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Defining a Transcriptional Fingerprint of Murine Splenic B-Cell Development

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Defining a Transcriptional Fingerprint of Murine Splenic B-Cell Development Genes and Immunity (2008) 9, 706–720 & 2008 Macmillan Publishers Limited All rights reserved 1466-4879/08 $32.00 www.nature.com/gene ORIGINAL ARTICLE Defining a transcriptional fingerprint of murine splenic B-cell development I Debnath1, KM Roundy1, DM Dunn2, RB Weiss2, JJ Weis1 and JH Weis1 1Division of Cell Biology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA and 2Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, USA B-cell development occurs in a stepwise fashion that can be followed by the expression of B cell-specific surface markers. In this study, we wished to identify proteins that could contribute to the changes in expression of such markers. By using RNA from freshly isolated B220 þ cells, we hoped to reduce the effect of artifacts that occur during the isolation and amplification steps necessary to use flow cytometry analysis-sorted subsets in microarray experiments. Analyses comparing expression patterns from B220 þ 2-week bone marrow (pro-B, pre-B, immature B cells), 2-week spleen (predominantly transitional cells) and 8-week spleen (mainly mature B cells) yielded hundreds of genes. We also examined the B cell-activating factor (BAFF)- dependent effects on immature splenic B cells by comparing expression patterns in the spleen between 2-week A/J vs 2-week A/WySnJ mice, which lack functional BAFF receptor signaling. Genes that showed the expression differences between spleen and bone marrow samples were then analyzed through quantitative PCR on B-cell subsets isolated using two different sorting protocols. A comparison of the results from our study with the results from other analyses showed not only some overlap of preferentially expressed genes but also an expansion of other genes potentially involved in B-cell development. Genes and Immunity (2008) 9, 706–720; doi:10.1038/gene.2008.70; published online 11 September 2008 Keywords: B cells; gene expression; microarray; CD21; CD23; BAFF Introduction The products of the mouse Cr2 (CD21) and Fcer2a (CD23) genes are widely used as cellular markers for The development of the B-cell lineage initiates in the differentiating splenic B-cell subsets.14–16 Our lab has a bone marrow when pro-B cells are generated from long-standing interest in understanding the immune common lymphoid progenitor cells.1 B cell-specific regulation and transcriptional control of the Cr2 and proteins such as the B-cell receptor (BCR), CD19, Pax5 Fcer2a genes. These two genes are expressed during the and the B220 form of CD45 allow for the further same window of B-cell development at the T1–T2 maturation of the B cell into a pre-B cell phenotype.2 transition phase, and their expression is extinguished Maturing B cells exit the marrow and colonize peripheral following the loss of Pax5, which occurs as B cells mature lymphatic tissues such as the spleen.3,4 Autoreactive B into terminally differentiated plasma cells.17,18 The Cr2 cells are eliminated, undergo receptor editing, or are and Fcer2a genes share a number of transcriptional anergized in the marrow5,6 and the spleen.7,8 The most control elements including those for Pax5, nuclear factor immature B cells of the spleen, termed transitional 1 cells (NF)-kB, nuclear factor of activated T cells family (T1),9 are susceptible to B-cell receptor-dependent cell members, E box protein E2A and the Notch signaling death/arrest. On the basis of the models proposed by family member retinol binding protein-Jk.19–22 Mature B Loder,10 Allman11 and others, T1 cells lead to another cells engineered to lack Pax5 (through a CD19-cre- transitional intermediate, the transition 2 (T2) cell. The dependent deletion) show decreased expression of the canonical B-cell differentiation pathway further suggests CR1/CR2 proteins and CD23.23 Earlier we have demon- that marginal zone (MZ) cells and follicular mature (FM) strated that Pax5 binds equally to the Cr2 and Fcer2a cells are the direct descendents of T2 B cells.10–12 This genes in expressing mature splenic B cells as well as highly ordered pathway of marrow and splenic B-cell marrow B cells, which do not express Cr2 and Fcer2a.19 development relies upon a complex network of various This finding suggests that these two genes either lack a environmental signals and transcription factor regula- key activating transcription factor to allow their expres- tory pathways.13 sion at the T1/T2 stages or are blocked from transcrip- tion in marrow B cells by a transcriptional silencing protein whose function is lost during B-cell differentia- Correspondence: Dr JH Weis, Department of Pathology, University tion. of Utah School of Medicine, Cell Biology and Immunology, 15 North In addition, it is known that the T1–T2 transition of Medical Drive East, Salt Lake City, UT 84112, USA. E-mail: [email protected] splenic B cells is dependent upon the cell survival and Received 11 July 2008; revised 5 August 2008; accepted 6 August maturation signals generated by the B cell-activating 2008; published online 11 September 2008 factor of the tumor necrosis factor family (BAFF).24–27 Gene expression analysis of splenic B-cell maturation I Debnath et al 707 BAFF binding to its receptor, BAFF-R, induces activation guidelines of the National Institutes of Health for the care of an alternative NF-kB signaling pathway (utilizing p52 and use of laboratory animals. Mice examined were age- homodimers)28–31 and downregulates the pro-apoptotic matched males and females, unless otherwise specified. protein Bim through a heightened activation of extra- cellular signal-regulated kinase.32 Animals deficient in Antibodies BAFF or BAFF-R show a profound loss in peripheral Fluorescein isothiocyanate-conjugated anti-mouse CD21 mature B-cell populations, but B-cell development in the (clone 7G6), phycoerythrin (PE)-conjugated anti-mouse marrow and the migration of T1 B cells into the spleen CD24 (clone M1/69), streptavidin (SA) PE-Cy5 (BD are not altered.15,24,27 The BAFF-R mutant A/WySnJ Phamingen, Inc., San Diego, CA, USA), biotin- and PE- mouse possesses a mutation within the cytoplasmic tail conjugated anti-mouse CD23 (clone B3B4) (eBioscience, of this receptor that eliminates its ability to bind to the San Diego, CA, USA), PE-conjugated anti-mouse AA4.1 intracellular signaling molecule tumor necrosis factor (CD93) (eBioscience) and Cy5-conjugated polyclonal anti- receptor-associated factor 3.33–37 These mice have defi- mouse IgM (Biomedia Corporation, Foster City, CA, USA) ciencies in T2, MZ and FM B-cell populations but their were used for flow cytometry analyses (FACS). To-Pro-3 bone marrow B-cell developmental stages and splenic T1 and 4’,6-diamidino-2-phenylindole were used for viability populations are normal and similar to those of BAFF- staining (Invitrogen Corporation, Carlsbad, CA, USA). deficient animals.24,27 The expression of Cr2 and Fcer2a has been suggested to be a direct effect of BAFF signaling Isolation of B-cell populations independent of the B-cell survival function of the Total splenocytes or marrow cell populations were cytokine16 (presumably through the enhanced transloca- isolated from various strains of mice at various ages as tion of NF-kB-p52 into the nucleus). However, we have indicated in figure legends. After red blood cell lysis, a B found BAFF-dependent expression levels of CD21 and cell-enriched population was obtained using B220 þ CD23 to be similar to those of CD19 and other B-cell magnetic beads depletion according to the manufac- marker proteins whose expression is not directly turer’s protocol (Miltenyi Biotech, Inc., Auburn, CA, dependent upon NF-kB-p52. Furthermore, NF-kB-p52 USA). B220 þ cells were stained with CD21-FITC, CD24- chromatin complexes are evident within Cr2 and Fcer2a PE, CD23-biotin/SA-PE-Cy5 or with IgM Cy5, AA4.1 PE genes from BAFF-R-defective and normal B cells.38 and CD23 PE-Cy7, for flow cytometry analyses and In this study, we sought to identify the key transcrip- resuspended at 5 Â 106 cells per ml for cell sorting. tional regulatory proteins that control Cr2 and Fcer2a Magnetic sorting with B220 routinely provided samples expression during B-cell maturation. We utilized cells 95% or greater homogeneity. from wild-type A/J bone marrow, immature spleen and Various B-cell subsets (see Results) were sorted and mature spleen to create overlapping genomic screens to isolated using FACSVantage SE at the FACS Core Facility, identify gene products preferentially lost or enhanced University of Utah. Two different approaches to isolate during B-cell development. This approach allowed us to the T1, T2, MZ and FM splenic cells were utilized1,11,40 enrich for populations that vary in their expression of and described in figure legends. CD21 and CD23 instead of isolating marrow and splenic B-cell subsets and using amplification to obtain enough Microarray analyses RNA. We also compared A/J mice with the mutant A/ Total RNA was isolated from B220 þ B cells from 2-week WySnJ to investigate the downstream targets/genes of marrow (20 animals), 2-week (four animals)- or 8-week BAFF signaling that could contribute to CD21 and CD23 (four animals)-old spleen from A/J, or 2-week spleen expression. We have identified a number of immuno- from A/WySnJ mice. Pools of cells from five mice were modulatory proteins whose expression and function are used per sample. Total RNA from the pools was linked to B-cell development by obtaining gene grid quantified and used for Affymetrix array hybridization array data from a variety of similar models and according to the manufacturer’s instructions (Affymetrix, comparing those data with known positive and negative Santa Clara, CA, USA).
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