Transcriptomic signature of the CD24hiCD38hi transitional B cells associated with an immunoregulatory phenotype in renal transplant recipient.
ABSTRACT: The role of B cells after transplant regarding allograft rejection or tolerance has become a topic of major interest. Recently, in renal transplant recipients, a B cell signature characterized by the overexpression of CD19+CD38hiCD24hi transitional B cells has been observed in operationally tolerant patients and in Belatacept treated patients with significant lower incidence of donor specific antibodies. The phenotypic and functional characterization of these transitional B cells is far to be exhaustive. We present the first transcriptomic and phenotypic analysis associated with this phenotype. Three populations were studied and compared: (i) transitional CD24hiCD38hi (ii) CD24+CD38- and (iii) CD24intCD38int populations. Peripheral blood mononuclear cells were isolated from 5 healthy donors and CD20+ B cells were isolated by magnetic beads. Three B cells populations were then sorted by flow cytometry: CD19+CD24hiCD38hi (test), CD19+CD24+CD38- (control 1) and CD19+CD24intCD38int (control 2)
Project description:Analysis of Hoechst dye 33342-effluxing side population (SP) cells from B-CLL peripheral blood mononuclear cells. 9 biological replicates from B-CLL patients sorted into CD5+CD19+ SP and non-SP subsets. Two color comparative gene expression using Agilent microarrays.
Project description:Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here we show that endoglin (Eng), a receptor for the TGFβ superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng+Flk1+ mesodermal precursor population at E7.5, a cell fraction also endowed with endothelial potential. In Eng knockout embryos, hematopoietic colony activity and numbers of CD71+Ter119+ erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key BMP target genes including the hematopoietic regulators Scl, Gata1, Gata2 and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng+Flk-1+ progenitors co-expressed TGFβ and BMP receptors and target genes. Furthermore, Eng+Flk-1+ cells presented high levels of phospho-SMAD1/5, indicating active TGFβ and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of endoglin-expressing cells was dependent on TGFβ superfamily ligands: BMP4, BMP2, or TGF-β1. These data demonstrate that the E+F+ fraction at E7.5 represents mesodermal cells competent to respond to TGFb1, BMP4, or BMP2, shaping their hematopoietic development, and that endoglin is a critical regulator in this process by modulating TGF/BMP signaling. E7.5 pooled embryos (25 litters; 300 embryos approximately) were dissected and 3,000 cells were sorted in triplicate for Eng-Flk1-, Eng-Flk1+, Eng+Flk1+, and Eng+Flk1- fractions. Microarray results were analyzed with GeneSpring GX software.
Project description:During a T cell response, naïve CD8 T cells differentiate into effector cells. Subsequently, a subset of effector cells termed memory precursor effector cells (MPECs) further differentiates into functionally mature memory CD8 T cells. The transcriptional network underlying this carefully scripted process is not well understood. Here, we report that the transcription factor FoxO1 plays an integral role in facilitating effector to memory transition and functional maturation of memory CD4 and CD8 T cells. We find that FoxO1 is not required for differentiation of effector cells, but in the absence of FoxO1, memory CD8 T cells displayed features of scenescence and progressive attrition in polyfunctionality, which in turn led to impared recall responses and poor protective immunity. These data suggest that FoxO1 is essential for active maintenance of functional CD8 T cell memory and protective immunity. Under competing conditions in bone marrow Single-cell suspensions from splenocytes of eight samples WT (control) and FoxO1-/- (experimental) LCMV-immune mice were prepared using standard procedures. CD8 T cells were then isoloated using Thy1.2 (CD90.2) (30-H12) microbeads (Miltenyi Biotec). Cells were then stained with anti-CD8, anti-CD44 and Db/NP396 MHC class I tetramer. Activated (CD8+CD44hi), naive (CD8+CD44lo), and virus-specific CD8 T cells were sorted using FACSAria II instrument (BD Biosciences). The purity of the cells was >95%. Total RNA was extracted from the sorted cells by Trizol Reagent. RNA samples were reverse transcribed and Cy3-labeled cDNAs were hyrbidized to Agilent whole Mouse Genome Oligo Microarrays. Fluorscence signals were detected using Agilent's Microarray Scanner system, data was analyzed using the Rosetta Resolver gene expression data analysis system and genes with a fold change < and p-values <0.01 were identified. Microarray data discussed in the paper is focused on virus-specific memory CD8 T cells from samples WT_Tet_2 vs KO_Tet_2.
Project description:In order to identify cell-autonomous factors that might be regulating the neurogenic niche, type 1 and 2 progenitors were FACS sorted from P7 and P28 animals and their cDNA was hybridized to a stem cell-specific microarray. Keywords: differential gene expression analysis Two samples per microarray (P7 and P28). Each microarray experiment was done in triplicate. P7 is the control sample.
Project description:RNA microarray profiling analysis was performed on 2 different ex-vivo splenic B cell subsets: IgD+CD27+ (marginal zone B cells) and switched memory IgG+CD27+ cells, isolated from splenic samples of 4 children (from 4 to 6 yr of age), 3 adults and 3 (or 4) seniors of more than 75 yr.
Project description:We applied a novel approach of parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations in a genetically defined subset of AML (AML with monosomy 7). We isolated phenotypic long-term HSC (LT-HSC), short-term HSC (ST-HSC), and committed granulocyte-monocyte progenitors (GMP) from individual patients with AML, and measured gene expression profiles of each population, and in comparison to their phenotypic counterparts from age-matched healthy controls. Bone marrow samples from AML patients bearing monosomy 7 and age-matched healthy controls were used in this study. Hematopoietic stem and progenitor compartments were purified by multiparameter-high speed fluorescence-activated cell sorting (FACS) from CD34+ enriched bone marrow to isolate LT-HSC (Lin-/CD34+/CD38-/CD90+), ST-HSC (Lin-/CD34+/CD38-/CD90-), and GMP (Lin-/CD34+/CD38+/CD123+/CD45R+).
Project description:We applied a novel approach of parallel transcriptional analysis of multiple, highly fractionated stem and progenitor populations from patients with acute myeloid leukemia (AML) and a normal karyotype. We isolated phenotypic long-term HSC (LT-HSC), short-term HSC (ST-HSC), and committed granulocyte-monocyte progenitors (GMP) from individual patients, and measured gene expression profiles of each population, and in comparison to their phenotypic counterparts from age-matched healthy controls. Bone marrow samples from AML patients with normal karyotype and age-matched healthy controls were used in this study. Hematopoietic stem and progenitor compartments were purified by multiparameter-high speed fluorescence-activated cell sorting (FACS) from CD34+ enriched bone marrow to isolate LT-HSC (Lin-/CD34+/CD38-/CD90+), ST-HSC (Lin-/CD34+/CD38-/CD90-), and GMP (Lin-/CD34+/CD38+/CD123+/CD45R+).
Project description:Objective: Production of pathogenic autoantibodies by self-reactive plasma cells (PC) is a hallmark of autoimmune diseases. Investigating the prevalence of PC in autoimmune disease and their relationship with known pathogenic pathways may increase our understanding of the role of PC in disease progression and treatment response. Methods: We developed a sensitive gene expression based method to overcome the challenges of measuring PC using flow cytometry. Whole genome microarray analysis of sorted cellular fractions identified a panel of genes, IGHA, IGJ, IGKC, IGKV, and TNFRSF17, expressed predominantly in PC. The sensitivity of the PC signature score created from the combined expression levels of these genes was assessed through ex vivo experiments with sorted cells. This PC gene expression signature was used for monitoring changes in PC levels following anti-CD19 therapy; evaluating the relationship between PC and other autoimmune disease-related genes; and estimating PC levels in affected blood and tissue from multiple autoimmune diseases. Results: The PC signature was highly sensitive and capable of detecting as few as 300 PCs. The PC signature was reduced over 90% in scleroderma patients following anti-CD19 treatment and this reduction was highly correlated (r = 0.77) with inhibition of collagen gene expression. Evaluation of multiple autoimmune diseases revealed 30-35% of lupus, rheumatoid arthritis, and scleroderma patients with increased PC levels. Conclusion: This newly developed PC signature provides a robust and accurate method to measure PC levels in the clinic. Our results highlight subsets of patients across multiple autoimmune diseases that may benefit from PC depleting therapy. To examine gene expression in purified cellular fractions, normal human blood was collected from 2 donors as per institutional policy. The granulocyte (CD15+), monocyte (CD14+), T cell (CD3+), B cell (Non-PC gated, CD19+), and PC (CD27++CD38++) fractions from peripheral blood were separated. White blood cells were washed with FACS buffer (PBS + 0.5%BSA + 2mM EDTA (Gibco)) and incubated with 20% heat-inactivated FBS for 10-15 minutes on ice. The following mAbs were added directly to the cells: CD15 (HI98); CD14 (M5E2), CD3 (UCHT1), CD27 (M-T271), CD38 (HB7), and DAPI (Molecular probes). Cells were sorted on a Becton Dickinson FACS Aria II flow cytometer. All sorted fractions were collected in FACS buffer, centrifuged, and the resulting cell pellet was suspended in RNA lysis buffer (Ambion).
Project description:To date nothing is known about the specificity of regulatory B cells. Here we analyzed the B cell repertoire at the Ig gene level of regulatory transitional CD24(high)CD38(high) B cells of patients with multiple sclerosis (MS) in comparison with healthy donors. 5 patients with highly active MS, 4 patients with benign MS and 6 healthy donors were examined. Mononuclear cells from peripheral blood were stained against CD19, CD24 and CD38 surface markers. Subpopulation CD19(+)CD24(high)CD38(high) and total pool of CD19(+) cells were sorted individually for subsequent RNA isolation and BCR sequences comparison. Variable heavy (VH) and light (VL) chains genes of each patient were amplified from cDNA and deep sequencing of the VH and VL genes was carried out.