scholarly journals T-bet, a Th1 transcription factor, is up-regulated in T cells from patients with aplastic anemia

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3983-3991 ◽  
Author(s):  
Elena E. Solomou ◽  
Keyvan Keyvanfar ◽  
Neal S. Young
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Aplastic Anemia ◽  
Kinase Inhibitors ◽  
Bone Marrow Failure ◽  
Hematopoietic Cells ◽  
T Box ◽  
Proximal Site ◽  
Active Transcription ◽  
Ifn Γ

In aplastic anemia, immune destruction of hematopoietic cells results in bone marrow failure. Type 1 cytokines, especially IFN-γ, have been implicated in the pathophysiology of T-cell–mediated, Fas-mediated stem cell apoptosis of hematopoietic cells. Here, we show that the transcription factor T-bet (T-box expressed in T cells) is increased in T cells from patients with aplastic anemia. Patients' T-bet bound directly to the proximal site of the IFN-γ promoter without any prior stimulation, in contrast to healthy controls. Increased levels of Itk kinase participated in T-bet up-regulation and active transcription of the IFN-γ gene observed in these patients. Blocking PKC-θ, a kinase that lies downstream of Itk kinase, decreased T-bet protein and IFN-γ intracellular levels. These data suggest that the increased IFN-γ levels observed in aplastic anemia patients are the result of active transcription of the IFN-γ gene by T-bet. Blocking the transcription of the IFN-γ gene with kinase inhibitors might lead to the development of novel therapeutic agents for patients with aplastic anemia and other autoimmune diseases.

T-bet Over-Expression in T Cells from Patients with Aplastic Anemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 832-832
Author(s):  
Elena E. Solomou ◽  
Keyvan Keyvanfar ◽  
Elaine M. Sloand ◽  
Barbara Weinstein ◽  
Olga Nunez ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Hematopoietic Stem ◽  
T Box ◽  
Protein Levels ◽  
Proximal Site ◽  
Ifn Γ ◽  
Bet Protein ◽  
Normal Controls

Abstract Acquired aplastic anemia (AA) is a bone marrow failure syndrome characterized by immune-mediated destruction of hematopoietic stem cells. T cells from patients with AA overproduce IFN-γ, a cytokine that inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. In T cells, regulation of IFN-γ production occurs primarily at the level of transcription. A proximal site of the IFN-γ gene (−75 to −45bp of the IFN-γ promoter) is a binding site for different transcription factors including NFAT, AP-1, ATF, CREB, and T-bet. T-bet is a member of the T-box family of transcription factors, this family contains a highly conserved DNA binding domain, the T-box, that binds to a specific sequence in the promoter of different genes, including the IFN-γ promoter. T-bet is found in Th1 but not in Th2 cells and is the key regulator of Th1 development and function (Rengarajan et al., Immunol Today2000; 21: 479). The inducible expression of T-bet is mediated in part by Itk kinase (Sjabo et al., Science2005; 307: 430). In the present study, we examined T-bet protein levels in T cells from patients with AA. Samples from 17 of 20 patients examined (85%) by immunoblot showed increased T-bet protein levels in unstimulated T cells compared to normal controls (p = 0.0001). Normal controls showed undetectable T-bet protein levels in unstimulated T cells but T-bet expression was induced after 24 hrs of stimulation with PMA and ionomycin. In electrophoretical mobility shift assays, we observed increased T-bet binding to the proximal site of the IFN-γ promoter in T cells from patients with AA; no binding was detected in unstimulated T cells from healthy controls, but binding was present after stimulation for at least 24 hrs. T-bet protein levels correlated with disease activity. Patients with increased T-bet protein levels showed increased intracellular IFN-g levels compared to controls, as detected by flow cytometry (p<0.05). Patients that expressed increased T-bet protein levels also showed increased levels of the Itk kinase (p=0.02). We examined if other kinases that lie downstream of Itk in the signal transduction activation cascade in T cells affected the inducible expression of T-bet. In normal T cells, rottlerin, a PKC-theta (PKC-𝛉) inhibitor, decreased T-bet protein levels by 50%; in AA T cells, rottlerin also decreased T-bet protein levels and IFN-γ intracellular levels by 50%. Our results suggest that the increased IFN-γ levels observed in AA are the result of activation of transcription of the IFN-γ gene by the regulator T-bet. Blocking of transcription of the IFN-γ gene by kinase inhibitors might represent a therapuetic strategy for AA and other human T cell-mediated autoimmune diseases.

SAP (SH2D1A), the Immunomodulator Deficient in X-Linked Lymphoproliferative Syndrome (XLP), Is Profoundly Decreased in Aplastic Anemia: An Immunologic Link between Constitutional and Acquired Bone Marrow Failure.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1141-1141
Author(s):  
Elena E. Solomou ◽  
Valeria Visconte ◽  
Federica Gibellini ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bone Marrow Failure ◽  
Protein Levels ◽  
Ifn Γ ◽  
Th1 Polarization

Abstract Ligation of the signaling lymphocyte activation molecule (SLAM), a member of the immunoglobulin superfamily expressed in T and B cells, results in T cell activation and Th1 cytokine production. SAP is a small cytoplasmic protein expressed in T- and NK cells that controls the activation signals mediated by SLAM. On T cell activation, SAP binds to Fyn kinase; Fyn is activated and phosphorylates tyrosine residues on SLAM; phosphorylation results in the formation of a complex that selectively down-regulates co-stimulatory signals in activated T cells, resulting in inhibition of IFN-γ production. Thus SAP acts as a natural suppressor of SLAM-mediated T cell activation, and, in the absence of SAP, T cells are constitutively activated and overproduce IFN-γ. Mutations in the SAP gene lead to abnormal T cell activation and enhanced Th1 cytokine production in mouse models and in humans: about half of patients with X-linked lympoproliferative disease (XLP) have functionally disabling SAP mutations. Acquired aplastic anemia (AA) is a bone marrow failure syndrome in which hematopoietic cell destruction is effected by cytotoxic T cells and type 1 cytokines. We have recently shown that T cells from patients with AA have increased protein levels of T-bet, resulting in IFN-γ overproduction (Solomou EE et al, Blood2006; 107:3983). IFN-γ inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. We examined SAP expression as an explanation for aberrant T cell activation and extreme Th1 polarization. SAP protein expression on immunoblot was very low to absent in unstimulated T cells from 16 of 20 AA patients examined, as compared to normal levels of expression in equivalent numbers of healthy donors (p<0.001). No significant differences were detected in Fyn and SLAM protein levels between AA and controls. SAP mRNA levels were also significantly decreased in T cells from those AA patients with low SAP protein levels, as determined by RT-PCR. Peripheral blood DNA samples from 18 patients with AA were analyzed for SAP mutations: three novel intronic mutations, not present in controls, were identified among 7 unrelated patients: one mutation was in the promoter region of SAP (position 106, C to T; 3 patients), and two mutations in the intron-exon junction between exons 1 and 2 (position 38975, C toT; 3 patients) and 3 and 4 (position 62771, C to A; 1 patient). IFN-γ, as measured by ELISA, in three patients with undetectable SAP protein levels was significantly increased compared to healthy controls (n=5, p<0.001). Increased IFN-γ levels and Th1 polarization in AA can in part be explained by functional SAP deficiency. SAP-deficient T cells in AA would be unable to block co-stimulatory signals, leading to an activated T cell phenotype and ultimately hematopoietic cell destruction and bone marrow failure. The SAP-deficient phenotype in T cells from patients with aplastic anemia may be secondary to subtle genetic alteration in the gene’s regulation (abnormal promoter binding sites or epigenetic modulation due to mutations in introns) or as yet unidentified aberrant upstream pathways (Ets-1 and Ets-2, the transcription factors that regulate SAP expression).

Experiment Therapy of Aplastic Anemia Using Cyclosporin A Combined with IDO Agonist.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1695-1695
Author(s):  
Lifang Huang ◽  
Min Dai ◽  
Wenli Liu ◽  
Jianfeng Zhou ◽  
Hanying Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cyclosporin A ◽  
Aplastic Anemia ◽  
Immune Tolerance ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Combination Group ◽  
Ifn Γ

Abstract Aplastic anemia(AA) is an unusual hematologic disease and the paradigm of the human bone marrow failure syndromes. Abnormal immunological responses have been found in most aplastic patients. Activated type 1 cytotoxic T cells may be the main contributing factor of pathogenesis. IDO is a rate-limiting enzyme in Tryptophan(Trp) metabolism. The proliferation of Th1 cells is specifically inhibited by the over-expression of IDO, which can degrade the Trp in local environment, hamper the conductioin of the activating signal in T cell and induce immune tolerance. 3,4-DAA is an IDO agonist and can activate IDO. In our experiment, Balb/c mice were irradiated (5.0Gy of 60Co), and then infused with 5×106 lymph node(LN) cells from DBA/2 mice in 4 hours. Blood count was monitored and marrow damage was assessed by histogical study. Concentrations of serum IFN-γ were measured by ELISA. The levels of Tryptophan and kynurenine were evaluated by high performance liquid chromatography (HPLC). CD4+CD25+ T cells in spleen were analyzed by flow cytometry. The level of Foxp3 in CD4+CD25+ T cell was measured by RT-PCR. Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and bone marrow hypoplasia. Bone marrow of affected mice showed lymphocyte infiltration. Serum IFN-γ concentration increased 3.7 fold at d6 post infusion. The recipient mice were divided into 4 different treating groups as follow: 0.9% Sodium Chloride as control; Cyclosporin A (CsA) (50ug/g/d ×5d, peritoneal injection); 3,4-DAA(5mg/d, orally daily); the combination of CsA and 3,4-DAA. The effects of CsA, IDO agonist and CsA combined IDO agonist were analyzed at day 6,10,14,21,24 and 28 after LN cells infusion. The white cell and the platelet recovered to near normal, respectively (4.2±0.32)×109, (937±190.47)×1012 at d21 in the combination group. Early stage treatment with CsA can improve periphery blood cells and BM nucleated cells, but long term effect was not remarkable. In contrast, the 3,4-DAA group exhibited slow and gradually enhanced role. Periphery blood cells and BM nucleated cells were improved remarkably in the combination group. The number and function of CD4+CD25+T cells also increased remarkably. In the treatment of AA, abnormal immune response in bone marrow was inhibited by CsA, meanwhile immune tolerance was induced through up-regulating the regulatory T cells(Treg) by 3,4-DAA. In this way, the balance of immune in bone marrow could be reestablished quickly. CsA combined IDO agonist could provide a new strategy for the management of AA.

scholarly journals Transcriptional Control of Impaired Th1 Responses in Patent Lymphatic Filariasis by T-Box Expressed in T Cells and Suppressor of Cytokine Signaling Genes

2005 ◽  
Vol 73 (6) ◽  
pp. 3394-3401 ◽  
Author(s):  
Subash Babu ◽  
V. Kumaraswami ◽  
Thomas B. Nutman
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Mrna Expression ◽  
Lymphatic Filariasis ◽  
Th2 Cells ◽  
Cytokine Signaling ◽  
Suppressor Of Cytokine Signaling ◽  
T Box ◽  
Ifn Γ

ABSTRACT T-bet (T-box expressed in T cells) and GATA-3 are transcription factors that play a critical role in the development of Th1 and Th2 cells, as do genes of the SOCS (suppressor of cytokine signaling) family, albeit indirectly. Another transcription factor, Foxp3, is a master regulator of natural regulatory T cells (Tregs). To identify the role of these factors in impaired Th1 responses of patent filarial infection, analysis of cytokine, SOCS, and transcription factor mRNA expression was performed on purified T cells of filaria-infected individuals (n = 6) and uninfected controls (n = 6). As expected (and in contrast to cells of uninfected individuals), there was a significant depression of gamma interferon (IFN-γ) and a concomitant increase in interleukin-4 (IL-4), IL-5, and IL-10 mRNA expression following stimulation with parasite antigen (BmA) but not with a polyclonal T-cell (anti-CD3) stimulus. T-bet (but not GATA-3) was expressed at significantly lower levels in cells of filaria-infected individuals in response to BmA compared with those from the uninfected group, accounting, at least partially, for the diminished IFN-γ expression. Second, we found no significant differences in expression of Foxp3 between the two groups, although induction of Foxp3 expression correlated with induced expression levels of IL-10, implicating Tregs in the IL-10 expression seen. Finally, parasite-specific T-cell expression of SOCS-1, SOCS-5, and SOCS-7 was significantly diminished among infected patients; in contrast, expression of SOCS-3 increased. Our data therefore indicate that the impaired Th1 responses observed in patent lymphatic filariasis are associated with decreased expression of T-bet, SOCS-1, SOCS-5, and SOCS-7 and increased expression of SOCS-3 in T cells.

scholarly journals Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia

Blood ◽  
2018 ◽  
Vol 132 (26) ◽  
pp. 2730-2743 ◽  
Author(s):  
Wanling Sun ◽  
Zhijie Wu ◽  
Zenghua Lin ◽  
Maile Hollinger ◽  
Jichun Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Aplastic Anemia ◽  
Bone Marrow Failure ◽  
Murine Bone Marrow ◽  
Immune Mediated ◽  
Tnf Α ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α−/− donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR−/− recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α−/− recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α−/− recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR−/− donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α–producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.

scholarly journals The role of the Th1 transcription factor T-bet in a mouse model of immune-mediated bone-marrow failure

Blood ◽  
2010 ◽  
Vol 115 (3) ◽  
pp. 541-548 ◽  
Author(s):  
Yong Tang ◽  
Marie J. Desierto ◽  
Jichun Chen ◽  
Neal S. Young
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Transcription Factor ◽  
Transforming Growth Factor ◽  
Bone Marrow Failure ◽  
Critical Role ◽  
Interferon Γ ◽  
Interleukin 17 ◽  
Immune Mediated

Abstract The transcription factor T-bet is a key regulator of type 1 immune responses. We examined the role of T-bet in an animal model of immune-mediated bone marrow (BM) failure using mice carrying a germline T-bet gene deletion (T-bet−/−). In comparison with normal C57BL6 (B6) control mice, T-bet−/− mice had normal cellular composition in lymphohematopoietic tissues, but T-bet−/− lymphocytes were functionally defective. Infusion of 5 × 106 T-bet−/− lymph node (LN) cells into sublethally irradiated, major histocompatibility complex–mismatched CByB6F1 (F1) recipients failed to induce the severe marrow hypoplasia and fatal pancytopenia that is produced by injection of similar numbers of B6 LN cells. Increasing T-bet−/− LN-cell dose to 10 to 23 × 106 per recipient led to only mild hematopoietic deficiency. Recipients of T-bet−/− LN cells had no expansion in T cells or interferon-γ–producing T cells but showed a significant increase in Lin−Sca1+CD117+CD34− BM cells. Plasma transforming growth factor-β and interleukin-17 concentrations were increased in T-bet−/− LN-cell recipients, possibly a compensatory up-regulation of the Th17 immune response. Continuous infusion of interferon-γ resulted in hematopoietic suppression but did not cause T-bet−/− LN-cell expansion or BM destruction. Our data provided fresh evidence demonstrating a critical role of T-bet in immune-mediated BM failure.

scholarly journals STAT3 mutations indicate the presence of subclinical T-cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients

Blood ◽  
2013 ◽  
Vol 122 (14) ◽  
pp. 2453-2459 ◽  
Author(s):  
Andres Jerez ◽  
Michael J. Clemente ◽  
Hideki Makishima ◽  
Hanna Rajala ◽  
Ines Gómez-Seguí ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Somatic Mutation ◽  
Bone Marrow Failure ◽  
Cell Clones ◽  
Key Points ◽  
Stat3 Mutations

Key PointsSTAT3+ T cells are found not only in detected concomitant LGL-BMFs, but in cases in which an LGL expansion was not suspected. Transformation via acquisition of a somatic mutation in T cells may be a mechanism of immune, mainly hypoplastic, bone marrow failure.

scholarly journals MIP1 alpha nuclear protein (MNP), a novel transcription factor expressed in hematopoietic cells that is crucial for transcription of the human MIP-1 alpha gene.

1995 ◽  
Vol 15 (6) ◽  
pp. 3110-3118 ◽  
Author(s):  
L M Ritter ◽  
M Bryans ◽  
O Abdo ◽  
V Sharma ◽  
N M Wilkie
Keyword(s):  
T Cells ◽  
Transcription Factor ◽  
Binding Site ◽  
Nuclear Protein ◽  
Inflammatory Responses ◽  
Hematopoietic Cells ◽  
Jurkat Cells ◽  
Negative Regulator ◽  
U937 Cells ◽  
Hematopoietic Stem

Murine macrophage inflammatory protein 1 alpha (MIP-1 alpha) and its human equivalent (GOS19, LD78, or AT464) are members of the -C-C family of low-molecular-weight chemokines. Secreted from activated T cells and macrophages, bone marrow-derived MIP-1 alpha/GOS19 inhibits primitive hematopoietic stem cells and appears to be involved in the homeostatic control of stem cell proliferation. It also induces chemotaxis and inflammatory responses in mature cell types. Therefore, it is important to understand the mechanisms which control the expression of MIP-1 alpha/GOS19. Previous work has shown that in Jurkat T cells, a set of widely expressed transcription factors (the ICK-1 family) affect the GOS19 promoter. One member, ICK-1A, behaves as a strong negative regulator. In this communication, we provide evidence that the pathway of induction in the macrophage cell line U937 is different from that in Jurkat cells. Furthermore, we show that the ICK-1 binding site does not confer negative regulation in U937 cells. We provide evidence for an additional binding site, the MIP-1 alpha nuclear protein (MNP) site, which overlaps the ICK-1 site. Interaction of nuclear extracts from various cell lines and tissue with the MNP site leads to the formation of fast-migrating protein-DNA complexes with similar but distinct electrophoretic mobilities. A mutation of the MNP site which does not abrogate ICK-1 binding inactivates the GOS19.1 promoter in U937 cells and reduces its activity by fourfold in Jurkat cells. We propose that the MNP protein(s) binding at the MNP site constitutes a novel transcription factor(s) expressed in hematopoietic cells.

Effects of EGF-R on Transcription Factor Activation in Hematopoietic Cells: EGF-R Activates Multiple Transcription Factors Which Induce Proliferation in the Absence of Autocrine Cytokines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4185-4185
Author(s):  
John G. Shelton ◽  
Linda S. Steelman ◽  
Martin McMahon ◽  
James A. McCubrey
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Kinase Inhibitors ◽  
Hematopoietic Cells ◽  
Growth Factor Receptor ◽  
Biological Properties ◽  
Small Molecular Weight ◽  
Erythroid Progenitor ◽  
Jnk Inhibitors ◽  
Induced Apoptosis

Abstract v-ErbB is an oncogene related to the Epidermal Growth Factor Receptor (EGF-R) that was initially discovered in the genome of avian erythoblastosis virus. v-ErbB will abrogate the requirement of erythroid progenitor cells for erythropoietin and stem cell factor and block terminal differentiation. EGF-R overexpression has been observed in many pathological situations and the EGF-R gene is amplified in certain tumors. Moreover, there is a truncated form of EGF-R referred to as EGFvIII which resembles v-ErbB in biological properties. One problem frequently encountered in studying the effects of EGF-R overexpression in many tumors is that EGF-R expression is often constitutive and in the presence of increased expression of other oncogenes or in the absence of certain tumor suppressor genes. To circumvent these problems, we subcloned v-ErbB into a vector which contained the estrogen receptor hormone binding domain which renders the v-ErbB protein dependent upon the addition of beta-estradiol or 4 OH tamoxifen for activity. The v-ErbB:ER oncogene will conditionally abrogate the cytokine dependence of human (TF-1) and murine (FL5.12 and FDC-P1) hematopoietic cells efficiently. This construct has allowed us to examine the transcription factors pathways activated by v-ErbB:ER in hematopoietic cells which are required for proliferation in the absence of previously required cytokines. By determining which signal transduction pathways were activated in response to either v-ErbB:ER activation or IL-3 addition in the presence and absence of specific small molecular weight membrane permeable kinase inhibitors, we could ascertain that v-ErbB:ER expression activated the STAT5, Elk, CREB, Jun, and Forkhead (Foxo) family of transcription factors in FL/v-ErbB:ER cells. The activation of these transcription factors was blocked by the respective kinase inhibitors. Thus v-ErbB:ER activated a broad spectrum of transcription factors. Treatment of v-ErbB:ER cells with the EGF-R inhibitor AG1478 very efficiently induced apoptosis in these cells at 100 to 1000 fold lower concentrations than MEK, PI3K or JNK inhibitors and activation of all these transcription factor inhibitors was inhibited. In contrast, when the cells were treated with MEK, PI3K or JNK inhibitors, only the transcription factors specific for the individual pathways were inhibited. FL5.12 cells conditionally transformed to grow in response to activated Raf and Akt, (FL/Akt:ER+Raf-1:AR) were also isolated. Activation of STAT5 by either Raf or Akt did not occur in FL/Akt:ER+Raf-1:AR cells, but did occur when the cells were treated with IL-3. Furthermore, Elk activation occurred in response to Raf activation but not IL-3 stimulation in the FL/Akt:ER+Raf-1:AR cells which grew in response to Raf and Akt. Thus oncogenes such as v-ErbB:ER, which are more effective in their ability to transform hematopoietic cells than oncogenes such as Raf and Akt, can induce multiple transcription factors, only some of which are required for growth in tissue culture systems.

Transcription Factor BACH2 Inhibits AP1 Proteins JunB and FosL1 in Umbilical Cord Blood (UCB) CD4+ T-Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1743-1743
Author(s):  
Mathew L. Lesniewski ◽  
Laura R. Fanning ◽  
Margeret Kozik ◽  
Richard P. Weitzel ◽  
Yeal Hegerfeldt ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Transcription Factor ◽  
T Cell ◽  
Cd4 T Cells ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Tnf Α ◽  
Ifn Γ ◽  
Sirna Knockdown

Abstract Introduction: Umbilical cord blood (UCB) CD4+ T-cells have been shown to express significant levels of BACH2 transcription factor protein compared to adult blood (AB) CD4+ T-cells. Previously, NFAT1 siRNA knockdown of UCB T-cells exhibited a significantly higher BACH2 mRNA expression, and IFN-γ, TNF-α. and CTLA-4 mRNA levels were significantly suppressed. BACH2, a member of the b-Zip family, has been shown to act as a heterodimer with the bZip protein MafK, as a transcriptional inhibitor via recruitment of a histone deacetylase class II complex (HDAC II) in differentiating B-cells, and neurons. Due to observed inverse expression of BACH2 and NFAT1 in UCB CD4+ T-cells, we hypothesized that BACH2 may regulate transcription factors known to bind with NFAT1 including AP-1 proteins JunB and FosL1. We tested this by siRNA knockdown of BACH2 in primary UCB-derived CD4+ T-cells. Key developmental transcription factors JUNB, FosL1, NFAT1 and downstream IFN-γ, and TNF-α were mRNA analyzed. Methods: UCB T-cells were purified using autoMACs system (Miltenyi). After overnight culture, T-cells were transfected with BACH2 siRNA (Dharmacon) using Amaxa Nucleofector system (Amaxa Inc). Both siRNA treated and control cells were incubated in media for 18 hours, and then stimulated using anti-CD3/anti-CD28 antibodies (BD BioScience). Aliquots of cells were collected at specified time points post-stimulation for protein and total RNA isolation. The relative change in mRNA levels for BACH2, JUNB, FosL1, IFN-γ, NFAT1, and TNF-α were determined by Lightcycler SybrGreen real time RT-PCR system (Roche). siRNA knockdown of BACH2 protein in transfected UCB T-cells was confirmed by western blot. Results: Real-time RT-PCR of BACH2 siRNA treated UCB CD4+ T-cells stimulated with anti-CD3/CD28 antibodies and analyzed after 6 hrs of stimulation showed a 4 log increase in FosL1 and NFAT1 mRNA, a 3 log increase in JunB mRNA, a 5 log increase in IFN-γ as compared to stimulated control UCB T-cells. TNF-α mRNA was decreased by 5 logs in BACH2 siRNA treated UCB T-cells as compared to control. CD3/CD28 stimulated untransfected UCB T-cells were previously shown to have decrease expression of NFAT1, JunB, FosL1, IFN-γ, and TNF-α, and in UCB T-cells compared to stimulated AB T-cells. Conclusions: BACH2 expression correlates with an inhibition of expression of AP1 transcription regulatory proteins in UCB T-cells during primary CD3/CD28 stimulation. The complete activation of the T-cell requires the activation of AP1 by CD28 pathway otherwise the antigen presenting cell signals the T-cell to enter anergy. In UCB CD4+ T-cells express BACH2, which acts as a transcriptional inhibitor of two critical AP1 genes, JUNB and FosL1, which mediate the CD28 co-stimulatory pathway. These results further suggests that expression of BACH2 in UCB T-cells may contribute to lower incidence of alloreactivity observed in leukemia patients receiving UCB stem cells compared to AB bone marrow stem cells and thus leads to low GVHD, and contribute to the weak Th1 response seen in stimulated UCB T-cells by reduced amounts of AP1 protein available for activating the T-cell.

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