ATP, transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis. Although the steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic purinergic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumour necrosis factor α (TNFα) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of nuclear factor of activated T-cells (NFAT) transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our results suggest that pulsatile stimulation of P2X4 via micromolar ATP may be sufficient to promote TNFα production, whereas high-amplitude ATP exposure is necessary for production via P2X7. Furthermore, under conditions that increase P2X4 expression, for instance, following activation by pathogen-associated molecular factors, P2X4-associated TNFα production is greatly enhanced. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

Clinical and biological relevance of genetic alterations in pediatric T-cell acute lymphoblastic leukemia in Taiwan.

The leukemogenesis of T-cell acute lymphoblastic leukemia (T-ALL) involves multistep processes of genetic alterations. We aimed to determine the genetic alterations including common fusion transcripts, overexpression of T-cell transcription factor oncogenes, and deletion or mutation of targeted genes in pediatric T-ALL in Taiwan as well as their impact on outcomes in those treated with the Taiwan Pediatric Oncology Group-ALL-2002 protocol. Between 1995 and 2015, bone marrow samples obtained from 102 children aged <18 years consecutively diagnosed with T-ALL were examined. Thirty-two genetic alterations were examined by reverse transcription polymerase chain reaction (PCR) assays-PCR-based assays-followed by direct sequencing, real time quantitative PCR with TaqMan assays, or multiplex ligase probe amplification. TAL1 overexpression, CDKN2A/2B deletions, and NOTCH1 mutation were the most frequent aberrations while none had NF1, SUZ12 deletion, JAK1 or JAK2 mutations, or NUP214-ABL1 fusion in our cohort. The most frequent cooperating occurrence of genetic alterations included CDKN2A/2B and MTAP, MTAP and CDKN2B, LEF1 and PTPN2, and HOX11L2 and PHF6 mutation/deletion. NOTCH1 mutations conferred a favorable overall survival, whereas SIL-TAL1 fusion, TAL overexpression, LEF1 deletion, and PHF6 deletion/mutation were associated with an inferior outcome. By multivariate analysis, PHF6 mutation/deletion was the only independent predictor for inferior overall survival. The present study showed that the frequencies of genetic alterations in Taiwanese children with T-ALL differed considerably from those reported in Western countries. PHF6 mutation/deletion was an independently adverse predictor.

Prophylactic treatment against GM-CSF, but not IL-17, abolishes relapses in a chronic murine model of multiple sclerosis.

The pathogenic role of IL-17 and GM-CSF has been unravelled in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). However, in most models, EAE is characterised by a monophasic attack which is not representative of the relapsing nature nor the chronicity displayed in MS. Here, we used proteolipid protein peptide (PLP139-151 ) to trigger EAE-relapses (EAE-II) in SJL mice that had recovered from a primary-EAE episode (EAE-I). This procedure resulted in severe and irreversible disease that, unlike EAE-I, was not abolished by anti-IL-17-mAb. In contrast, prophylactic anti-GM-CSF-mAb treatment prevented EAE-I and -II. Strikingly, the expression of T-cell transcription factors and cytokines/chemokines in mice treated with anti-GM-CSF during both EAE episodes was silenced. Anti-GM-CSF-mAb treatment administered only during EAE-II did not completely prevent relapses but mice ultimately reached full recovery. Anti-GM-CSF treatment also strongly impaired and ultimately resolved monophasic MOG35-55 -induced EAE in C57Bl/6 mice. In such protected mice, anti-GM-CSF treatment also prevented a further relapse induced by MOG-revaccination. These results underscore the critical role of GM-CSF on pro-inflammatory mediator production. Furthermore, we observed a strong preventive and curative effect of anti-GM-CSF neutralisation in two EAE models, relapsing and chronic. Altogether these findings are relevant for further MS research.