RNAseq of the small intestine of mice 6 days after they were transplanted with either wildtype allogeneic T cells or glucocorticoid-resistant allogeneic T cells (mouse model of acute graft-versus-host disease
ABSTRACT: Given that mice transplanted with glucocorticoid-resistant T cells display more severe disease symptoms, the idea was that an RNA-seq comparison between mice transplanted with either wildtype or glucocorticoid-resistant T cells could yield genes involved in disease progression and potential therapeutic targets.
Project description:Glucocorticoids (GCs) and topoisomerase II inhibitors are used in the treatment of acute lymphoblastic leukaemia (ALL) due to their ability to induce cell death in lymphoid cells. GC-induced apoptosis is mediated by the glucocorticoid receptor (GR), whereas topoisomerase II inhibitors cause DNA damage and activate sensors of DNA damage including the tumour suppressor p53. In order to shed light on the role of the microenvironment in cell death and identify determinants of drug sensitivity we performed transcriptomic analysis in ALL cells treated with the synthetic glucocorticoid dexamethasone, and the topoisomerase II inhibitor etoposide combined with bone marrow-derived conditioned media (CM).
Project description:Introduction. Glucocorticoids are critical drugs used to treat acute lymphoblastic leukemia, and response to glucocorticoids is highly predictive of outcome. Here we report a study evaluating the NOD/SCID xenograft mouse model to investigate glucocorticoid-induced gene expression. Methods. NOD/SCID mice were inoculated with ALL-3, a glucocorticoid-sensitive xenograft, and when highly engrafted were randomised to either dexamethasone 15mg/kg or vehicle control IP. Cells were harvested at 0, 8, 24 or 48 hours thereafter, RNA was extracted and hybridised onto Illumina WG-6_V3 chips. Results. The 8 hour dexamethasone-treated timepoint had the highest number of significantly differentially expressed genes with minimal changes seen across the time-matched controls. Replicate analysis revealed that using data from 3 replicates instead of 4 resulted in excellent recovery scores of >0.9 at timepoints with high signal. When assessed at the level of pathways, gene expression changes in the 8 hour xenograft samples were similar to patients treated with glucocorticoids. Conclusions. The NOD/SCID xenograft mouse model provides a reproducible experimental model system in which to investigate clinically-relevant mechanisms of in vivo glucocorticoid-induced gene regulation in ALL; the 8 hour timepoint provides the highest number of significantly differentially expressed genes; time-matched controls are redundant and excellent recovery scores can be obtained with 3 replicates. At 0, 8, 24 or 48 hours, NOD/SCID xenograft mice were treated with vehicle control, or dexamethasone. We used 4 biological replicates per time point (3 at the 48hour time point), each of which went onto an Illumina HumanWG-6_V3_0_R1_11282955_A microarray. Samples from each of the 7 groups were divided across a total of 5 microarray slides
Project description:Regulatory T (Treg) cells play an important role in the induction and maintenance of peripheral tolerance. Treg cells also suppress a variety of other immune responses, including anti-tumor and alloimmune responses. We have previously reported that tumor-activated Treg cells express granzyme B and that granzyme B is important for Treg cell-mediated suppression of anti-tumor immune responses (GSE13409). Here, we report that allogeneic mismatch also induces the expression of granzyme B. Granzyme B-deficient mice challenged with fully mismatched allogeneic P815 mastocytoma cells have markedly improved survival compared to WT and other granzyme- or perforin-deficient mice, suggesting an immunoregulatory role for granzyme B in this setting. Treg cells harvested from the tumor environment of P815-challenged mice express granzyme B. Treg cells also express granzyme B in vitro during mixed lymphocyte reactions and in vivo in a mouse model of graft-versus-host disease (GVHD). However, in contrast to findings from our previously published tumor model, granzyme B is not required for the suppression of effector T cell (Teff) proliferation in in vitro Treg suppression assays stimulated by either Concanavalin A or allogeneic antigen presenting cells. Additionally, in an ex vivo assay, sort-purified in vivo-activated CD4+Foxp3+ Treg cells from mice with active GVHD -- under conditions known to induce granzyme B expression in Treg cells -- suppressed Teff cell proliferation in a granzyme B-independent manner. Adoptive transfer of naive granzyme B-deficient CD4+CD25+ Treg cells into a mouse model of GVHD rescued hosts from lethatlity equivalently to naive wild-type Treg cells. Serum analysis of GVHD-associated cytokine production in these recipients also demonstrated that Treg cells suppressed production of IL-2, IL-4, IL-5, GM-CSF, and IFN-gamma in a granzyme B-independent manner. In order to determine whether the context in which Treg cells are activated alters the intrinsic properties of Treg cells, we used Foxp3 reporter mice to obtain gene expression profiles of CD4+Foxp3+ Treg cells purifed from naive resting spleens, spleens from mice with acute GVHD, and from ascites fluid of mice challenged intraperitoneally with allogeneic P815 tumor cells. Unsupervised analyses revealed distinct activation signatures of Treg cells among the 3 experimental groups. Taken together, these findings demonstrate that granzyme B is not required for Treg cell-mediated suppression of GVHD, which is in contrast to what we have previously reported for Treg cell function in the setting of tumor challenge. Cell intrinsic differences could partially account for these differential phenotypes. These data also suggest the therapeutic potential of targeting specific Treg cell suppressive functions in order to segregate GVHD and graft-versus-tumor effector functions. Experiment Overall Design: Six replicates of Naive CD4+Foxp3+ Treg cells were purified from resting spleens, five replicates of allogeneic tumor-activated Treg cells and three samples of GVHD-activated Treg cells. Experiment Overall Design: Naive reps 1-3 are controls for the GVHD-activated samples. Experiment Overall Design: Naive reps 4-6 are controls for the Allogeneic tumor-activated samples.
Project description:The purpose of this study was to identify direct targets of the glucocorticoid receptor (GR) using an orthogonal analysis. An expression study of mouse livers in the presence or absence of exogenous glucocorticoid complemented a genome-wide location analysis on chromatin from the same livers. These were hybridized to the BCBC Mouse PancChip 5.0 and the Mouse PromoterChip BCBC-3.0 respectively.
Project description:In this study, we examined transcriptional profiles from 3 different microarray platforms, across 103 peripheral blood samples with and without acute rejection, to find a critical gene-set for the diagnosis of acute renal rejection that matched biopsy diagnosis, irrespective of patient demographics, clinical confounders, concomitant infection, immunosuppression usage or sample processing methods. We hypothesized that changes in peripheral blood expression profiles correlate with biopsy-proven rejection, and that these changes could be used as biomarkers for the diagnosis and prediction of acute rejection. This SuperSeries is composed of the SubSeries listed below. We performed cross-sectional microarray analysis of 103 matched peripheral blood samples collected at a single time point, timed with a biopsy where acute rejection was either confirmed as present (60 AR samples) or absent (62 STA samples). The samples were hybridized to one of 3 microarray platforms: Affymetrix (n=75 the 54K HG-U133_Plus2 Array), Agilent (n=26, 44K oligo Array) and cDNA array (n=21, ~30K cDNA). Of 103 samples, 14 were used on both Affy and Agilent arrays; 1 was used in cDNA and Agilent array and 2 were used across 3 array platforms. Microarray data generated from 3 array platforms were cross-compared, by mapping common and overlapping transcripts to Human Gene Organization (HUGO) gene names. Significant gene lists on each platform were identified using Significant Analysis of Microarray (SAM, ref) with a common significance threshold of a false discovery rate (FDR) of <10%.This set contains the data for samples hybridized to Affymetrix arrays. Refer to individual Series
Project description:In this study, we examined transcriptional profiles from 3 different microarray platforms, across 103 peripheral blood samples with and without acute rejection, to find a critical gene-set for the diagnosis of acute renal rejection that matched biopsy diagnosis, irrespective of patient demographics, clinical confounders, concomitant infection, immunosuppression usage or sample processing methods. We hypothesized that changes in peripheral blood expression profiles correlate with biopsy-proven rejection, and that these changes could be used as biomarkers for the diagnosis and prediction of acute rejection. Disease State: Acute Rejection (AR#sampleid) or Stable (S#sampleid) samples We performed cross-sectional microarray analysis of 103 matched peripheral blood samples collected at a single time point, timed with a biopsy where acute rejection was either confirmed as present (60 AR samples) or absent (62 STA samples). The samples were hybridized to one of 3 microarray platforms: Affymetrix (n=75 the 54K HG-U133_Plus2 Array), Agilent (n=26, 44K oligo Array) and cDNA array (n=21, ~30K cDNA). Of 103 samples, 14 were used on both Affy and Agilent arrays; 1 was used in cDNA and Agilent array and 2 were used across 3 array platforms. Microarray data generated from 3 array platforms were cross-compared, by mapping common and overlapping transcripts to Human Gene Organization (HUGO) gene names. Significant gene lists on each platform were identified using Significant Analysis of Microarray (SAM, ref) with a common significance threshold of a false discovery rate (FDR) of <10%. This set contains the data for samples hybridized to cDNA arrays.
Project description:Mammalian lung development during the saccular and alveolar stages is dependent upon antagonistic molecular signalling by endogenous retinoic acid (RA) and glucocorticoids (GCs) which regulate gene expression via the retinoic acid receptor (RAR) family and the glucocorticoid receptor (GR), respectively. The genomic mechanism of this antagonism was investigated with in vitro distal lung explant cultures from E18.5 GR-null (GR-/-) mice treated with all-trans-RA (atRA) for 2h . Whole mouse genome microarray analysis from lung explant tissue identified a small number of gene targets which were not only significantly induced by atRA in the wildtype lung, but also significantly stimulated to levels greater than atRA-treated wildtype lungs in GR-/- lungs.
Project description:This study compared gene expression in murine bcr-abl positive acute lymphoblastic leukemia cells in vivo in allogeneic BMT recipients compared to syngneneic BMT recipients. Experiment Overall Design: The goal of the experiment was to compare gene expression in leukemia cells that were in either an allogeneic transplant (5 samples) or syngeneic transplant (5 samples) immune environment in vivo. The bone marrow transplant model involved preparation of C57BL/6 recipients with total body irradiation and 5-fluoruracil. These recipients were then infused IV with either allogeneic C3.SW marrow and spleen cells or syngeneic C57BL/6 marrow and spleen cells. The leukemia cells were mixed in with the marrow and spleen cells. After a few weeks (2-3) the C57BL/6 leukemia cells were purified by flow cytometry and passed into other freshly transplanted animals. After three serial transplants the leukemia cells were flow purified and RNA prepared. Leukemia was C57BL/6 background (cells contain the human p210 bcr/abl oncogenic fusion gene and also have a deletion in the Ink4a/Arf locus, see: Biology of Blood and Marrow Transplantation. 14:622-630, 2008).
Project description:LREX' are a LnCAP/AR subline with natural expresison of the glucocorticoid receptor. We used the model to compare the AR and GR cistromes in prostate tissue. We determined the GR cistrone in the presence of Dex (100nM) treatment and the AR cistrome in the presene of DHT (1nM) treatment.
Project description:Xenotransplantation holds the promise of providing an unlimited supply of donor organs for terminal patients with organ failure. The gal carbohydrate results in rejection of wild type pig grafts, however, chimerism established by expression of the GalT gene prior to transplantation in GalT knockout mice results in tolerance to Gal+ heart grafts. We used microarrays in order to further understand the early events that occur within grafts that demonstrate tolerance. Experiment Overall Design: The GalT BMT recipient is a GalT knockout mouse which recieved GalT gene transduced allo-bone marrow cells transplantation after sublethal irradiation. A heart of wild type C57BL/6 was heterotopically transplanted into the recipient after GalT BMT. Syngeneic Control recipient is a wild type C57BL/6 transplanted a heart of wild C57BL/6.