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1
Tilburgs, Tamara, Ângela C. Crespo, Anita van der Zwan, et al. "Human HLA-G+ extravillous trophoblasts: Immune-activating cells that interact with decidual leukocytes." Proceedings of the National Academy of Sciences 112, no. 23 (2015): 7219–24. http://dx.doi.org/10.1073/pnas.1507977112.
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Invading human leukocyte antigen-G+ (HLA‐G+) extravillous trophoblasts (EVT) are rare cells that are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. Here highly purified HLA‐G+ EVT and HLA‐G− villous trophoblasts (VT) were isolated. Culture on fibronectin that EVT encounter on invading the uterus increased HLA‐G, EGF-Receptor-2, and LIF-Receptor expression on EVT, presumably representing a further differentiation state. Microarray and functional gene set enrichment analysis revealed a striking immune-activating potential for EVT that was absent in VT. Cocultures of HLA‐G+ EVT with sample matched decidual natural killer cells (dNK), macrophages, and CD4+ and CD8+ T cells were established. Interaction of EVT with CD4+ T cells resulted in increased numbers of CD4+CD25HIFOXP3+CD45RA+ resting regulatory T cells (Treg) and increased the expression level of the Treg-specific transcription factor FOXP3 in these cells. However, EVT did not enhance cytokine secretion in dNK, whereas stimulation of dNK with mitogens or classical natural killer targets confirmed the distinct cytokine secretion profiles of dNK and peripheral blood NK cells (pNK). EVT are specialized cells involved in maternal–fetal tolerance, the properties of which are not imitated by HLA‐G–expressing surrogate cell lines.
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Wang, Yingjie, and Yun Wang. "Palmitic Acid Upregulates CD96 Expression to Mediate Maternal–Foetal Interface Immune Tolerance by Inhibiting Cytotoxic Activity and Promoting Adhesion Function in Human Decidual Natural Killer Cells." Bioengineering 10, no. 9 (2023): 1008. http://dx.doi.org/10.3390/bioengineering10091008.
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Decidual natural killer cells (dNK cells) are an essential component of the immune cells present at the maternal–foetal interface during early pregnancy, and they play a vital role in various physiological processes. Abnormalities in the ratio or function of dNK cells have been linked to recurrent miscarriages. CD96 has been previously shown to regulate NK cell function in the tumour microenvironment; however, its role and mechanism at the maternal–foetal interface remains unclear. The present study aimed to investigate the immunomodulatory role of CD96 in dNK cells and its function at the maternal–foetal interface. Immunofluorescence staining and flow cytometry were used to detect the expression of cellular markers such as CD96. Furthermore, the secretory function, adhesion-function-related molecules, and cell proliferation markers of CD96+ and CD96− dNK cells were detected using flow cytometry. In addition, we performed cell culture experiments via the magnetic bead sorting of NK cells to detect changes in the expression of the aforementioned functional molecules in dNK cells after the CD96 blockade. Furthermore, we examined the functional characteristics of dNK cells after palmitic acid treatment at a concentration of 10 μM. We also examined the changes in dNK cell function when subjected to the combined effect of palmitic acid and CD96 antagonists. The results indicated that CD96, TIGIT, CD155, and CD112 were highly expressed at the maternal–foetal interface, with dNK cells predominantly expressing CD96, whereas TIGIT was mainly expressed on T cells, and CD155 and CD112 were mainly present in metaphase stromal and trophoblast cells. CD96+ dNK cells displayed low cytotoxic activity and a high adhesion phenotype, which mediated the immunosuppressive effect on dNK cells at the maternal–foetal interface. Palmitic acid upregulated CD96 expression on the surface of dNK cells in the coculture system, inhibiting dNK cell activity and increasing their adhesion molecule expression. CD96 antagonist treatment blocked the inhibitory effect of trophoblasts on dNK cells, resulting in enhanced cytokine secretion and reduced adhesion. The results of this study provide valuable insight into the immunomodulatory role of CD96 in dNK cells and its mechanism at the maternal–foetal interface, particularly in metaphase NK cells. This study sheds light on the mechanisms of immune regulation at the maternal–foetal interface and their implications for the study of recurrent miscarriages of unknown origin.
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Danilova, M. S., R. A. Bontsevich, and M. L. Maksimov. "Some etiological features of spontaneous abortions (literature review)." Hirurg (Surgeon), no. 4 (August 2, 2024): 15–25. http://dx.doi.org/10.33920/med-15-2404-02.
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The review article examines some etiological features of spontaneous abortions. There are many mechanisms of this pathology, and the article presents some of them: a shift in the balance of decidual natural killer cells (dNK), natural killer T (NKT) cells, regulatory T cells (Tregs), monocytes, macrophages, lymphocytes, and dendritic cells at the fetal-maternal border. There are several pathologies associated with Tregs: expression of soluble Tim-3, imbalance between Th1/Th2 cells and Th17/Tregs at different stages of pregnancy. Spontaneous abortions are associated with defects in fetal sHLA class I, which affect placental vessels and maternal immune cells through dNK and INF-γ, as well as defects in decidual cells, which are precursors of immune cells at the fetal-maternal border. The B7‑H4 protein, a regulator of T cell activity, also plays an important role. In addition, some researchers have noted the presence of immune reactions against the Y chromosome of the fetus. Poorly studied mechanisms of spontaneous abortions are defective LIF and CD95/CD95 ligand system.
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Tilburgs, Tamara, Angela Crespo, Anita van der Zwan, Ashley Moffett, and Jack Strominger. "Human HLA-G+ extravillous trophoblasts: immune activating cells that interact with decidual leucocytes (MUC2P.921)." Journal of Immunology 194, no. 1_Supplement (2015): 65.4. http://dx.doi.org/10.4049/jimmunol.194.supp.65.4.
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Abstract Invading HLA-G+ extravillous trophoblasts (EVT) are believed to play a key role in the prevention of a maternal immune attack on foreign fetal tissues. EVT are difficult to study due to their low frequency and lack of proliferative capacity. Here highly purified HLA-G+ EVT and HLA-G- villous trophoblast (VT) were isolated. Culture on fibronectin increased HLA-G expression on EVT but differentiation from VT into EVT was not observed. Moreover microarray analysis demonstrated that VT and EVT have more than 4200 differentially expressed genes. Functional gene set enrichment analysis (GSEA) revealed a striking immune activating potential for EVT that is absent in VT. Co-culture of HLA-G+ EVT with sample matched decidual NK, macrophages, CD4+ and CD8+ T cells were established and demonstrated interactions of all leukocyte types with EVT. Interaction of EVT with CD4+ T cells resulted in increased proportion of CD4+CD25hiFOXP3+ Tregs and increased FOXP3 protein level in these cells. However, EVT did not enhance cytokine secretion in dNK whereas stimulation of dNK using mitogens or classical NK targets confirmed the distinct cytokine profiles of dNK and pNK. Thus EVT are specialized cells with an immune activating profile whose properties are not imitated by HLA-G expressing cell lines. Careful validation of EVT-leukocyte interactions using primary HLA-G+ EVT needs to be carried out to understand the unique contribution of EVT to the decidual immune response in human pregnancy.
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Tilburgs, Tamara, and Jack Strominger. "Molecular analysis of human HLA-G+ trophoblasts reveals their strong immune regulatory potential (P2172)." Journal of Immunology 190, no. 1_Supplement (2013): 69.24. http://dx.doi.org/10.4049/jimmunol.190.supp.69.24.
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Abstract INTRODUCTION: During pregnancy HLA-G+ extravillous trophoblasts (EVTs) play a key role in the prevention of a maternal immune attack to allogeneic fetal tissue. Human EVTs are extremely difficult to study due to their low frequency and lack of proliferative capacity. In this study we obtained robust villous trophoblast (VT) and EVT preparations and performed microarray analysis on RNA isolates. RESULTS: Comparison of the expression profiles revealed >4000 differentially expressed genes based on a 2-fold difference. To identify functional differences a Gene Set Enrichment Analysis (GSEA) was performed. In EVTs, 14 functional gene sets out of the 20 most significantly enriched gene sets were associated with direct immune activation. The core genes of these immune pathways include cytokines (e.g. EBi3, TGFβ and IL8) and cell surface molecules (e.g. B7-H3 and CRTAM) which can all directly influence lymphocyte binding and activation. To evaluate the implications of this immune activating profile, co-cultures of EVT with decidual NK (dNK) cells and decidual T (dT) cells were established. Low percentages of dNK and dT cells form immune synapses with HLA-G+ EVTs. Preliminary analysis of dNK-EVT and dT-EVT co-cultures demonstrates that EVTs influence the expression of activation markers on decidual lymphocytes. CONCLUSION: Our data supports the hypothesis that proper immune activation, rather than general immune suppression, facilitates the establishment of fetal-maternal tolerance.
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Zhang, Jianhong, Caroline E. Dunk, Rebecca L. Jones, et al. "Human dNK and regulatory T cells communication in the fetal-maternal interface: a cellular identity and explanation of preeclampsia." Placenta 35, no. 9 (2014): A60. http://dx.doi.org/10.1016/j.placenta.2014.06.195.
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Patel, Anjali, Giuseppe Maiocco, Catherine Mirto, Donald S. Torry та Andrew Wilber. "Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ)." Journal of Clinical Oncology 42, № 16_suppl (2024): e14557-e14557. http://dx.doi.org/10.1200/jco.2024.42.16_suppl.e14557.
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e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.
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Klemen, Nicholas, Raul Vizcardo, Linda Tran, and Nicholas P. Restifo. "Precocious Differentiation of Somatic and Pluripotent Stem Cells Bearing Pre-Arranged TCR." Blood 126, no. 23 (2015): 847. http://dx.doi.org/10.1182/blood.v126.23.847.847.
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Abstract Adoptive cell transfer (ACT) of mutation-specific T lymphocytes may represent the future of immunotherapy for cancer. Current strategies utilize mature T cells isolated from tumors, but this approach may be hampered by limited persistence, advanced differentiation status, and exhaustion of transferred cells. Cellular rejuvenation is needed, and this could be accomplished through induced pluripotent stem cell (iPS) technology. IPS cells can be generated from various types of somatic cells using the four Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc), and we have shown that iPS generated from T cells (T-iPS) retain their original TCR specificity upon re-differentiation into T cell lineage. With existing technology, tumor antigen-specific progenitor cells can be derived from T-iPS. These cells could have superior potential to engraft, proliferate, and persist for long periods of time. To explore this concept, we have generated a murine iPS cell line from our Pmel transgenic mouse model, in which T cells bear a pre-rearranged TCR specific for the melanoma antigen GP100. Differentiation in vitro employs the OP9-DL1 co-culture system. We characterize the DN1-DN4 stages by flow cytometry of T cell lineage differentiation markers, such as c-kit, CD3, CD4, CD8, CD25, TCRvb, and TCRvb13 (present in the Pmel transgene). To study somatic stem cell differentiation, we used lineage negative bone marrow from femurs of wild type C57BL/6 (WT) and Pmel TCR transgenic (Pmel) mice. After 10 days of culture, cells were harvested and analyzed. Frequencies of cells in the DN1-DN4 stages for WT were 2%, 41%, 55%, and 2%; for Pmel they were 5%, 31%, 39%, and 25%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 2%. In contrast, CD3 expression in Pmel cells was 85% for cells in DN1/DN2 and 95% for cells in DN3/DN4. To study pluripotent stem cell differentiation, we used WT embryonic stem cells (ES) and Pmel iPS. After 21 days of culture, cells were harvested and analyzed. The double positive compartment was 9% in WT ES and 30% in Pmel iPS; the double negative compartment was 60% and 37%. Frequencies of cells in DN1-DN4 stages for WT ES were 4%, 6%, 45%, and 45%; for Pmel they were 1%, 7%, 69%, and 23%. Expression of CD3 in WT cells in the DN1/DN2 stage was 1%, and for the DN3/DN4 stage was 13%. In contrast, CD3 expression in Pmel cells was 76% for cells in DN1/DN2 and 91% for cells in DN3/DN4. These data show for the first time that in vitro differentiation of pluripotent stem-derived progenitor cells bearing a pre-rearranged TCR will undergo precocious expression of the CD3 molecule and enhanced progression to DP stage as compared to WT controls. The pattern is consistent with that previously seen during in vitro differentiation of somatic stem cells. It appears that the presence of a pre-rearranged TCR is driving precocious development of T cells in vitro, regardless of whether the source of progenitors is from somatic (bone marrow) or pluripotent (ES/iPS) stem cells. Our ultimate goal is to intricately characterize the nature of iPS-derived T cells, and study their antitumor efficiency in vitro and in vivo, in order to evaluate their suitability for clinical use. Disclosures No relevant conflicts of interest to declare.
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Yang, Xiuhua, Yingying Tian, Linlin Zheng, Thanh Luu, and Joanne Kwak-Kim. "The Update Immune-Regulatory Role of Pro- and Anti-Inflammatory Cytokines in Recurrent Pregnancy Losses." International Journal of Molecular Sciences 24, no. 1 (2022): 132. http://dx.doi.org/10.3390/ijms24010132.
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Recurrent pregnancy losses (RPL) is a common reproductive disorder with various underlying etiologies. In recent years, rapid progress has been made in exploring the immunological mechanisms for RPL. A propensity toward Th2 over Th1 and regulatory T (Treg) over Th17 immune responses may be advantageous for reproductive success. In women with RPL and animals prone to abortion, an inordinate expression of cytokines associated with implantation and early embryo development is present in the endometrium or decidua secreted from immune and non-immune cells. Hence, an adverse cytokine milieu at the maternal-fetal interface assaults immunological tolerance, leading to fetal rejection. Similar to T cells, NK cells can be categorized based on the characteristics of cytokines they secrete. Decidual NK (dNK) cells of RPL patients exhibited an increased NK1/NK2 ratio (IFN-γ/IL-4 producing NK cell ratios), leading to pro-inflammatory cytokine milieu and increased NK cell cytotoxicity. Genetic polymorphism may be the underlying etiologies for Th1 and Th17 propensity since it alters cytokine production. In addition, various hormones participate in cytokine regulations, including progesterone and estrogen, controlling cytokine balance in favor of the Th2 type. Consequently, the intricate regulation of cytokines and hormones may prevent the RPL of immune etiologies. Local or systemic administration of cytokines or their antagonists might help maintain adequate cytokine milieu, favoring Th2 over Th1 response or Treg over Th17 immune response in women with RPL. Herein, we provided an updated comprehensive review regarding the immune-regulatory role of pro- and anti-inflammatory cytokines in RPL. Understanding the roles of cytokines involved in RPL might significantly advance the early diagnosis, monitoring, and treatment of RPL.
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Goričar, Katja, Viljem Kovač, Janez Jazbec, Janez Lamovec, and Vita Dolžan. "Homologous Recombination Repair Polymorphisms and the Risk for Osteosarcoma / Polimorfizam Gena Odgovornih Za Reparaciju Dnk Homolognom Rekombinacijom I Rizikza Pojavu Osteosarkoma." Journal of Medical Biochemistry 34, no. 2 (2015): 200–206. http://dx.doi.org/10.2478/jomb-2014-0031.
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Summary Background: DNA repair mechanisms are essential for maintaining genome stability, and genetic variability in DNA repair genes may contribute to cancer susceptibility. Our aim was to evaluate the influence of polymorphisms in the homologous recombination repair genes XRCC3, RAD51, and NBN on the risk for osteosarcoma. Methods: In total, 79 osteosarcoma cases and 373 controls were genotyped for eight single nucleotide polymorphisms (SNPs) in XRCC3, RAD51, and NBN. Logistic regression was used to determine the association of these SNPs with risk for osteosarcoma. Results: None of the investigated SNPs was associated with risk for osteosarcoma in the whole cohort of patients, however, in patients diagnosed before the age of thirty years XRCC3 rs861539 C>T and NBN rs1805794 G>C were associated with significantly decreased risk for osteosarcoma (P=0.047, OR=0.54, 95% CI=0.30-0.99 and P=0.036, OR=0.42, 95% CI=0.19-0.94, respectively). Moreover, in the carriers of a combination of polymorphic alleles in both SNPs risk for osteosarcoma was decreased even more significantly (Ptrend=0.007). The risk for developing osteosarcoma was the lowest in patients with no wild-type alleles for both SNPs (P=0.039, OR=0.31, 95% CI=0.10-0.94). Conclusions: Our results suggest that polymorphisms in homologous recombination repair genes might contribute to risk for osteosarcoma in patients diagnosed below the age of thirty years.
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Hathcock, karen S., Joseph An, Max Xu, and Richard J. Hodes. "Differential survival requirements for Lck- and CD3e-dependent signaling defines two populations of ATMKO thymic T cell lymphomas." Journal of Immunology 204, no. 1_Supplement (2020): 154.1. http://dx.doi.org/10.4049/jimmunol.204.supp.154.1.
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Abstract The product of theataxia-telangiectasia mutated gene (ATM) is a serine-threonine kinase thatcommunicates between molecules that detect dangerous DNA double strand breaksand effector mechanisms that maintain genomic integrity. ATM deficiency in bothhumans and mice is associated with genomic instability and increased malignancies. ATMKO mice exclusively develop thymic T cell lymphomas that resemble thymically-derivedT-ALL that occur in human ATM-deficient ataxia telangiectasia patients. Flowcytometry identified two subsets of ATMKO T cell lymphomas based on surfaceexpression of CD3ɛ, TCRβ and pre-Tα:Cells that express surface CD3ɛ, TCRβ and pre-Tα(surface receptor high; sRhi) and cells that do not express detectable surface CD3ɛ, TCRβ orpre-Tα(sRlo). When assayed for CD25 and CD44 expression sRhi lymphomas resemble DN3or DN4 cells while sRlo lymphomas resemble more immature DN1 or DN2 cells. Pre-TCRsignaling in normal DN3 and DN4 thymocytes provides an essential survivalsignal that is proposed to be constitutive and ligand-independent. Therefore, weasked if surface sRhi and sRlo cells are dependent on pre-TCR signaling byincubating cells with either Lck inhibitor, CAS 213743-31-8, or CD3ɛshRNA to inhibit pre-TCR signaling. Both treatments significantly inhibitedsurvival of sRhi lymphomas, while survival of sRlo cells was largely unaffected. These results suggest that ATMKO T celllymphomas arise from cells transformed at different stages of DN development andthat sRhigh cells, like normal DN3 and DN4 thymocytes, retain apre-TCR-dependent survival requirement. These results also suggest that pre-TCRsignaling may be a useful target for clinical intervention in treatment ofhuman T-ALL that express a pre-TCR.
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Sashida, Goro, Changshan Wang, Daisuke Sato, Motohiko Oshima, and Atsushi Iwama. "Ezh2 Loss Promotes the Transformation of Early T Cell Precursor Leukemia Via Suppressing Critical Genes for T-Cell Differentiation." Blood 126, no. 23 (2015): 308. http://dx.doi.org/10.1182/blood.v126.23.308.308.
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Abstract Early T cell precursor (ETP) acute lymphoblastic leukemia (ALL) has been identified as a new pathologic entity with poor outcome in patients with T-ALL. In contrast to cortical T-ALL, ETP-ALL has been characterized by the activating mutations in genes regulating cytokine signaling and the inactivating mutations in the polycomb repressor complex 2 (PRC2) component genes including EZH2. EZH2 catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3) that mediates silencing of critical genes for cell proliferation and differentiation. Given that loss-of-function mutations in EZH2 are often found in ETP-ALL, EZH2 may play a tumor suppressor role in the pathogenesis of ETP-ALL. To determine how EZH2 dysfunction promotes the development of ETP-ALL in vivo, we generated a novel mouse model of ETP-ALL by utilizing Ezh2 and p53 conditional knockout mice. We harvested bone marrow cells from Cre-ERT (WT), Cre-ERT;Ezh2flox/flox (Ezh2 KO), Cre-ERT;Trp53 flox/flox (p53 KO) and Cre-ERT;Trp53 flox/flox;Ezh2flox/flox (DKO) mice, and transplanted them into lethally-irradiated Ly5.1+ recipient mice. We deleted p53 and Ezh2 via administration of tamoxifen 4 weeks post-transplantation. We observed that p53 KO mice died dominantly due to CD3+ CD8+ cortical T-ALL (median survival; 137 days), as previously reported. While 2 out of 8 DKO mice developed CD8+ cortical T-ALL, we found that 6 out of 8 DKO mice developed CD4- CD8- (DN) T-ALL with a longer latency (median survival; 189 days). At the time of sacrifice, DKO mice showed significantly enlarged thymus due to the expansion of c-Kit+ CD44+ CD25- DN1 and c-Kit+ CD44+ CD25+ DN2 cells, which were markedly compromised in differentiation into DN3 and further down to CD4+ CD8+ (DP) cells. Histological analysis demonstrated the expansion of cytoplasmic CD3+ (cyCD3) tumor cells in the thymus of DKO mice with DN T-ALL. Thus, DKO mice faithfully recapitulated the phenotypic features of human ETP-ALL, indicating that the loss of Ezh2 is prerequisite to the development of c-Kit+ CD44+ CD25+ cyCD3+ DN ALL in mice lacking p53. To understand the underlying molecular mechanism in the pathogenesis of Ezh2 loss-induced ETP-ALL, we performed gene expression analysis in DN1, DN2 and DN3 cells isolated from WT and DKO mice at pre-disease and ETP-ALL stages. Since NOTCH1 activating mutations are not frequently found in patients with ETP-ALL, we observed no significant difference in activation of Notch1-target genes between WT and DKO ETP-ALL cells. In contrast, Ras-target genes were significantly activated in DKO ETP-ALL DN2 cells relative to WT DN2 cells. Thus, our ETP-ALL mouse model shows active Ras signaling but lacks Notch1 activation, consistent with the molecular features of human ETP-ALL. Furthermore, we found that the genes critical for T-cell commitment, including Tcf7 or Bcl11b, were transcriptionally repressed in DKO ETP-ALL DN2 cells compared to WT DN2 cells, while the signature genes of both HSCs and myeloid cells were retained in DKO ETP-ALL DN2 cells. Taken together, Ezh2 and p53 deletions cooperate to activate the function of HSCs and impede the transcriptional program of T-cell differentiation at the DN2 stage with sustaining myeloid potential. To determine how H3K27me3 modification contributed to induce ETP-ALL in the absence of Ezh2, we performed H3K27me3-chromatin immunoprecipitation (ChIP) sequencing in WT and DKO ETP-ALL DN1/2 cells. We found that H3K27me3 marks were lost or kept at low levels at the promoter regions of T-cell differentiation regulators in ETP-ALL cells. Thus, we sought to determine whether altered DNA hypermethylation contributed to silencing the expression of T-cell differentiation regulators. The transduction of either Tcf7 or Bcl11b expression alone was not sufficient to induce differentiation of DKO DN1/2 cells in vitro, however, we found that treatment of decitabine, a demethylating agent, clearly induced the differentiation of DKO DN1/2 cells beyond the DN3 stage in vitro, implying that Ezh2 loss and p53 loss cooperatively induced aberrant DNA hypermethylation, thereby impeding the differentiation of DN1/2 cells. In conclusion, we demonstrated that combined deletion of Ezh2 and p53 altered the epigenetic regulation to an extent not seen in either deletion alone, and induced highly penetrant ETP-ALL characterized by the molecular profile similar to that in patients with ETP-ALL harboring mutations in the PRC2 components. Disclosures No relevant conflicts of interest to declare.
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Li, Long, and Ellen Rothenberg. "Bcl11b: a novel regulator of T-lineage commitment (36.50)." Journal of Immunology 184, no. 1_Supplement (2010): 36.50. http://dx.doi.org/10.4049/jimmunol.184.supp.36.50.
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Abstract Commitment of HSC to a T-lineage fate is driven by a network of transcription factors and environment cues. Transcription factor Bcl11b is expected to be a crucial component of the network. Bcl11b is an essential regulator for T-cell development. However, little is known about how Bcl11b works in the commitment stage of T-cell development. Using an in vitro growth system, we have found that under certain conditions Bcl11b-deficient pre-T cells fail to pass the DN2/DN3 transition, which is the lineage commitment step in T-cell development. Instead, Bcl11b-deficient cells continuously grow at DN1-like and DN2A-like stages for more than twenty days, while wild-type controls can develop normally to DN3 and DN4 cells. Unlike their wild-type counterparts, the long-term Bcl11b-deficient cells still possess developmental plasticity with an IKDC-like population growing out from the cells. Gene expression profile reveals that Bcl11b-deficient cells express T-cell identity genes including Cd3e and Lck, and continue to silence some important genes associated with major developmental alternatives, such as B-cell factors; however, they fail to turn off stem cell genes and some lineage-regulators, such as Scl, Lyl1, Gfi1b, Id2, Il2rb, Zbtb16 and Bcl11a. The results indicate that Bcl11b plays a major role in T-lineage commitment by controlling the correct exit of T-cell precursors from a progenitor cell state. No other factor has been shown to have this distinctive function.
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Wang, Yinghua, Xuan Pan, Mei Yu, and Renren Wen. "Polycomb Group Protein Yin Yang 1 Controls DNA Methylation in Murine T Lymphocyte Development." Blood 142, Supplement 1 (2023): 3928. http://dx.doi.org/10.1182/blood-2023-190263.
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Transcription factors and epigenetic regulators play key functions in T lymphocyte lineage commitment and development. Mammalian PcG proteins are major negative regulators of gene expression that typically bind as large complexes (such as Polycomb Repressive Complex 1 (PRC1) and PRC2) to gene regulatory regions selectively targeted by an as yet unknown mechanism. While nearly all mammalian PcG proteins are lack of specific DNA binding capacities, transcription factor Yin Yang 1 (YY1) can bind at the DNA sites specifically and recruit other PcG proteins to specific DNA sequences. A small 25 amino acid YY1 domain (the REPO domain) is necessary and sufficient for the recruitment of PcG proteins, PcG-mediated stable transcriptional repression and histone modification. While YY1ΔREPO is competent for DNA binding, transcriptional activation, transient transcriptional repression and interaction with transcriptional coregulators such as HDACs, YY1ΔREPO is defective in all YY1 PcG functions. Our prior study demonstrated that deletion of YY1 in the hematopoietic system leads to T cell development blockage at the double-negative 3 (DN3) stage. Interestingly, ectopic expression of YY1∆REPO in YY1 null background by retroviral bone marrow transplantation could not rescue YY1 deficiency mediated T cell survival defect. This suggests that the YY1 REPO domain/PcG function plays a vital role in T cell development. Herein, we generated the YY1 REPO domain conditional knockout mouse model ( Yy1 f/∆REPO Vav-Cre) by CRISPR/Cas9 to further dissect the YY1 REPO domain/PcG function in T cell development. Yy1 f/∆REPO Vav-Cre ( Yy1 -/∆REPO) mice have reduced thymus weight and cellularity with medulla aplasia. Compared with wild-type (WT) and YY1 heterozygous ( Yy1 +/-) mice, Yy1 -/∆REPO mice have an increased percentage of DN3 cells with decreased DN4 percentage supporting a developmental blockage at the DN3 stage. Yy1 -/∆REPO DN T cells fail to go through TCRβ rearrangement with increased apoptosis and reduction of TCRβ + cells in the thymus compared with WT and Yy1 +/-cells. Interestingly, RNA-seq analysis in sorted primary DN3 cells revealed the genetic network governing DNA methylation, histone methylation, chromosome maintenance, and translation initiation complex formation are deregulated in Yy1 -/∆REPO mice compared to WT and Yy1 +/-mice. DN T cell development requires precise and dynamic regulation of histone and DNA methylation and demethylation. Interestingly, the DNA demethylase enzymes ten-eleven translocation (TET) family proteins Tet1 and Tet2 are downregulated in Yy1 -/∆REPO DN3 thymocytes compared with WT and Yy1 +/- DN3 cells. Our data show that the YY1 REPO domain/PcG function is required for DN3-DN4 transition via regulating TCRβ rearrangement and the YY1 REPO domain/PcG function plays a critical role in proper DNA methylation status in early T cell development. As PcG proteins modify histone via trimethylation of histone H3 on lysine 27, we will further assess the impact of YY1 REPO domain/PcG function on histone methylation in T cell development. By utilizing a powerful mouse model for identifying YY1 functions as PcG domain-dependent or PcG domain-independent, our study has generated fundamental new insights into the epigenetic landscape in T cell development.
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Saidi, Amal, Tangliang Li, Falk Weih, Patrick Concannon, and Zhao-Qi Wang. "Dual Functions of Nbs1 in the Repair of DNA Breaks and Proliferation Ensure Proper V(D)J Recombination and T-Cell Development." Molecular and Cellular Biology 30, no. 23 (2010): 5572–81. http://dx.doi.org/10.1128/mcb.00917-10.
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ABSTRACT Immunodeficiency and lymphoid malignancy are hallmarks of the human disease Nijmegen breakage syndrome (NBS; OMIM 251260), which is caused by NBS1 mutations. Although NBS1 has been shown to bind to the T-cell receptor alpha (TCRα) locus, its role in TCRβ rearrangement is unclear. Hypomorphic mutations of Nbs1 in mice and patients result in relatively mild T-cell deficiencies, raising the question of whether the truncated Nbs1 protein might have clouded a certain function of NBS1 in T-cell development. Here we show that the deletion of the entire Nbs1 protein in T-cell precursors (Nbs1 T-del) results in severe lymphopenia and a hindrance to the double-negative 3 (DN3)-to-DN4 transition in early T-cell development, due to abnormal TCRβ coding and signal joints as well as the functions of Nbs1 in T-cell expansion. Chromatin immunoprecipitation (ChIP) analysis of the TCR loci reveals that Nbs1 depletion compromises the loading of Mre11/Rad50 to V(D)J-generated DNA double-strand breaks (DSBs) and thereby affects resection of DNA termini and chromatin conformation of the postcleavage complex. Although a p53 deficiency relieves the DN3→DN4 transition block, neither a p53 deficiency nor ectopic expression of TCRαβ rescues the major T-cell loss in Nbs1 T-del mice. All together, these results demonstrate that Nbs1's functions in both repair of V(D)J-generated DSBs and proliferation are essential for T-cell development.
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Scripture-Adams, Deirdre, Alexandra Arias, Koorosh Elihu, I.-Cheng Ho, and Ellen Rothenberg. "Extreme GATA-3 dose dependence in early T cell development (36.28)." Journal of Immunology 184, no. 1_Supplement (2010): 36.28. http://dx.doi.org/10.4049/jimmunol.184.supp.36.28.
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Abstract GATA-3 expression first reaches peak levels during T-lineage commitment, but a specific requirement for GATA-3 at each of the early DN stages in T development has not been proven. Using retroviral expression of shRNA to reduce GATA-3 protein at each of the earliest stages of T-cell differentiation (DN1, DN2, DN3), we identify two dose-dependent effects: a defect in survival or expansion at DN1, and a separable developmental arrest of cells at DN2, which was cell-autonomous, and not relieved by a Bcl2 transgene. The DN2 arrest phenotype was extremely robust; we were able to document this developmental block even in T development assay cultures initiated with a single fetal liver derived precursor. The DN2-stage developmental block was confirmed using transduced Cre to induce deletion of a floxed Gata3 allele. Reduction of GATA-3 also increased PU.1 levels in pro-T cells, despite relatively normal T-lineage gene induction. To test roles of GATA-3 in exclusion of non-T fates, we forced expression of wild type, obligate repressor, and tamoxifen-inducible GATA-3 forms in precursors undergoing B and myeloid development. GATA-3 blocked B cell development via direct repression under conditions which remained permissive for myeloid development. Thus GATA-3 is required for at least two steps of T-cell progression to commitment, and restricts two different alternative fates by distinct mechanisms.
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Ratiu, Jeremy J., William Barclay, Qun Wang, Mari L. Shinohara та Yuan Zhuang. "Loss of Zfp335 triggers cGAS/STING-dependent apoptosis of post-β selection pre-T cells". Journal of Immunology 208, № 1_Supplement (2022): 107.18. http://dx.doi.org/10.4049/jimmunol.208.supp.107.18.
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Abstract Production of a diverse peripheral T cell compartment requires massive expansion of the bone marrow progenitors that seed the thymus. There are two main phases of expansion during T cell development, following T lineage commitment at the DN2 stage and following successful rearrangement and selection for functional TCRβ chains in DN3 thymocytes, which promotes development of DN4 cells to the DP stage. Signals driving expansion of DN2 thymocytes are well studied, however, factors regulating the proliferation and survival of DN4 cells remain poorly understood. Here, we uncover an unexpected link between the transcription factor Zfp335 and control of cGAS/STING-dependent cell death in post-β-selection DN4 thymocytes. Zfp335 controls survival by sustaining expression of Ankle2 and regulating phosphorylation of Baf which functions to promote nuclear envelope integrity and suppress cGAS/STING-dependent cell death. Together, this study identifies Zfp335 as a key transcription factor controlling the survival of proliferating post-β-selection thymocytes and demonstrates a key role for the cGAS/STING pathway driving apoptosis of developing T cells. Supported by grants from NIH R01-GM059638, P01-AI102853, RO1-AI099100, RO1-NS120417, and RO1-AI160737
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Saba, Ingrid, Christian Kosan, and Tarik Möröy. "Miz-1 is required to coordinate the pre-TCR-dependent transitional step in DN3/DN4 thymocytes. (160.1)." Journal of Immunology 186, no. 1_Supplement (2011): 160.1. http://dx.doi.org/10.4049/jimmunol.186.supp.160.1.
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Abstract Miz-1 (Myc-interacting zinc finger-1) is a BTB/POZ domain transcription factor that can activate or repress gene transcription. To explore the function of Miz-1, we have generated mice that express a non-functional protein lacking the POZ domain, Miz-1ΔPOZ. These mice show a very small thymus owing to a dramatic reduction of early T cell progenitors and DN4 pre-T cells. DN1-3 cells have higher apoptotic rate indicating that Miz-1 is coordinating survival signals. Miz-1ΔPOZ mice also have a DN3/DN4 transitional block and DN3 cells showed a dramtic up-regulation of the cell cycle regulator p21Waf1. Surprisingly, neither the introduction of a Bcl-2 transgene nor the deletion of p21Waf1 rescued the DN3/DN4 block in Miz-1ΔPOZ mice. The block at the pre-TCR-dependent selection stage rather suggested that Miz-1 might regulate pre-TCR signaling. Although the pre-TCR signaling does not seem to be altered by the expression of a non-functional Miz-1, the intracellular expression of the TCRβ is reduced. Introduction of a rearranged TCR transgene rescued the DN3/DN4 cell numbers and their function in Miz-1ΔPOZ mice. Our data suggest that Miz-1 is required for the transport or the stability of the pre-TCR in order to support the proliferative burst of DN3 cells. Comparative gene expression analysis will determine which are the direct effector genes of Miz-1 and contribute to a better understanding of the critical factors involved in this precise step of T cell development.
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Kassar, Nahed El, Baishakhi Choudhury, Francis Flomerfelt, et al. "IL-7 Effects on Thymocyte Progenitors." Blood 106, no. 11 (2005): 3318. http://dx.doi.org/10.1182/blood.v106.11.3318.3318.
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Abstract IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.
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Maillard, Ivan, Laleh Talebian, Zhe Li, et al. "A Hypomorphic Cbfb Allele Reveals a Critical Dosage-Sensitive Function of Core Binding Factors at the Earliest Stages of T Cell Development." Blood 106, no. 11 (2005): 124. http://dx.doi.org/10.1182/blood.v106.11.124.124.
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Abstract The family of core binding factors includes the DNA-binding subunits Runx1-3 and the common non-DNA binding partner CBFβ. Runx1 and CBFβ are essential for the emergence of hematopoietic stem cells during fetal development, but not for stem cell maintenance during later ontogeny. Runx1 is also required for megakaryocyte differentiation, B cell development, and for the DN2 to DN3 transition in thymocyte development. Runx2/CBFβ are critical for normal osteogenesis, and Runx3 for CD4 silencing in CD8+ T cells, but their contribution to other steps of hematopoietic development is unknown. To examine the collective role of core binding factors in hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss). CBFβ protein levels were reduced by approximately 2–3 fold in fetuses homozygous for the Cbfbrss allele (Cbfbrss/rss), and 3–4 fold in fetuses carrying one hypomorphic and one knockout allele (Cbfbrss/−). Cbfbrss/rss and Cbfbrss/− fetuses had normal erythroid and B cell development, and relatively mild abnormalities in megakaryocyte and granulocyte differentiation. In contrast, T cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in Cbfbrss/rss fetuses, and virtually absent in Cbfbrss/−fetuses. We next assessed the development of Cbfbrss/rss and Cbfbrss/− fetal liver progenitors after transplantation to irradiated adult recipients, in competition with wild-type (wt) bone marrow cells. Wt, Cbfbrss/rss and Cbfbrss/− fetal progenitors replenished the erythroid, myeloid and B cell compartments equally well. The overall development of Cbfbrss/rss T cells was preserved, although CD4 expression was derepressed in double negative thymocytes. In Cbfbrss/− chimeras, mature thymocytes were entirely derived from competitor cells. Furthermore, the developmental block in Cbfbrss/− progenitors was present at the earliest stages of T cell development within the DN1 (ETP) and DN2 subsets. Our data define a critical CBFβ threshold for normal T cell development, and they situate an essential role of core binding factors during the earliest stages of T cell development. In addition, early thymopoiesis appeared more severely affected by reduced CBFβ dosage than by the lack of Runx1 (Ichikawa et al., Nat Med 2004; Growney et al., Blood 2005), suggesting that Runx2/3 may contribute to core binding factor activity in the T cell lineage.
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Scripture-Adams, Deirdre, Constantin Georgescu, and Ellen Rothenberg. "Transcription factor expression pattern differences are present in OP9 culture derived fetal liver origin early thymocytes when compared to fetal and adult thymocytes (64.22)." Journal of Immunology 186, no. 1_Supplement (2011): 64.22. http://dx.doi.org/10.4049/jimmunol.186.supp.64.22.
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Abstract The advent of the OP9 stromal culture system has tremendously improved our ability to replicate T cell development in vitro. While giving unprecedented access and numerical advantage, these cells may not mirror real thymocytes in their developmental capacity and transcription factor expression patterns. We have examined the pattern of expression of 65 developmentally regulated gene transcripts across the early thymocyte stages of DN1, DN2, DN3 and DN4, in fetal liver derived thymocytes (FLDN) derived from co-culture on OP9 DL-1 cells, and compared them to those found in fetal and adult thymocytes using real time quantitative PCR. Our assessment suggests an overall pattern of similarity between FLDN expression patterns and “real” freshly isolated thymocytes, but some classes of genes are not regulated similarly, and these may have important consequences for development and progression. We present a cluster analysis identifying transcription factors which are differentially regulated in FLDN relative to adult and fetal populations, and identify groups of factors which track with those differentially regulated transcripts. We additionally analyze the effect of long vs. short term culture on these changes in the transcription factor control network that drives T cell development.
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Ryu, Hak-Seung, Na-Yeon Ryoo, Ki-Hong Jung, Gynheung An, and Jong-Seong Jeon. "Rice functional genomics using T-DNA mutants." Journal of Plant Biotechnology 37, no. 2 (2010): 133–43. http://dx.doi.org/10.5010/jpb.2010.37.2.133.
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Yu, Shuyang, Dongmei Zhao, Raja Jothi, and Hai-hui Xue. "GABPalpha is required for normal T cell development (36.67)." Journal of Immunology 184, no. 1_Supplement (2010): 36.67. http://dx.doi.org/10.4049/jimmunol.184.supp.36.67.
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Abstract GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knockout mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative (DN)-3 stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of pre-arranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively GABP is critically required for TCR rearrangements and hence normal T cell development.
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Ichii, Michiko, Natsuko Fujita, Daisuke Okuzaki, et al. "The Role of Signal-Transducing Adaptor Protein-2 in Early T Lymphopoiesis in Thymus." Blood 124, no. 21 (2014): 752. http://dx.doi.org/10.1182/blood.v124.21.752.752.
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Abstract Immune cells show quick responses to infection. Many studies concerning cellular and humoral immunity have focused on the regulation of mature lymphocyte function. However, recent studies revealed that the early stage of hematopoiesis plays an important role in the immune system. In bone marrow, hematopoietic stem/ progenitors (HSPC) are targets of pathogen products and danger signals. After the exposure to Gram-negative lipopolysaccharide, the ligand of Toll-like receptor 4, hematopoietic stem cells enter cell-cycle and differentiate into myeloid lineage cells while B lymphopoiesis almost arrests. Little has been known how early T lymphopoiesis in thymus changes. Previously, we cloned signal-transducing adaptor protein-2 (STAP-2) as a c-fms/M-CSFR interacting protein, and found STAP-2 in T lymphocytes or macrophages is crucial for immune responses. The function of STAP-2 is generally recognized under inflammatory condition, interacting with a variety of signaling or transcriptional molecules. We reported that STAP-2 binds to STAT5 for regulation of T cell proliferation, and in macrophages, STAP-2 combines with MyD88 and IκB kinase to activate NF-κB and enhances the production of IL-6 and TNFα. In this study, the effects of STAP-2 on early T progenitors were evaluated using gene-modified mice. All experimental procedures were conducted under specific pathogen-free conditions, according to protocols approved by Institutional Animal Care and Use Committees of Osaka University. We first evaluated the expression level of STAP-2 in murine thymus with quantitative PCR. STAP-2 mRNA was ubiquitously observed all through T cell development, including the CD4- CD8- double-negative (DN) stage. To test the influences on T lymphopoiesis, we generated knock-out and transgenic mice (Tg) that are modified STAP-2 gene expression. In Tg mice, STAP-2 was overexpressed under the control of the Lck proximal promoter. The promoter could drive expression of the inserted cDNA in T lineage cells from the late of DN 2 stage (CD44+ CD25+). We found that thymus was significantly enlarged in Tg mice (46.7 ± 11.15 mg in WT vs 88.1 ± 25.2 mg in Tg), while the number of T lymphocytes in periphery was comparable to wild-type mice (WT). Results from flow cytometric analysis showed STAP-2 enhanced the percentages of DN2 and DN3 (CD44- CD25+) T progenitors, and the actual numbers of DN2, DN3, DN4 (CD44- CD25-) and CD4+ CD8+ double-positive subpopulations. There were no differences between control and knock-out mice in thymus and peripheral bloods. When Lin- Sca1+ cKitHigh HSPC derived from Tg mice were cultured with Delta-like 1-transduced OP9 stromal cells (OP9-DL1) under T cell generation condition, the development of DN4 cells was accelerated (26.9 ± 6.2 % in WT vs 35.2 ± 4.1 % in Tg). Co-cultures from Lin- CD44+ CD25- cKitHigh early T cell progenitors showed the same tendency. These results indicate that STAP-2 regulates the proliferation and differentiation of T progenitors during DN3 to DN4 stage. To elucidate the signaling regulated by STAP-2, microarray experiment with DN3 T progenitors was conducted. The bioinformative approach with Ingenuity Pathway Analysis showed the canonical pathways related with IL-12 signaling, 4-1BB (CD137) signaling and helper T cell differentiation were significantly influenced. Interestingly, we found that STAP-2 affected the distribution of functional T lymphocytes. The ratio of helper CD4+ cells to suppressor CD8+cells in peripheral bloods was lower in Tg mice than that in WT. In summary, we found that STAP-2 regulates the early T lymphopoiesis in thymus. DN2 to DN4 stages of T progenitors increased in STAP-2 transgenic mice, and STAP-2 promoted the differentiation in vitro. Moreover, STAP-2 affected the cell decision in development to helper CD4+ cells or suppressor CD8+ cells. Our study indicates the up-regulation of STAP-2 under inflammatory condition might be crucial for immune response at the early stage of T lymphopoiesis. Further study would clarify the precise molecular mechanisms of the enhancement of T lymphopoiesis by STAP-2. Disclosures No relevant conflicts of interest to declare.
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Hager-Theodorides, Ariadne L., Johannes T. Dessens, Susan V. Outram, and Tessa Crompton. "The transcription factor Gli3 regulates differentiation of fetal CD4–CD8– double-negative thymocytes." Blood 106, no. 4 (2005): 1296–304. http://dx.doi.org/10.1182/blood-2005-03-0998.
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AbstractGlioblastoma 3 (Gli3) is a transcription factor involved in patterning and oncogenesis. Here, we demonstrate a role for Gli3 in thymocyte development. Gli3 is differentially expressed in fetal CD4–CD8– double-negative (DN) thymocytes and is most highly expressed at the CD44+ CD25– DN (DN1) and CD44–CD25– (DN4) stages of development but was not detected in adult thymocytes. Analysis of null mutants showed that Gli3 is involved at the transitions from DN1 to CD44+ CD25+ DN (DN2) cell and from DN to CD4+CD8+ double-positive (DP) cell. Gli3 is required for differentiation from DN to DP thymocyte, after pre–T-cell receptor (TCR) signaling but is not necessary for pre-TCR–induced proliferation or survival. The effect of Gli3 was dose dependent, suggesting its direct involvement in the transcriptional regulation of genes controlling T-cell differentiation during fetal development.
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Manesso, Erica, Vijay Chickarmane, Hao Yuan Kueh, Ellen V. Rothenberg, and Carsten Peterson. "Computational modelling of T-cell formation kinetics: output regulated by initial proliferation-linked deferral of developmental competence." Journal of The Royal Society Interface 10, no. 78 (2013): 20120774. http://dx.doi.org/10.1098/rsif.2012.0774.
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Bone-marrow-derived progenitors must continually enter the thymus of an adult mouse to sustain T-cell homeostasis, yet only a few input cells per day are sufficient to support a yield of 5 × 10 7 immature T-cells per day and an eventual output of 1–2 × 10 6 mature cells per day. While substantial progress has been made to delineate the developmental pathway of T-cell lineage commitment, still little is known about the relationship between differentiation competence and the remarkable expansion of the earliest (DN1 stage) T-cell progenitors. To address this question, we developed computational models where the probability to progress to the next stage (DN2) is related to division number. To satisfy differentiation kinetics and overall cell yield data, our models require that adult DN1 cells divide multiple times before becoming competent to progress into DN2 stage. Our findings were subsequently tested by in vitro experiments, where putative early and later-stage DN1 progenitors from the thymus were purified and their progression into DN2 was measured. These experiments showed that the two DN1 sub-populations divided with similar rates, but progressed to the DN2 stage with different rates, thus providing experimental evidence that DN1 cells increase their commitment probability in a cell-intrinsic manner as they undergo cell division. Proliferation-linked shifts in eligibility of DN1 cells to undergo specification thus control kinetics of T-cell generation.
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Zhu, Liang, Zi Hu, John Heidemann, Duane Wessels, Allison Mankin, and Nikita Somaiya. "T-DNS." ACM SIGCOMM Computer Communication Review 44, no. 4 (2015): 379–80. http://dx.doi.org/10.1145/2740070.2631442.
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Simon, Camille, Jalila Chagraoui, Jana Krosl, Josee Hebert, and Guy Sauvageau. "Ezh2 Is An Essential Regulator Of T-Cell Development and Oncogenic Transformation." Blood 122, no. 21 (2013): 3729. http://dx.doi.org/10.1182/blood.v122.21.3729.3729.
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Abstract Enhancer of zeste homolog 2 (EZH2) catalyzes di- and trimethylation of lysine 27 on histone H3 (H3K27me2/3) and establishes chromatin marks associated with gene silencing. We and others have recently shown that Ezh2 and its partners act as tumour suppressor genes in mouse and likely human lymphoblastic leukemia. Moreover some studies also suggest that Ezh2 is strongly required during B and T cell differentiation. However, the function of EZH2 during these processes remains unclear. For functional study we exploited an Ezh2 conditional knockout mouse model. The Cre-mediated deletion generates a mutated Ezh2Δ allele and abrogates production of EZH2 protein. Upon gene inactivation we monitored T-cell maturation and cancer development. We found that Ezh2 inactivation induces a block at the DN3-DN4 transition of TCRab+T-cells while TCRγδ T-cells were increased by 5 fold compared to wild type animals. Cell cycle analysis revealed increase in the proportions of TCRγδ+T-cells in the G2 phase compared to TCRβ+T-cells and wild type controls. This observation suggested a possibility of G2/M checkpoint activation resulting from either improper DNA replication, or a non-repaired DNA damage. Moreover we found that the Ezh2 deficient TCRγδ+ leukemia were prone to genomic instability. A majority of leukemias analyzed were aneuploid, and ∼50% were near-tetraploid. These observations were confirmed by Spectral Karyotyping (SKY), which also enabled detection of several chromosomal rearrangements. Consistent with these observations, analysis of global gene expression data from various RNA-Seq-derived datasets revealed that the genes having the highest correlation factor with Ezh2 are involved in cell division, DNA replication and DNA damage repair. Together, these studies show that Ezh2 is an essential regulator of the TCRγδ T-cell state, and prevents T-cell transformation, likely through regulation of DNA replication, cell division or DNA damage repair. Disclosures: No relevant conflicts of interest to declare.
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Colpitts, Sara, Thomas Stoklasek та Leo Lefrançois. "Developmental queues dictate IL-15 expression in γδ T cells (115.4)". Journal of Immunology 188, № 1_Supplement (2012): 115.4. http://dx.doi.org/10.4049/jimmunol.188.supp.115.4.
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Abstract Interleukin-15 (IL-15) is a homeostatic cytokine largely produced by myeloid cells. Surprisingly, analysis of a recently generated transgenic reporter mouse using emerald GFP expression as a surrogate for IL-15 promoter activity (EmGFP/IL-15) revealed unexpected expression in lymphoid γδ T cells. Similar to myeloid cells, in which EmGFP/IL-15 is developmentally regulated in the bone marrow, we found that EmGFP/IL-15 expression was downregulated as EmGFP/IL-15high thymic progenitors committed to a mature T cell fate. EmGFP/IL-15 was significantly reduced between DN2 and DN3 cells and silenced in DN4 and single positive thymocytes. However, γδ T cells and their precursors (CD25+ pre-γδ T cells) maintained low levels of EmGFP/IL-15, which was similar to DN3 cells. Interestingly, we found that intestinal intraepithelial γδ T cells were EmGFP/IL-15neg while a small subset of γδ T cells located in the peripheral lymph nodes expressed elevated levels of EmGFP/IL-15. These cells had a CD44highCD62LlowCD27lowCCR6+ phenotype, which corresponded to the Vγ2+ subset of IL-17-producing γδ T cells. These findings suggest that EmGFP/IL-15 is regulated concomitantly with the waves of γδ T cell development occurring perinatally and that transcriptional regulation at the IL-15 locus may overlap with that of IL-17-inducing machinery. Current studies aim to determine if the novel production of IL-15 by γδ T cells contributes to the overall homeostasis of IL-15-dependent cells in vivo.
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Wang, Hong-Cheng, S. Scott Perry, and Xiao-Hong Sun. "Id1 Attenuates Notch Signaling and Impairs T-Cell Commitment by Elevating Deltex1 Expression." Molecular and Cellular Biology 29, no. 17 (2009): 4640–52. http://dx.doi.org/10.1128/mcb.00119-09.
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ABSTRACT Complete inhibition of E protein transcription factors by Id1 blocks the developmental transition of CD4/CD8 double-negative 1 (DN1; CD44+ CD25−) thymocytes to the DN2 (CD44+ CD25+) stage. To understand the underlying mechanisms, we observed that mRNA levels of Deltex1, as well as Deltex4, were dramatically elevated in Id1-expressing thymocytes, which could result in developmental arrest by attenuating Notch function. In support of this hypothesis, we found that Deltex1 ablation enabled Id1-expressing progenitors to differentiate to the DN3 (CD44− CD25+) stage, which was accompanied by enhanced Notch1 expression in T-cell progenitors. Consistently, constitutive activation of Notch1 drove the differentiation of Id1-expressing progenitors to the DN3 stage. Furthermore, we showed that Gfi1b levels decreased, whereas GATA3 levels increased in Id1 transgenic thymocytes. When overexpressed, GATA3 was able to upregulate Deltex1 transcription. Thus, T-cell commitment may be controlled by the interplay among E proteins, Gfi1b, and GATA3 transcription regulators, which influence Notch function through the expression of Deltex1.
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Hesterberg, Rebecca S., and Pearlie K. Epling-Burnette. "Genetic Ablation of Cereblon (CRBN) Increases Long-Lived Memory T Cells." Blood 126, no. 23 (2015): 3440. http://dx.doi.org/10.1182/blood.v126.23.3440.3440.
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Background: Immunomodulatory drugs (IMiDs) consist of thalidomide and derivatives, pomalidomide and lenalidomide. IMiDs target the E3 ubiquitin ligase substrate receptor, cereblon (CRBN), which forms a complex with DDB1-Cul4A, to induce anti-proliferative effects in tumor cells and increase the activation of T cells. Using crbn deficient T-cells, we have shown that CRBN is a negative regulator of T cell activation in mice. Dynamic changes in the T-cell repertoire occur after thymic involution. With the reduction of thymic output with age, there is a loss of naïve T-cells in the peripheral blood, and the accumulation of a memory-like T-cell population that arises through homeostatic proliferation. In this study, we demonstrate the role of CRBN in peripheral homeostatic regulation during aging. Methods: In this study, C57BL6 crbn -/- mice were housed in pathogen-free conditions to limit foreign antigen exposure. Splenocytes were stained with T cell markers to define various memory populations from 3, 8, 10 and 13 month old crbn -/- and age-matched wild-type mice. Expression of CD8, CD44, CD127 (IL-7 receptor) and KLRG1 were determined by flow cytometry. CD44-CD127+ KLRG1- CD8+ T cells were considered naive in this analysis. Consistent with previous reports, CD44+ CD127+ KLRG1- T cells represent long-term memory T-cells while CD44+CD127-KLRG1+ T cells are effector memory cells. To better understand age-related changes in the thymus, the thymus was dissected and the absolute number of thymocytes was determined by trypan blue staining. Thymic subpopulations were defined using CD4 and CD8 as single positive, double positive, and double negative cells in 13 month old crbn -/- and WT mice. We further characterized double negative (DN) populations using CD44 and CD25 to define DN1, DN2, DN3, and DN4. Results: Splenocytes in 3 and 8 month old crbn -/- and WT mice have no significant differences in CD44+ memory cells, naïve cells (p=0.7850 and p=0.5061) or recently activated cells [CD69+ or CD25+]. Changes related to thymic involution in WT mice become evident at 10 months of age. Crbn -/- mice exhibit a similar percentage and absolute number of total memory cells (p=0.8194) at 10 months of age, as defined by CD44 expression. Evaluation of long-lived versus short-lived sub-populations of memory cells in these older mice showed that crbn deficiency is associated with significantly more CD44+ CD127+ KLRG1- CD8+ [long-lived] memory T cells compared to WT mice (p<0.001). Evaluation of the thymus revealed no difference in absolute numbers of thymocytes in younger mice. Moreover, crbn deficient mice have normal distribution of SP, DP, and DN populations, with only slight changes in DN1, DN2, DN3, and DN4 subpopulations. Absolute numbers of thymocytes, however, were significantly higher in the crbn -/- mice at 10 months of age compared to age-matched controls (p=0.0182). Conclusions: The fate of developing T cells is regulated by positive and negative selection in the thymus where the coordination and selection of self-tolerant repertoire is maintained. Age-related changes caused by thymic involution impacts the protective responses of these cells against foreign pathogens and tumor cells. Our data show that the genetic germline depletion of crbn leads to an increase in long-lived memory T cells in naturally aged animals. Universally in cancer patients, and especially in MDS, the lymphocyte compartment is characterized by premature age-related changes due possibly to reduction in lymphopoiesis at the stem cell level or due to chronic antigen stimulation. Given that the CD44+ CD127+ KLRG1- population is significantly higher in aged crbn -/- mice, these mice may be more resistant to viral infection and or development of malignancy. The mechanism responsible for this phenotypic difference could be due to cell intrinsic signaling or metabolic properties. However, a role for CRBN in thymic epithelial cells must also be considered given the use of germline deficient animals in this study. Taken together, we have demonstrated that ablation of crbn results in an increase in long-lived CD8+ T cells during aging, which suggests that targeting CRBN and/or treating with lenalidomide may functionally improve memory T cell capacity in the elderly. Disclosures No relevant conflicts of interest to declare.
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Do, Jeongsu, Pamela Fink, Lily Li та ін. "Spontaneous development of IL-17-producing γδ T cells in the thymus occurs via a TGFβ1-dependent mechanism (36.37)". Journal of Immunology 184, № 1_Supplement (2010): 36.37. http://dx.doi.org/10.4049/jimmunol.184.supp.36.37.
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Abstract In naive animals γδ T cells are innate sources of IL17. However, mechanism(s) involved in the generation of these cells remains unexplored. We show here that some γδ T cells spontaneously differentiate into IL17+ cells in vivo and IL17-competent γδ T cells were preferentially found in the peripheral LN but not in the mesenteric LN. Ontogeny study revealed that γδ T cell IL17 differentiation mainly occurs in the thymus particularly during the postnatal period, e.g. prior to 7 days of age; ~40% of γδ TCR+ thymocytes in 5-day old mice expressed IL17 upon stimulation. IL17+ γδ TCR thymocytes were mainly found to be CD44highCD25low, ‘DN1 phenotype’ cells. Because γδ TCR is expressed after the DN2/3 stage of the thymic development pathway, this finding strongly suggest that IL17+ CD44highCD25low cells may be the DN4 γδ TCR+ thymocytes that have upregulated CD44 expression and acquired IL17 expression. In deed, in vitro culture of FACS sorted DN4 γδ TCR+ cells with OP9-DL4 stromal cells confirmed this possibility. Using gene-deficient mice, we also found that TGFβ is the primary factor mediating IL17 differentiation of γδ T cells in the thymus. However, other cytokines involved in Th17 differentiation including IL23, IL6, and IL21 played little or no roles as IL17+ γδ T cells were unaffected in these strains of mice. Taken together, our results identify a novel developmental pathway during which IL17-competent γδ T cells arise in the thymus by a TGFβ1-dependent mechanism
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Buza-Vidas, Natalija, Henrik Ahlenius, Corrado M. Cilio, et al. "Critical and Complementary Role of FLT3 and Interleukin 7-Receptor Alpha Signaling in T Lymphocyte Development." Blood 104, no. 11 (2004): 112. http://dx.doi.org/10.1182/blood.v104.11.112.112.
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Abstract We recently demonstrated that signaling through the cytokine tyrosine kinase receptor flt3 and interleukin-7 receptor a (IL-7Ra) is indispensable for fetal and adult B cell commitment and development (Sitnicka et al., J. Exp. Med. 198: 1495, 2003). These receptors are also implicated to be important in regulation of T cell development, but their potential interdependence remains unexplored. We recently showed that flt3 ligand (FL)-deficient mice have reduced levels of early thymic progenitors as well as the common lymphoid progenitor (CLP) (Sitnicka et al., Immunity, 17:463, 2002). In the present study we investigated T cell development in mice deficient in FL and IL-7Ra expression. Strikingly, when compared to FL−/− and IL-7Ra−/− mice, FL−/−xIL-7Ra−/− (double deficient) mice (8-10 week old) lack visible lymph nodes and Peyer’s Patches. Thymic cellularity was dramatically reduced to only 0.3% of FL−/− and wild type (WT) controls and to only 4% of IL-7Ra−/− mice. In agreement with previous studies, IL-7Ra−/− thymocytes revealed a partial block at the progression from the DN2 (CD4−CD8−CD44+CD25+) to DN3 (CD4−CD8−CD44−CD25+) stage, while in FL−/−xIL-7Ra−/− mice DN1 (CD4−CD8−CD44+CD25−), DN2 and DN3 thymic progenitors were undetectable. Thus, severe reductions in early thymocyte development in FL−/−xIL-7Ra−/− mice support a similar role for cross talk between these two signaling pathways in T cell development as recently demonstrated for B cell genesis.
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Ahn, Hong-Il, Kong-Sik Shin, Hee-Jong Woo, et al. "Analysis of right border flanking sequence in transgenic chinese cabbage harboring integrated T-DNA." Journal of Plant Biotechnology 38, no. 1 (2011): 15–21. http://dx.doi.org/10.5010/jpb.2011.38.1.015.
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Yang, X., X. Li F Zhang, K. Liu, et al. "Integration and characterization of T-DNA insertion in upland cotton." Czech Journal of Genetics and Plant Breeding 49, No. 2 (2013): 51–57. http://dx.doi.org/10.17221/217/2012-cjgpb.
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Copy numbers were evaluated by real-time quantitative PCR, and 149 junctions of T-DNA were isolated by thermal asymmetric interlaced PCR from 92 independent transgenic cotton lines transformed by Agrobacterium tumefaciens strain LBA4404. Real-time quantitative PCR results showed that 46% had integration of one or two T-DNA copies, 54% had three or more copies. Among 63 amplified products at LB junctions, 51% showed co-transformation of the vector backbone, 30% retained a portion of LB ranging from 3 to 23 bp, and 19% showed deletions ranging from 1 to 148 bp from the LB inner end. In contrast, all of the cleavage sites were located in the inner region of RB. The distribution of T-DNA insertions in upland cotton genome included coding sequences, transposons, plastid-derived sequences and microsatellites.
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Isoda, Takeshi, Masatoshi Takagi, Jinhua Piao та ін. "T-Cell Development Failure At β-Selection Checkpoint and TCRα/δ Locus Break Formation Associated with Chromosome 14 Translocation in Ataxia-Telangiectagia Mutated Deficient Mice". Blood 118, № 21 (2011): 184. http://dx.doi.org/10.1182/blood.v118.21.184.184.
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Abstract Abstract 184FN2 Ataxia Telangiectagia (AT) is an autosomal recessive immunodeficiency, caused by mutation of ataxia telangiectagia mutated gene (ATM). ATM plays a crucial role for responding to DNA damages by extrinsic and intrinsic factors, and is a master regulator for maintaining DNA integrity. VDJ recombination and class switch recombination during lymphocyte maturation are the steps of intrinsic DNA damage response where ATM stabilizes DNA ends during recombination. ATM deficiency (ATM−/−) is known to predispose to T-cell lymphopenia and T-lineage lymphoma development. ATM−/− mouse has been shown to have a failure of T-cell development at the stage from double positive (DP) to single positive (SP) differentiation, which is due to a failure of T-cell receptor a (TCRa) recombination. Thymic lymphomas in ATM−/− mice have recently been shown to have a chromosome 14 translocation involving TCRd locus, suggesting that the first event for translocation arises during TCRd locus recombination at double negative (DN) stage. However, phenotypic features of T-cell development at DN phase and the timing of chromosome 14 translocation formation in ATM−/− are not fully elucidated. Here we demonstrate that T cells of ATM−/− mice show a failure at the transition from DN3a to DN3b at b and gd-selection checkpoints due to multiple TCR recombination failure in-vivo. Consistent with in-vivo developmental profiles of ATM−/− mice thymocytes, long term hematopoietic stem cells (LTR-HSCs) of ATM−/− mice cultured with OP9-DLL1 show a delay at b-selection checkpoint in chronological order. In this culture system, failures in gd-T-cell development are also observed in ATM−/− LTR-HSCs. Involvement of thymic stromas in the failure of this transition was ruled out by bone-marrow transplantation (BMT) of ATM−/− donor to WT recipient mice, where thymocytes reconstitution showed the same transition failure at b-selection checkpoint. Thymocytes in RAG2−/− mice are arrested at DN3 stage by a failure of cleavage of TCR genes, but the arrested thymocytes are known to progress to DP phase by anti-CD3e antibody stimulation. This experiment enables to analyze pre-TCR dependent differentiation signal machinery. Then anti-CD3e antibody was injected into RAG2−/−ATM−/− mouse and DN3 cells were shown to be led to DP phase, indicating that ATM itself is not involved in the differentiation program during DN to DP phase. These results suggested loss of ATM attenuates T cell differentiation at DN3a to DN3b transition due to inefficient TCRg, d and b locus recombination. Thus differentiation failure from DN3a to DN3b in ATM deficiency is presumably the primary cause of T cell lymphopenia at the stage prior to positive-selection. We next investigated when of the differentiation stages chromosome 14 translocation involving TCRa/d locus monitored. When the LTR-HSCs is cultured on the OP9-DLL1 cells with high-dose cytokine including 10 ng/ml of Flt3-L, IL-7 and SCF, differentiation of LTR-HSCs to T cells halt at DN2-3a phase before b-selection. Then, by reducing the Flt3-L and IL7 to 5 ng/ml and 1 ng/ml, respectively, the differentiation arrest is released and Tcell differentiation progresses from DN3a to DN3b. No detectable chromosome break at TCRad locus was observed at DN2-3a in wild type, while 5% of ATM−/− cells carried TCRad break, associated with chromosome 14 translocation in approximately 0.8 % of DN2-3a cells. After progression to DN3b-4 phase, TCRad locus break was still observed in AT cells at the frequency of 1%, and chromosome 14 translocations involving TCRad locus was observed in 12% of ATM−/− cells, which was in contrast to none in wild type cell. Mono- or bi-allelic TCRa/d breaks, chromosome 14 dicentric, and t (12:14) were also observed in minor population of ATM−/− cells. These results suggest that critical point for generation of chromosome 14 translocations involving TCRa/d locus lies at DN2-3a to 3b stages corresponding during b and gd selection checkpoint in ATM deficient thymocytes. Our findings revealed that developmental failure of T-cells in AT arises during b and gd–selection checkpoint, which leads to the breaks of TCRa/d locus and subsequent chromosome 14 translocation formation. Thus we propose T-lymphopenia and predisposition to T cell leukemia/lymphoma are tightly connected in ATM deficient condition. Disclosures: No relevant conflicts of interest to declare.
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Sitnicka, Ewa, Natalija Buza-Vidas, Henrik Ahlenius, et al. "Critical Role of flt3 in Fetal and Adult T Lymphopoiesis." Blood 106, no. 11 (2005): 339. http://dx.doi.org/10.1182/blood.v106.11.339.339.
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Abstract We recently demonstrated that signalling through the cytokine tyrosine kinase receptor flt3 and interleukin-7 receptor α (IL-7Rα ) is indispensable for fetal and adult B cell commitment and development (Sitnicka et al., J. Exp. Med. 198: 1495, 2003). We have also shown that flt3 ligand (FL)-deficient mice have reduced levels of the common lymphoid progenitor (CLP) but normal levels of mature T cells (Sitnicka et al., Immunity, 17:463, 2002). However, previous studies failed to demonstrate a role of flt3 and its ligand in T lymphopoiesis. Herein, although having normal numbers of thymocytes, FL-deficient mice were shown to have distinct reductions in the earliest double negative (DN) progenitors in the fetal, postnatal and adult thymus. A critical role of flt3 in thymocyte development was most evident in the absence of Interleukin-7 receptor a (IL-7Rα ) signalling. Strikingly, in adult FL−/−IL-7Rα −/− (double deficient) mice thymic cellularity was reduced 400 fold as compared to FL−/− and wild type (WT) controls and 30 fold as compared to IL-7Rα −/− mice. In agreement with previous studies, IL-7Rα −/− thymocytes revealed a partial block at the progression from the DN2 (CD4−CD8−CD44+CD25+) to DN3 (CD4−CD8−CD44−CD25+) stage, while in FL−/−IL-7Rα −/− mice DN1 (CD4−CD8−CD44+CD25−), DN2 and DN3 thymic progenitors were undetectable. The DP CD4+CD8+ thymocytes were reduced more than 1000 fold as compared to IL-7Rα −/− mice and low levels of peripheral T cells remaining in FL−/−IL-7Rα −/− mice had exclusively an activated/memory phenotype. Furthermore, FL−/−IL-7Rα −/− bone marrow cells lacked T cell reconstituting potential after transplantation into lethally irradiated recipients. Taken together these data demonstrate indispensable and complementary roles of flt3 and IL-7Rα in T lymphopoiesis.
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Wang, Hong-Cheng, and Xiao-Hong Sun. "Id1 attenuates Notch signaling and impairs T cell commitment by elevating Deltex1 expression (85.3)." Journal of Immunology 182, no. 1_Supplement (2009): 85.3. http://dx.doi.org/10.4049/jimmunol.182.supp.85.3.
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Abstract Complete inhibition of E protein transcription factors by Id1 blocks the developmental transition of CD4/CD8 double negative 1 (DN1, CD44+CD25-) thymocytes to the DN2 (CD44+CD25+) stage. To understand the underlying mechanisms, we observed that mRNA levels of Deltex1, as well as Deltex4, were elevated in Id1-expressing thymocytes, which could result in developmental arrest by attenuating Notch function. In support of this hypothesis, we found that Deltex1 ablation enabled Id1-expressing progenitors to differentiate to the DN3 (CD44-CD25+) stage, which was accompanied by enhanced Notch1 expression in T cell progenitors. Consistently, constitutive activation of Notch1 drove the differentiation of Id1-expressing progenitors to the DN3 stage. Furthermore, we showed that Gfi1b levels decreased whereas GATA3 levels increased in Id1 transgenic thymocytes, and GATA3 expression up-regulated Deltex1 transcription. Thus, T cell commitment may be controlled by the interplay among E proteins, Gfi1b and GATA3 transcription regulators, which influence Notch function through Deltex1 expression. This work was supported by grant from the Nation Institute of Health to X.-H. S. (CA77553).
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Oda, Shuji, Hiroshi Yamaguchi, Akira Ono, Chojiro Kojima, Yoshinori Kondo, and Yoshiyuki Tanaka. "2P174 Spectroscopic analysis of DNA duplex including T-Hg^-T base pairs(36. DNA to chromatin,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)." Seibutsu Butsuri 46, supplement2 (2006): S339. http://dx.doi.org/10.2142/biophys.46.s339_2.
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Hollenbeck, Anita, Stefanie Weber, Kathrin Händschke, et al. "Activation of the Hypoxia-Inducible Factor Pathway Expands Early Thymic Progenitors." Blood 124, no. 21 (2014): 2896. http://dx.doi.org/10.1182/blood.v124.21.2896.2896.
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Abstract Early thymic progenitors enter the thymus and are exposed to regional hypoxia while they develop in a step-wise manner to mature functional T-cells. Therefore, hypoxia might represent an important component of the highly specialized thymic microenvironment. On the molecular level the hypoxia-inducible factor pathway controls the cellular response to hypoxia. In this pathway, the von-Hippel-Lindau protein (pVHL) continuously mediates the destruction of the transcription factor hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. Under hypoxia HIF-1α degradation is inhibited leading to the activation of HIF-1α target genes. Others used lck-Cre transgene-mediated conditional in vivo deletion of the Vhl gene to study the role of the oxygen-sensing pathway in developing thymocytes and found normal numbers of early double-negative (DN; CD4-CD8-) thymocytes (Biju et al., Mol Cell Biol, 2004). However, lck-Cre deletion initiates at the DN3 (CD25+CD44-) stage leaving the Vhl locus of very early DN1 (CD25-CD44+), DN2 (CD25+CD44+) and DN3 thymocytes unaltered. Therefore, we here used the ubiquitous hematopoietic deleter strain vav-Cre to investigate the role of pVHL in very early thymocytes (vav-Cre;VhlloxP;loxP mice). Using a PCR-based strategy we confirmed complete deletion of the Vhl gene in this model. We observed unaltered DN1 and DN2 progenitor numbers, however in contrast to the published lck-cre-mediated system we consistently observed an up to twofold expansion of the DN3 cellular compartment. As the hypoxia-inducible factor pathway was shown to modulate NOTCH1 signaling we studied Notch1 expression on Vhl-deficient thymocytes. Strikingly, Notch1 expression was significantly increased on expanded Vhl null DN3 thymocytes. At the DN3 developmental stage selection of cells with an accurately re-arranged T-cell receptor β-locus occurs. Thus, we analyzed pre- and post-β-selection DN3 cells by CD28 staining. Interestingly, we found both pre- and post-β-selection DN3 subpopulations expanded. In order to investigate whether the progenitor expansion is mediated by the lack of HIF-1α inhibition in the Vhl-deficient context we studied DN3 thymocytes in a conditional hematopoietic HIF-1α gain-of-function model (vav-Cre;HIF1dPA). Overexpression of HIF-1α, which is insensitive to pVHL-mediated degradation in vav-Cre;HIF1dPAmice, also resulted in expanded DN3 thymocytes. In summary, we describe novel conditional models to genetically alter the hypoxia-inducible factor pathway within very early thymic progenitors. Genetic Vhl loss led to an expansion of DN3 thymocytes. This DN3 expansion is most likely due to the absence of HIF-1α-inhibition, because HIF-1α overexpression phenocopied the Vhl-deficient DN3 thymocyte expansion. Disclosures Dührsen: Celgene: Honoraria, Research Funding.
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Jovanovic, Goran, Nikola Buric, and Milos Tijanic. "Stimulation of mucoperiostal slice epithelization by small power laser after the primary plastic of oroantral communication." Medical review 63, no. 3-4 (2010): 188–93. http://dx.doi.org/10.2298/mpns1004188j.
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Introduction The oroantral communication is a pathologic communication between the oral cavity and maxillary sinus. It originates with extraction of the upper lateral teeth. Primary plastics communication, which is more extensive than 5 mm has been unsuccessful in 16%. Small power lasers have positive reaction on wounds healing. The aim of this work was to determine the lasers effects on slice epithelization after the plastics more extensive than 5 mm. Material and methods The experimental research was done on dogs with extraction of the upper second premolars on both sides, and formed oroantral communication having diameter of 10 mm. After the plastics of sinus, the left side slices were exposed to radiation for 7 days, and the opposite slices healed spontaneously. Eight points (8x1J) were treated for 100s by GaAlAs laser, power 10 mW and wavelength 670 nm. Biopses of the slices connections were taken on the 14th day to be laboratory treated and examined microscopially. The study included 36 examinees with communication diameter of 10 mm and performed plastics of sinuses. In half of the examinees wounds were exposed to radiation for 7 days, and in other examinees they healed spontaneously. The results were analyzed on the fourteenth day according to the scale: 1 - complete healing, without dehiscention; 2 - incomplete healing, with minimal dehiscention; 3 - communication did not heal, with partial dehiscention; 4 - open communication, with significant dehiscention. Results and discussion The microscopic analysis shows that no wounds exposed to radiation were overcast with mucous membrane which had smooth sub epithelia chronic inflammation and inflammable infiltrate, and strong fibroplasias and granulations. Wounds exposed to radiation had mucous membrane without any signals of inflammation. Laser radiation causes anti-inflammatory reaction, i.e. it provokes reduction of exudation, alteration and proliferation, it blocks cyclo- and lipo-oxygenation by delaying the synthesis of prostaglandin, stimulates neutrophyll, macrophage and lissome activity and it activates the function of immune complex T and B lymphocytes, so this difference could be primary referred to the action of laser. Our clinical study shows that complete healing of oroantral communication was recorded in 88,8% of the examinees who were exposed to radiation in relation to 50%, of those who did not receive radiation therapy which is statistically much higher percentage (?2 test < 0, 05). The surgery was repeated in 5,6% of those who had received radiation therapy and in 16,7% of those who had not been exposed to radiation. Laser radiation stimulates changing of ADP in ATP and it accelerates cells metabolism, it increases microcirculation and accelerates substance exchange of cells, it increases DNK and RNK synthesis and stimulates cells division, which cause quicker regeneration of epithelia, i.e. it accelerates the process of wound healing. Conclusion It can be concluded that small power laser can be used successfully as additional method of treatment, after closing of oroantral communication surgically.
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REZAEIZADEH, Rezvan, El Hadı Omar SWESE Ettahır, and Aybaba HANÇERLİOĞULLARI. "Pv/T Systems For Energy Efficiency By Using Advanced Deep Neural Network (DNN) And Nanofluid In Solar Systems." Inspiring Technologies and Innovations 2, no. 2 (2023): 25–30. https://doi.org/10.5281/zenodo.10555897.
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Today, solar energy is a very popular alternative energy source due to its enormous availability in nature. In this study, focusing on the electro-mechanical production industry of advanced PV/T solar panels, studies carried out on the development of new methodological methods for the efficiency of existing asset management practices of the infrastructure of this industry and the optimal improvement. For this, it is to integrate a power-generating PV/T panel and a solar thermal heating panel within the same collection surface. PV/T systems are one of the subjects that scientific studies have focused on in recent years. The main reasons for this are to increase the electricity generation performance of PVs, as well as to obtain thermally hot fluid from the system. In this research, it was implemented using a new roof-mounted PV/T multi-reflection panel, which not only increases the power output of the PV/T panel, but most importantly, the aesthetic aspect is a major barrier to large-scale uptake of PV/TIn this study, we developed a new advanced MPPT (maximum power point tracking) algorithm such as Deep Neural Network (DNN) controller especially for photovoltaic system. The proposed DNN based MPPT algorithm is developed PV/T voltage, current and corresponding duty cycle.
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Choi, Chul Won, Yang Jo Chung, Christopher Slape, and Peter D. Aplan. "A NUP98-HOXD13 Fusion Gene Impairs Differentiation of T and B Lymphocytes." Blood 110, no. 11 (2007): 2650. http://dx.doi.org/10.1182/blood.v110.11.2650.2650.
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Abstract Chromosomal translocations leading to NUP98 gene fusions are associated with a wide range of hematologic malignancies, including acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), and myelodysplastic syndrome (MDS). The NUP98-HOXD13 (NHD13) gene fusion was first identified in a patient with a MDS that progressed to AML. Recently, we used an NHD13 fusion gene to develop a mouse model of MDS that recapitulates all of the key findings of the human disease, including ineffective hematopoiesis leading to peripheral blood cytopenias, dysplasia, and progression to AML. In addition to the features noted above, we observed that the NHD13 mice were lymphopenic, and 10–30% of these mice, depending on the strain background, progressed to T-ALL. These findings prompted us to define the lymphocyte development of NHD13 mice. CBCs obtained from clinically healthy NHD13 mice showed lymphopenia (2.21 vs. 8.72 K/μL, p&lt;0.01) compared with wild type (WT) littermate controls. This lymphopenia was due to a decrease in both T and B cells, as FACS analysis of peripheral blood (PB) from NHD13 mice revealed a marked decrease in CD4 single positive (SP) and B220+/IgM+ cells (p&lt;0.05) compared to WT controls; similar findings were observed in the spleen (p&lt;0.01). The percentage of CD8 SP cells was not different between the NHD13 and WT mice in either the PB or spleen. To investigate the cause for the B-cell lymphopenia, we determined the Hardy fractions of bone marrow B cells. Although the pro-B cell (B220+/CD43+, Hardy fractions A–C) populations showed no difference between NHD13 and WT BM, the NHD13 BM showed decreased pre-B and B cell (B220+/CD43−, Hardy fractions D–F) populations, suggesting impaired differentiation at the pro-B to pre-B stage. Thymi from NHD13 mice (n=7, median age=7 months) showed grossly decreased size and decreased total number of thymocytes (1.75×107 vs. 9.21×107, p&lt;0.01). The CD4/CD8 DN population was markedly increased (p&lt;0.001) and the CD4/CD8 DP population markedly decreased (p&lt;0.001) in NHD13 compared to WT mice. In addition, there was a variable increase in the DN1 and DN2 population, as well as a decrease in the DN3 and DN4 population in thymi from the NHD13 thymus, suggesting a partial block at the DN2 to DN3 transition. To determine clonality of the NHD13 thymocyte population, we used degenerate RT-PCR to identify clonal TCRβ gene rearrangements. As expected, the WT thymi showed polyclonal TCRβ gene rearrangements. However, 5 of 6 NHD13 thymus samples showed clonal DJ rearrangements, with over half of the TCRβ rearrangements in the thymus showing an identical D-J junction, but distinct V-D junctions. This finding suggested that there was a massive clonal expansion of DN2 cells that had undergone a DJ rearrangement, but not completed a VDJ rearrangement, further supporting the contention of a partial block at the DN2 to DN3 transition. Interestingly, analysis of TCRβ gene rearrangements in NHD13 spleens showed no evidence of cells with clonal DJ rearrangements, suggesting that the thymocytes with clonal DJ rearrangements did not mature and migrate from the thymus. Finally, we noted that although there is a marked increase in DN thymocytes from the NHD13 mice, the T-ALL that developed in these mice were typically DP or CD4 SP, suggesting that a rare cell that “escapes” the T-cell differentiation block is susceptible to leukemic transformation. Taken together, these findings demonstrate that the NHD13 transgene inhibits lymphoid as well as myeloid and erythroid differentiation, and is oncogenic in lymphoid as well as myeloid cells.
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Zhang, Shuangmin, Yi Zheng, Richard Lang, and Fukun Guo. "Gene Targeting of RhoA Reveals Its Essential Role In Lymphopoiesis." Blood 116, no. 21 (2010): 282. http://dx.doi.org/10.1182/blood.v116.21.282.282.
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Abstract Abstract 282 RhoA GTPase is an intracellular signal transducer capable of regulating a wide range of cell functions including cytoskeleton dynamics, proliferation, and survival. In lymphocytes, studies by using dominant negative mutant or C3 transferase expressing transgenic mice suggest that RhoA is involved in TCR and BCR signaling and related T cell functions such as polarization, migration, survival, and proliferation. To date, the physiological role of RhoA in lymphocyte development remains unclear. In this study, we have achieved T cell, B cell, and hematopoietic stem cell-specific deletion of RhoA by conditional gene targeting with CD2, CD19 and Mx1 promoter-driven Cre expression, respectively, in the RhoAloxP/loxP mice. First, we found that RhoA gene disruption in early T cells caused a drastic decrease in thymocyte cellularity, with the numbers of CD4−CD8− double negative (DN), CD4+CD8+ double positive (DP), CD4+CD8− single positive (SP), and CD4−CD8+ SP T cells decreased by 88.8% ± 6.0%, 99.4% ± 1.0%, 99.3% ± 1.2%, and 98.6% ± 2.0%, respectively. Among DN subpopulations, CD44+CD25− (DN1), CD44+CD25+ (DN2), CD44−CD25+ (DN3), and CD44−CD25− (DN4) cells were reduced by 91.7% ± 6.0%, 54.9% ± 27.7%, 50.9% ± 33.3%, and 96.7% ± 3.4%, respectively. Further, RhoA knockout led to a significant loss of DP thymocytes at the initial stage (CD69highTCRint) of positive selection, suggesting that RhoA is required for positive selection. The decreased thymocyte cellularity in mutant mice is associated with increased apoptosis of all thymic T lineages. RhoA deficiency also resulted in a perturbation in thymocyte cell cycle progression as manifested by increased BrdU incorporation in DN1 and DN2 cells and decreased BrdU incorporation in DN4 and DP cells. Concomitantly, RhoA-deficient thymocytes showed a 59.8% ± 26.3% reduction in proliferative potential in response to TCR crosslinking. Western blot analysis revealed that the activities of ZAP70, LAT, Akt, Erk, and p38 were impaired in RhoA-/- thymocytes. In periphery, spleens of the RhoA null mice contained 7.4% ± 8.0% of CD4+ T cells and 3.7% ± 2.7% of CD8+ T cells compared with that of wild type (WT) mice. Loss of peripheral mature T cells in mutant mice is reflected by a marked reduction of naive T cells, whereas effector and memory phenotype cells were marginally affected by RhoA deficiency. RhoA-deficient naïve T cells were more susceptible to apoptosis, suggesting that homeostatic defect of naïve T cells in RhoA-/- mice is attributed to impaired cell survival. Abrogation of RhoA caused an increased in vivo BrdU incorporation in naïve T cell compartments. Thus, RhoA deficiency induces naïve T cell homeostatic proliferation, possibly due to a compensatory effect of lymphopenia. In contrast to that in thymocytes, Erk was constitutively activated in RhoA-deficient splenic T cells. These observations implicate RhoA in the multiple stages of T cell development and the proper assembly of early TCR signaling complex. Second, deletion of RhoA in pre-proB cells had no effect on early B cell development in bone marrow but significantly inhibited late B cell development in spleen, resulting in 78.2% ± 13.6%, 78.6% ± 16.9%, and 93.2% ± 3.4% reduction in transitional, follicular, and marginal zone B cells, respectively. Plasma cells in spleen were decreased by 50.9 % ± 25.9% in RhoA null mice. However, we did not detect any changes in survival of in vivo RhoA-/- B cells or RhoA-/- B cells cultured in vitro with survival factor BAFF. Distinct from previously characterized Cdc42 knockout mice, BAFF-R expression was not altered in RhoA-/- B cells. Moreover, RhoA-/- B cells appeared to be normal in proliferation and Akt and Erk activation in response to BCR crosslinking. These data suggest that RhoA is important for late B cell development through regulation of differentiation but not cell survival or proliferation. Finally, deletion of RhoA from hematopoietic stem cells did not affect common lymphoid progenitor production, indicating that RhoA is not required for early lymphoid progenitor commitment. Taken together, these lineage-specific mouse genetic studies demonstrate that RhoA critically regulates T and B cell development by distinct cellular mechanisms at multiple stages of lymphopoiesis. Disclosures: No relevant conflicts of interest to declare.
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Martin, Francis, Thomas Nicholson, Joseph Loureiro, Taiping Chen, and Qian Huang. "KMT1E is a regulator of T cell development (P4542)." Journal of Immunology 190, no. 1_Supplement (2013): 200.2. http://dx.doi.org/10.4049/jimmunol.190.supp.200.2.
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Abstract The histone methyltransferase KMT1E has a demonstrated role in pluripotency, embryonic stem cell development, and melanoma formation, yet its role in T cell development has not been investigated. As T-cell development involves many stages of lineage commitment, we hypothesized that KMT1E may have a role in regulating these commitment stages. KMT1E is dynamically regulated during the stages of T-cell development, peaking at the DN2 through DN4 stages and dramatically waning as lineage commitment occurs. In order to further query the role of KMT1E in thymocyte development, we created conditional knockout animals in which KMT1E is deleted specifically in the T cell compartment (where the Cre recombinase is driven by the Lck promoter). Deletion of KMT1E caused a profound reduction in the numbers of DN1 (Lin-/CD44+/CD25-) cells in the thymus and single positive CD4 and CD8 cells in the thymus and spleen. In order to elucidate the genetic changes induced by KMT1E that may influence the observed phenotype, we performed microarray experiments on thymocyte precursors from the wild type and conditional knockout animals. Deletion of KMT1E had a modest effect on global gene expression, with only a small subset of the genes being upregulated. Currently we are examining the role of these genes mediating KMT1E’s function in early T cell development.
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Schmitt, Thomas M., Maria Ciofani, Howard T. Petrie, and Juan Carlos Zúñiga-Pflücker. "Maintenance of T Cell Specification and Differentiation Requires Recurrent Notch Receptor–Ligand Interactions." Journal of Experimental Medicine 200, no. 4 (2004): 469–79. http://dx.doi.org/10.1084/jem.20040394.
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Notch signaling has been shown to play a pivotal role in inducing T lineage commitment. However, T cell progenitors are known to retain other lineage potential long after the first point at which Notch signaling is required. Thus, additional requirements for Notch signals and the timing of these events relative to intrathymic differentiation remain unknown. Here, we address this issue by culturing subsets of CD4 CD8 double negative (DN) thymocytes on control stromal cells or stromal cells expressing Delta-like 1 (Dll1). All DN subsets were found to require Notch signals to differentiate into CD4+ CD8+ T cells. Using clonal analyses, we show that CD44+ CD25+ (DN2) cells, which appeared committed to the T cell lineage when cultured on Dll1-expressing stromal cells, nonetheless gave rise to natural killer cells with a progenitor frequency similar to that of CD44+ CD25− (DN1) thymocytes when Notch signaling was absent. These data, together with the observation that Dll1 is expressed on stromal cells throughout the thymic cortex, indicates that Notch receptor–ligand interactions are necessary for induction and maintenance of T cell lineage specification at both the DN1 and DN2 stages of T cell development, suggesting that the Notch-induced repression of the B cell fate is temporally separate from Notch-induced commitment to the T lineage.
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Martineau, Belinda, Toni A. Voelker, and Rick A. Sanders. "On Defining T-DNA." Plant Cell 6, no. 8 (1994): 1032. http://dx.doi.org/10.2307/3869882.
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Tinland, Bruno. "Reassessing T-DNA insertions." Trends in Plant Science 3, no. 4 (1998): 125. http://dx.doi.org/10.1016/s1360-1385(98)01217-5.
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De Buck, Sylvie, Anni Jacobs, Marc Van Montagu, and Ann Depicker. "The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration." Plant Journal 20, no. 3 (1999): 295–304. http://dx.doi.org/10.1046/j.1365-313x.1999.t01-1-00602.x.
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Behar, S. M., S. A. Porcelli, E. M. Beckman, and M. B. Brenner. "A pathway of costimulation that prevents anergy in CD28- T cells: B7-independent costimulation of CD1-restricted T cells." Journal of Experimental Medicine 182, no. 6 (1995): 2007–18. http://dx.doi.org/10.1084/jem.182.6.2007.
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A class of molecules that is expressed on antigen presenting cells, exemplified by CD80 (B7), has been found to provide a necessary costimulatory signal for T cell activation and proliferation. CD28 and CTLA4 are the B7 counterreceptors and are expressed on the majority of human CD4+ T cells and many CD8+ T cells. The signal these molecules mediate is distinguished from other costimulatory signals by the finding that T cell recognition of antigen results in a prolonged state of T cell unresponsiveness or anergy, unless these costimulatory molecules are engaged. However, nearly half of the CD8+ and CD4-CD8- T cells lack CD28, and the costimulatory signals required for the activation of such cells are unknown. To understand the pathways of activation used by CD28- T cells, we have examined the costimulatory requirements of antigen-specific CD4-CD8- TCR(+)-alpha/beta circulating T cells that lack the expression of CD28. We have characterized two T cell lines, DN1 and DN6, that recognize a mycobacterial antigen, and are restricted not by major histocompatibility complex class I or II, but by CD1b or CD1c, two members of a family of major histocompatibility complex-related molecules that have been recently implicated in a distinct pathway for antigen presentation. Comparison of antigen-specific cytolytic responses of the DN1 and DN6 T cell lines against antigen-pulsed CD1+ monocytes or CD1+ B lymphoblastoid cell lines (B-LCL) demonstrated that these T cells recognized antigen presented by both types of cells. However, T cell proliferation occurred only when antigen was presented by CD1+ monocytes, indicating that the CD1+ monocytes expressed a costimulatory molecule that the B-LCL transfectants lacked. This hypothesis was confirmed by demonstrating that the T cells became anergic when incubated with the CD1(+)-transfected B-LCL in the presence of antigen, but not in the absence of antigen. The required costimulatory signal occurred by a CD28-independent mechanism since both the CD1+ monocytes and CD1+ B-LCL transfectants expressed B7-1 and B7-2, and DN1 and DN6 lacked surface expression of CD28. We propose that these data define a previously unrecognized pathway of costimulation for T cells distinct from that involving CD28 and its counterreceptors. We suggest that this B7-independent pathway plays a crucial role in the activation and maintenance of tolerance of at least a subset of CD28- T cells.