GSK3 inhibition prevents lethal GVHD in mice
2012; Elsevier BV; Volume: 41; Issue: 1 Linguagem: Inglês
10.1016/j.exphem.2012.09.005
ISSN1873-2399
AutoresGuy Klamer, Sylvie Shen, Emma Song, Alison Rice, Robert J. Knight, Robert Lindeman, Tracey O’Brien, Alla Dolnikov,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoGraft-versus-host disease (GVHD) is a major contributor to transplant-related mortality and morbidity after allogeneic stem cell transplantation. Despite advancements in tissue-typing techniques, conditioning regimens, and therapeutic intervention, the incidence rate of GVHD remains high. GVHD is caused by alloreactive donor T cells that infiltrate and destroy host tissues (e.g., skin, liver, and gut). Therefore, GVHD is prevented and treated with therapeutics that suppress proinflammatory cytokines and T-cell function (e.g., cyclosporine, glucocorticoids). Here we report that the small molecule inhibitor of glycogen synthase kinase 3, 6-bromoindirubin 3′-oxime (BIO), prevents lethal GVHD in a humanized xenograft model in mice. BIO treatment did not affect donor T-cell engraftment, but suppressed their activation and attenuated bone marrow and liver destruction mediated by activated donor T cells. Glycogen synthase kinase 3 inhibition modulated the Th1/Th2 cytokine profile in vitro and suppressed activation of signal transducers and activators of transcription 1 and 3 signaling pathways both in vitro and in vivo. Importantly, human T cells derived from BIO-treated mice were able to mediate anti-tumor effects in vitro, and BIO did not affect stem cell engraftment and multilineage reconstitution in a mouse model of transplantation. These data demonstrate that inhibition of glycogen synthase kinase 3 can potentially abrogate GVHD without compromising the efficacy of transplantation. Graft-versus-host disease (GVHD) is a major contributor to transplant-related mortality and morbidity after allogeneic stem cell transplantation. Despite advancements in tissue-typing techniques, conditioning regimens, and therapeutic intervention, the incidence rate of GVHD remains high. GVHD is caused by alloreactive donor T cells that infiltrate and destroy host tissues (e.g., skin, liver, and gut). Therefore, GVHD is prevented and treated with therapeutics that suppress proinflammatory cytokines and T-cell function (e.g., cyclosporine, glucocorticoids). Here we report that the small molecule inhibitor of glycogen synthase kinase 3, 6-bromoindirubin 3′-oxime (BIO), prevents lethal GVHD in a humanized xenograft model in mice. BIO treatment did not affect donor T-cell engraftment, but suppressed their activation and attenuated bone marrow and liver destruction mediated by activated donor T cells. Glycogen synthase kinase 3 inhibition modulated the Th1/Th2 cytokine profile in vitro and suppressed activation of signal transducers and activators of transcription 1 and 3 signaling pathways both in vitro and in vivo. Importantly, human T cells derived from BIO-treated mice were able to mediate anti-tumor effects in vitro, and BIO did not affect stem cell engraftment and multilineage reconstitution in a mouse model of transplantation. These data demonstrate that inhibition of glycogen synthase kinase 3 can potentially abrogate GVHD without compromising the efficacy of transplantation. Graft-versus-host disease (GVHD) remains a major cause of transplant-related mortality after allogeneic stem cell transplantation. The pathophysiology of GVHD is divided into three phases: preconditioning-induced secretion of proinflammatory cytokines, such as tumor necrosis factor (TNF)–α and interleukin (IL)-1β, as well as lipopolysaccharide from conditioning-damaged epithelium; antigen-presenting cell (APC)–mediated activation of donor T-cells; and donor T-cell–mediated destruction of host tissues, such as the skin, liver, gut, and bone marrow (BM) [1Ferrara J.L. Levine J.E. Reddy P. Holler E. Graft-versus-host disease.Lancet. 2009; 373: 1550-1561Abstract Full Text Full Text PDF PubMed Scopus (1879) Google Scholar, 2Ferrara J.L. Pathogenesis of acute graft-versus-host disease: cytokines and cellular effectors.J Hematother Stem Cell Res. 2000; 9: 299-306Crossref PubMed Scopus (142) Google Scholar]. Therapeutics that inhibit T-cell activation and proinflammatory cytokine signaling are administered to prevent GVHD, but the beneficial role of donor T-cells in mediating a graft-versus-leukemia (GVL) effect and post-transplantation immunity that are critical to ensure disease-free survival in patients limits the capacity to control GVHD. All the immunosuppressive agents that are currently used to prevent GVHD (e.g., cyclosporin A, mycophenolate mofetil, glucocorticosteroids, and T-cell depletion) increase the risk of leukemia relapse by inhibiting the GVL effect [1Ferrara J.L. Levine J.E. Reddy P. Holler E. Graft-versus-host disease.Lancet. 2009; 373: 1550-1561Abstract Full Text Full Text PDF PubMed Scopus (1879) Google Scholar]. There is a need to develop new treatments that do not increase the risk of leukemia relapse. Glycogen synthase kinase 3-beta (GSK3) is a multifunctional serine/threonine kinase that regulates inflammation [3Ko K.H. Holmes T. Palladinetti P. et al.GSK-3beta inhibition promotes engraftment of ex vivo expanded hematopoietic stem cells and modulates gene expression.Stem Cells. 2011 Jan; 29: 108-118https://doi.org/10.1002/stem.551Crossref PubMed Scopus (56) Google Scholar, 4Holmes T. O'Brien T.A. Knight R. et al.The role of glycogen synthase kinase-3beta in normal haematopoiesis, angiogenesis and leukaemia.Curr Med Chem. 2008; 15: 1493-1499Crossref PubMed Scopus (28) Google Scholar, 5Holmes T. O'Brien T.A. Knight R. et al.Glycogen synthase kinase-3beta inhibition preserves hematopoietic stem cell activity and inhibits leukemic cell growth.Stem Cells. 2008; 26: 1288-1297Crossref PubMed Scopus (61) Google Scholar, 6Klamer G. Song E. Ko K.H. O'Brien T.A. Dolnikov A. 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Michalek S.M. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3.Nat Immunol. 2005; 6: 777-784Crossref PubMed Scopus (939) Google Scholar]. In vivo administration of GSK3 inhibitors induced anti-inflammatory effects in mouse models of endotoxin shock, colitis, arthritis, and asthma [14Martin M. Rehani K. Jope R.S. Michalek S.M. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3.Nat Immunol. 2005; 6: 777-784Crossref PubMed Scopus (939) Google Scholar, 15Dugo L. Collin M. Allen D.A. et al.Insulin reduces the multiple organ injury and dysfunction caused by coadministration of lipopolysaccharide and peptidoglycan independently of blood glucose: role of glycogen synthase kinase-3beta inhibition.Crit Care Med. 2006; 34: 1489-1496Crossref PubMed Scopus (52) Google Scholar, 16Dugo L. Collin M. Allen D.A. et al.GSK-3beta inhibitors attenuate the organ injury/dysfunction caused by endotoxemia in the rat.Crit Care Med. 2005; 33: 1903-1912Crossref PubMed Scopus (140) Google Scholar, 17Cuzzocrea S. Mazzon E. Di Paola R. et al.Glycogen synthase kinase-3beta inhibition attenuates the degree of arthritis caused by type II collagen in the mouse.Clin Immunol. 2006; 120: 57-67Crossref PubMed Scopus (90) Google Scholar, 18Bao Z. Lim S. Liao W. et al.Glycogen synthase kinase-3beta inhibition attenuates asthma in mice.Am J Respir Crit Care Med. 2007; 176: 431-438Crossref PubMed Scopus (81) Google Scholar], however, their potential therapeutic effects on GVHD have not been examined. Using a human xenograft mouse model of GVHD, we demonstrate that in vivo administration of the small molecule ATP-competitive GSK3 inhibitor, 6-bromoindirubin 3′-oxime (BIO), prevents lethal GVHD that develops in mice injected with human peripheral blood mononuclear cells (PB MNC). BIO does not affect early donor T-cell engraftment, but reduces T-cell activation in the spleen and later T-cell infiltration in BM and liver, and attenuates tissue destruction mediated by activated donor T cells. In vitro analyses demonstrate that BIO modulates Th1/Th2 cytokine profile and inhibits effector T-cell function, potentially through inhibition of signal transducers and activators of transcription (STAT)1 and STAT3 signaling. Importantly, engrafted donor cytolytic T cells are able to mediate an anti-leukemic effect in vitro. BIO does not affect stem cell engraftment and multilineage reconstitution in mice transplanted with human cord blood CD34+ stem cells. These data demonstrate that GSK3 inhibition should be considered as a novel therapeutic approach in the management of GVHD. The aims of this project are to examine the potential of GSK3 inhibition as a novel preventative measure of GVHD in xenotransplanted mice and to understand the cellular and molecular mechanisms induced by GSK3 in human T cells that results in GVHD attenuation. Animal procedures were approved by University of New South Wales ethics research committee. Nonobese diabetic severe combined immune-deficient IL2Rγ mutant (NSG) mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and housed in a sterile environment. NSG mice were irradiated (2.5 Gy) and transplanted with 5 × 106 PB MNCs. A "humanized" model of GVHD was selected as all research was focused on the effects of GSK3 inhibition on human T-cell responses; something not possible with a mouse model of allogeneic transplantation. NSG mice were chosen because they provide better human cell engraftment, thus significantly fewer human cells were required to induce GVHD, as compared with nonobese diabetic severe combined immune-deficient mice [19Schroeder M.A. DiPersio J.F. Mouse models of graft-versus-host disease: advances and limitations.Dis Model Mech. 2011; 4: 318-333Crossref PubMed Scopus (200) Google Scholar, 20King M.A. Covassin L. Brehm M.A. et al.Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex.Clin Exp Immunol. 2009; 157: 104-118Crossref PubMed Scopus (292) Google Scholar, 21Gorin N.C. Piantadosi S. Stull M. et al.Increased risk of lethal graft-versus-host disease-like syndrome after transplantation into NOD/SCID mice of human mobilized peripheral blood stem cells, as compared to bone marrow or cord blood.J Hematother Stem Cell Res. 2002; 11: 277-292PubMed Google Scholar, 22Ito R. Katano I. Kawai K. et al.Highly sensitive model for xenogenic GVHD using severe immunodeficient NOG mice.Transplantation. 2009; 87: 1654-1658Crossref PubMed Scopus (109) Google Scholar, 23Ito M. Hiramatsu H. Kobayashi K. et al.NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.Blood. 2002; 100: 3175-3182Crossref PubMed Scopus (1171) Google Scholar]. The development of GVHD-like pathology in this model is dependent on human donor APC presenting mouse antigens, in the context of major histocompatibility complex class II molecules, to human donor CD4+ T cells and to a lesser degree, CD8+ T cells, which mediate the tissue damage indicative of GVHD [19Schroeder M.A. DiPersio J.F. Mouse models of graft-versus-host disease: advances and limitations.Dis Model Mech. 2011; 4: 318-333Crossref PubMed Scopus (200) Google Scholar]. Mice received sublethal 2.5-Gy x-ray irradiation previously shown to be sufficient for GVHD induction [19Schroeder M.A. DiPersio J.F. Mouse models of graft-versus-host disease: advances and limitations.Dis Model Mech. 2011; 4: 318-333Crossref PubMed Scopus (200) Google Scholar, 20King M.A. Covassin L. Brehm M.A. et al.Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex.Clin Exp Immunol. 2009; 157: 104-118Crossref PubMed Scopus (292) Google Scholar, 21Gorin N.C. Piantadosi S. Stull M. et al.Increased risk of lethal graft-versus-host disease-like syndrome after transplantation into NOD/SCID mice of human mobilized peripheral blood stem cells, as compared to bone marrow or cord blood.J Hematother Stem Cell Res. 2002; 11: 277-292PubMed Google Scholar, 22Ito R. Katano I. Kawai K. et al.Highly sensitive model for xenogenic GVHD using severe immunodeficient NOG mice.Transplantation. 2009; 87: 1654-1658Crossref PubMed Scopus (109) Google Scholar, 23Ito M. Hiramatsu H. Kobayashi K. et al.NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.Blood. 2002; 100: 3175-3182Crossref PubMed Scopus (1171) Google Scholar]. This dose is sufficient to induce host proinflammatory cytokines that activate APC, initiating the GVHD process [20King M.A. Covassin L. Brehm M.A. et al.Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex.Clin Exp Immunol. 2009; 157: 104-118Crossref PubMed Scopus (292) Google Scholar, 22Ito R. Katano I. Kawai K. et al.Highly sensitive model for xenogenic GVHD using severe immunodeficient NOG mice.Transplantation. 2009; 87: 1654-1658Crossref PubMed Scopus (109) Google Scholar]. Transplanted mice were treated with dimethyl sulfoxide (DMSO) vehicle control or BIO at a dosage of 30 mg/kg on days 0 to 3 and 3 mg/kg on days 5 to 8, 10 to 13, and 15 to 17 via intraperitoneal injection and monitored for signs of GVHD according to an established mouse GVHD grading system [24Wilson J. Cullup H. Lourie R. et al.Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease.J Exp Med. 2009; 206: 387-398PubMed Google Scholar]. Histopathological examination of BM, spleen, and liver was conducted postmortem to identify MNC infiltration. The Beckman Coulter Ac·T diff (Beckman Coulter, Inc., Brea, CA, USA) was used to perform full blood counts. Flow cytometric analysis of PB, BM, and spleen was performed to analyze human T-cell engraftment and T-cell subsets. To examine the effect of BIO on stem cell engraftment, NSG mice were transplanted with 1 × 105 umbilical cord blood (UCB)–derived CD34+ cells. Analysis of human CD45+ cells was performed weekly in PB. BIO or DMSO was administered at the dosing regimen described for the model of GVHD. CD34+ hematopoietic stem cell concentration was not measured in PB-derived samples used in vivo because we were using nonmobilized blood with low stem cell numbers that only marginally contribute to human cell engraftment in PB or BM of graft recipient mice. As for the CB samples used for the in vitro experiments, we used the CD34-negative fraction after CD34+ cell isolation. UCB was obtained from the Sydney Cord Blood Bank. PB was obtained from normal donors through the Australian Red Cross Blood Service. UCB and PB were obtained from consenting donors according to an institutional-approved ethics protocol. MNCs were isolated by density gradient centrifugation using Lymphoprep (1114544; Axis-Shield, Oslo, Norway). CD3+ T cells were isolated from MNCs by negative selection using a pan T-cell isolation kit (130-091-156; Miltenyi Biotec, Bergisch Gladbach, Germany). CD34+ cells were isolated from UCB by positive selection (130-046-702; Miltenyi Biotec). Naïve CD4+ and CD8+ T cells were isolated from UCB (130-094-131 and 131-093-244, respectively; Miltenyi Biotec). Regulatory T cells (CD4+CD25+CD127dim) were isolated from UCB (130-094-775; Miltenyi Biotec). T cells were seeded at 2 to 5 × 105 cells/mL in RPMI-1640 (22400; Invitrogen, GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal calf serum; 50 ng/mL recombinant human IL-2 (202-IL-050; R&D Systems, Minneapolis, MN, USA); and 1% penicillin, streptomycin, and gentamycin cocktail. Donor disparate CB MNCs were cocultured with an allogeneic source of MNCs or T cells at a ratio of 1:1 for 5 to 7 days. Stimulator cells were irradiated at 3 Gy. Responder cells were stained with carboxyfluorescein succinimidyl ester (CFSE). Mixed lymphocyte cultures (MLCs) were cultured in RPMI-1640 supplemented with 10% fetal calf serum; 50 ng/mL IL-2; and 1% penicillin, streptomycin, and gentamycin cocktail. Purified T cells were fluorescently labeled with 5 μM CFSE (C34554; Invitrogen) and stimulated with either 2.5 μg/mL phytohemagglutin (PHA, L1668; Sigma Aldrich, St Louis, MO, USA) or an allogeneic source of MNCs. CFSE-labeled cells were co-stained with anti-CD25 antibodies (MA1-12259; Thermo Scientific, Logan, UT, USA) to measure T-cell activation. Cells were stained in the dark for 45 min at room temperature with propidium iodide, 50 μg/mL (P4864; Sigma Aldrich), 1% Triton (N150; Sigma Aldrich), and 1% sodium citrate (S1804; Sigma Aldrich), and co-stained with anti–Ki-67 antibody (556027; BD Pharmingen) as per manufacturer's instructions. RNA was extracted from T cells using the Qiagen RNeasy Minikit (74104; Qiagen). Complementary DNA was synthesized using the Promega Reverse Transcriptase System (A3500; Promega, Madison, WI, USA) in a Bio-Rad iCycler Thermal Cycler (Bio-Rad, Hercules, CA, USA). Reverse transcriptase real-time polymerase chain reaction (RT q-PCR) products were quantified using the Bio-Rad IQ4 iCycler multicolour RT q-PCR detection system (Optical System Software version 3.1, Bio-Rad, Hercules, CA, USA). RT q-PCR primers for B2M, TNFα [25Kruse N. Pette M. Toyka K. Rieckmann P. Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood.J Immunol Methods. 1997; 210: 195-203Crossref PubMed Scopus (165) Google Scholar], IκBα [26Ohtsuka T. Buchsbaum D. Oliver P. et al.Synergistic induction of tumor cell apoptosis by death receptor antibody and chemotherapy agent through JNK/p38 and mitochondrial death pathway.Oncogene. 2003; 22: 2034-2044Crossref PubMed Scopus (145) Google Scholar], interferon (IFN)-γ [25Kruse N. Pette M. Toyka K. Rieckmann P. Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood.J Immunol Methods. 1997; 210: 195-203Crossref PubMed Scopus (165) Google Scholar], IL-10 [25Kruse N. Pette M. Toyka K. Rieckmann P. Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood.J Immunol Methods. 1997; 210: 195-203Crossref PubMed Scopus (165) Google Scholar], and CD25 [27Goto S. Okada N. Kaneko A. Isemura M. Different effects of all-trans-retinoic acid on phorbol ester-stimulated and phytohemagglutinin-stimulated interleukin-2 expression in human T-cell lymphoma HUT-78 cells.Cell Struct Funct. 2008; 33: 13-19PubMed Google Scholar] were purchased from Sigma Aldrich. Total RNA was biotin-labeled and amplified with the Illumina TotalPrep RNA Amplification Kit (AMIL1791; Applied Biosystems, Carlsbad, CA, USA). Gene expression was analyzed on the HumanHT-12 v4 Expression BeadChip Kit (BD-103-0204; Illumina, San Diego, CA, USA) on the Illumina BeadArray Reader (Illumina). Data were analyzed with Illumina GenomeStudio Software (Illumina). Significant modulation of a gene was characterized as a differentiation score ≤−13 or ≥13, which correspond to p values of ≤0.05. Donors 1 and 2 were treated with PHA for 72 hours with or without 2 μM BIO. Resting T-cell gene expression was also analyzed from donor 1 to observe genes modulated by PHA alone. Gene set enrichment analysis was performed using the online DAVID (Database for Annotation, Visualization and Integrated Discovery) database [28Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc. 2009; 4: 44-57Crossref PubMed Scopus (26846) Google Scholar]. Protein lysates were prepared as described previously [7Song E.Y. Palladinetti P. Klamer G. et al.Glycogen synthase kinase-3beta inhibitors suppress leukemia cell growth.Exp Hematol. 2010; 38: 908-921. e901Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. The following antibodies were used: rabbit-derived anti–human β-actin (A2006; Sigma), mouse-derived anti–β-catenin (610154; BD Pharmingen, San Diego, CA, USA), mouse-derived anti–TNFα (ab1793; Abcam, Cambridge, MA, USA), rabbit-derived anti–c-myc (#sc-764; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse-derived phospho-STAT1 (Ser727) (sc-81522; Santa Cruz Biotechnology), rabbit-derived phospho-STAT3 (Ser727) (sc-135649; Santa Cruz Biotechnology), mouse-derived STAT1 (sc-417 AC; Santa Cruz Biotechnology), rabbit-derived STAT3 (sc-482; Santa Cruz Biotechnology), anti-mouse horseradish peroxidase (NA931V dilution; GE Healthcare, Buckinghamshire, UK), anti-rabbit horseradish peroxidase (NA934V; GE Healthcare). Chemiluminescence was detected with the SuperSignal West Femto Maximum Sensitivity Substrate kit (34096; Thermo Scientific). QuantityOne (Bio-Rad) software was used to quantify protein expression. The human Th1/Th2 cytometric bead array kit was used to analyze cytokine expression in supernatant and serum (550749; BD, San Diego, CA, USA). Monensin (2 μM) (00-4505; eBioscience, San Diego, CA, USA) was added to cultures 24 hours before the end of the experiment for intracellular cytokine analysis. Human CD45+ cells were isolated from the spleens of BIO-treated and DMSO-treated mice from the GVHD model on day 13 post-transplantation by positive selection (130-045-801; Miltenyi Biotec) and cocultured with irradiated (30 Gy) human leukemia U937 cells for 7 days at ratios of 20:1, 10:1, and 5:1 (effector to target). IL-2 (50 ng/mL) was added to culture every 3 days. After 7 days, fresh irradiated (30 Gy) CFSE-labeled U937 cells were added to culture, at the ratios described, and apoptosis of target cells was determined by 7-amino-actinomycin D+ analysis in CFSE-gated cells after 18 hours of culture. β-catenin DNA reporter constructs (CCS-018G; SABiosciences, Valencia, CA, USA) were transfected into HEK293, as described previously, as transfection efficiency in primary human T cells was low. Normality of distribution was assessed by a frequency histogram. The p values were calculated using Student's t test for normal data and a Mann-Whitney test for non-normalized data. All p values are from replicate experiments, donors, or experiments (n ≥ 3). A Lowess curve was used to determine end points (20% weight loss) for in vivo experiments. The Log-rank (Mantel-Cox) test was used to calculate p values from survival curve data. Median, mean, and standard deviations were calculated with GraphPad Prism 5 software (San Diego, CA, USA). Twenty-four of twenty-four (100%) control mice from five individual experiments died of the GVHD-like syndrome within 3 to 5 weeks of human PB MNC transplantation (Fig. 1A). All control mice showed early onset (beginning from day 9) of GVHD symptoms, which included hunching, lethargy, and coat ruffling. Sustained weight loss was the strongest indicator of GVHD (Fig. 1B). In addition, it is relevant that irradiated, nontransplanted mice began to gain weight from week 2 (data not shown). Mice transplanted with PB MNCs showed persistent weight loss that continued beyond week 2 post-irradiation. Appropriate histology was conducted for all mice requiring sacrifice for GVHD-like symptoms after irradiation and PB transplantation, and it revealed significant destruction of the BM and liver; consistent with GVHD pathology. All DMSO vehicle–treated mice showed reduced red blood cells and platelets in the PB, suggestive of anemia and potential internal bleeding. This correlated with defective BM hematopoiesis demonstrated by histopathological analysis. All DMSO vehicle–treated mice had a relatively hypocellular BM compared with nonirradiated control. Early destruction of the BM after major histocompatibility complex–mismatched hematopoietic stem cell transplantation was recently reported in an allogeneic mouse model of GVHD [29Shono Y. Ueha S. Wang Y. et al.Bone marrow graft-versus-host disease: early destruction of hematopoietic niche after MHC-mismatched hematopoietic stem cell transplantation.Blood. 2010; 115: 5401-5411Crossref PubMed Scopus (144) Google Scholar].Figure 1BIO prevents lethal GVHD in NSG mice transplanted with human PB MNCs. (A) Kaplan-Meier survival curves. (B) Weight tracking of mice from experiment depicted as donor 2 in (A); p ≤ 0.01 for end-point weights (control: cull, day +51: BIO-treated). (C) Histopathological analysis (hematoxylin and eosin [H&E] stain) of BM harvested at the end of the experiment. Experiment depicted in (A) as donor 3. Histology represents histology observed across BM from three mice per group. 40× magnification. (D) Total BM cellularity of mice at the end point of experiment. Experiment depicted in (A) as donor 3. n ≥ 4 for each group. (E) Histopathological analysis (H&E stain of liver samples harvested from transplanted mice at the end of experiment, i.e., lethal GVHD or survival). Two representative mice from (A) donor 2 illustrated. Long arrow = donor MNC infiltration; short arrow = apoptotic body. (F) Total number of apoptotic bodies in liver, and megakaryocytes in BM, observed per field as viewed under a ×40 magnification objective lens; 30 fields from three mice per group were chosen randomly. (G) Western blot analysis of β-catenin in MNCs harvested from mice 6 hours after 30 mg/kg BIO treatment. BIO treatment occurred on day +13 in one mouse to allow sufficient engraftment and expansion of donor cells for Western blot analysis. (E–G) Data from experiments using intraperitoneal BIO 30 mg/kg (days 0–3) and 3 mg/kg (days 5–17 with drug-free days every 5th day. Vehicle control was 5% (30 mg/kg BIO) and 0.5% (3 mg/kg BIO) DMSO/phosphate-buffered saline.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1BIO prevents lethal GVHD in NSG mice transplanted with human PB MNCs. (A) Kaplan-Meier survival curves. (B) Weight tracking of mice from experiment depicted as donor 2 in (A); p ≤ 0.01 for end-point weights (control: cull, day +51: BIO-treated). (C) Histopathological analysis (hematoxylin and eosin [H&E] stain) of BM harvested at the end of the experiment. Experiment depicted in (A) as donor 3. Histology represents histology observed across BM from three mice per group. 40× magnification. (D) Total BM cellularity of mice at the end point of experiment. Experiment depicted in (A) as donor 3. n ≥ 4 for each group. (E) Histopathological analysis (H&E stain of liver samples harvested from transplanted mice at the end of experiment, i.e., lethal GVHD or survival). Two representative mice from (A) donor 2 illustrated. Long arrow = donor MNC infiltration; short arrow = apoptotic body. (F) Total number of apoptotic bodies in liver, and megakaryocytes in BM, observed per field as viewed under a ×40 magnification objective lens; 30 fields from three mice per group were chosen randomly. (G) Western blot analysis of β-catenin in MNCs harvested from mice 6 hours after 30 mg/kg BIO treatment. BIO treatment occurred on day +13 in one mouse to allow sufficient engraftment and expansion of donor cells for Western blot
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