Elsevier

Molecular Immunology

Volume 45, Issue 2, January 2008, Pages 362-375
Molecular Immunology

Transcriptome analysis in primary B lymphoid precursors following induction of the pre-B cell receptor

https://doi.org/10.1016/j.molimm.2007.06.154Get rights and content

Abstract

Pre-BCR signals are part of a checkpoint where early precursor (pre-) B cells with a pairing Ig μH chain (μHC) are clonally expanded before they differentiate into IgL-rearranging, resting pre-B cells. A pre-BCR consists of two μHCs, two surrogate L chains and the signal transducer Igα/Igβ. The molecular circuits by which the pre-BCR controls proliferation and differentiation of pre-B cells are poorly characterized. Therefore, we identified the differential transcriptome by genome-wide expression profiling in progenitor (pro-) B cells from a Rag2-deficient mouse, in which the expression of a transgenic μHC and thus a pre-BCR as well as pre-BCR-mediated clonal expansion can be controlled by tetracycline (μHC-inducible mouse). This analysis revealed that pre-BCR signals upregulate components of the BCR signalosome, open the IgL chain (LC) locus and induce the krüppel-like transcription factor KLF2, a key regulator of quiescence and lymphocyte migration. Hence, pre-BCR signals establish the molecular network for BCR signaling even before the production of an IgLC and induce the expression of KLF2, a candidate for controlling clonal expansion and migration of functional pre-B cells.

Introduction

The establishment of a large antibody repertoire through the assembly of functional exons encoding the variable regions (V) and thus the antigen binding site of an B cell receptor (BCR) from V, D and J gene segments first at the IgH and subsequently at the IgL locus is the hallmark of B cell development (Alt et al., 1984). In this scenario, signals provided by an immature Ig-like transmembrane complex, the so-called pre-B cell receptor, are part of a critical checkpoint where precursor B (pre-B) cells with a functional, i.e., an IgL chain-pairing μH chain (μHC) are clonally expanded (Bradl et al., 2007, Burrows et al., 2002, Keyna et al., 1995a, Keyna et al., 1995b, Kline et al., 1998, Martensson et al., 2002, Meffre et al., 2000, Muljo and Schlissel, 2000, Vettermann et al., 2006). Although all descendants from a clonally expanded pre-B cell produce the same VH idiotype, each of these cells will synthesize a different IgL chain (LC) at a later stage of differentiation and thus a different antigen specificity. Hence, pre-BCR signals are critical for increasing the diversity of the primary antibody repertoire by expanding the pool of pre-B cells that produce a functional μHC.

The extent of clonal expansion of a pre-B cell is determined by the strength of signal provided by the pre-BCR. This receptor consists of two μHCs, two surrogate light chains (SLCs), which are composed of the invariant and non-covalently associated Ig-like polypeptides VpreB and λ5, and the signal transducer Igα/Igβ. As pre-B cells pass the pre-BCR checkpoint, they undergo first a limited clonal expansion phase with two to six cell divisions and differentiate into small resting pre-B cells, which subsequently assemble a V and J gene segment at the IgL-locus into a VL exon (Bradl et al., 2007, Decker et al., 1991, Hendriks and Middendorp, 2004, Hess et al., 2001, Rolink et al., 2000, Vettermann et al., 2006) Molecular circuits by which the pre-BCR controls the limited clonal expansion of early pre-B cells and rearrangements at the IgL locus are still poorly understood.

Therefore, we investigated the effect of pre-BCR signals on the transcriptome and the opening of the Igκ locus in the μHC-inducible mouse (Hess et al., 2001, Schuh et al., 2003), a double-transgenic (dTg) mouse that allows us to induce de novo the expression of a μHC and thus a pre-BCR by tetracycline (Tet) in freshly isolated primary pro-B cells. Affymetrix-microarray analyses identified 231 genes that were significantly up- or downregulated upon pre-BCR expression. Besides known pre-B cell signature genes like CD2, CD25, c-kit and TdT we found that pre-BCR signals also opened the Igκ locus and upregulated signature genes of immature B cells, such as CD20 and molecules involved in BCR signaling and antigen presentation. Hence, pre-BCR signals establish the molecular network for BCR signaling even before IgL rearrangement has been completed. Most importantly, we identified a large group of novel pre-BCR target genes, including LKLF/KLF2, a transcription factor of the krüppel-like family known to be a key regulator in controlling quiescence and migration of T cells (Carlson et al., 2006, Kuo et al., 1997a). KLF2 as well as its target gene sphingosine-1-phosphate receptor-1 (S1P1) (Carlson et al., 2006) were both strongly upregulated after pre-BCR induction; as expected, the highest expression of KLF2 and S1P1 was detected in freshly isolated and primary small, resting pre-B cells. Hence, KLF2 is a new pre-BCR target, which may play a critical role in controlling clonal expansion and cell migration of functional pre-B cells.

Section snippets

Animals

Rag2−/−/dTg mice, Rag2−/−/tTA mice (Hess et al., 2001, Schuh et al., 2003) and C57Bl/6 mice (Charles River, Sulzfeld, Germany) were maintained under pathogen-free conditions in the animal facility of the Nikolaus-Fiebiger-Center (Erlangen, Germany). Transgenic animals were genotyped by PCR using DNA from tail biopsies.

Flow cytometry

For surface staining, 5 × 105 cells were stained with the respective antibodies as described (Schuh et al., 2003). For cytoplasmic staining, 5 × 105 cells were fixed in 1%

Experimental system: primary pro-B cells with Tet-regulated expression of a transgenic μHC

To determine the impact of pre-BCR signals on the mRNA transcriptome in B-lymphoid precursors, we isolated CD19+ B-lymphoid cells from the bone marrow of Rag2−/− mice, in which the expression of a transgenic μHC (tetop-μHC) can be controlled by a transgenic Tet-controllable transactivator (tTA). In the absence of Tet, μHC and thus pre-BCR expression is turned on, whereas in the presence of Tet, μHC expression is turned off (Fig. 1A; Hess et al., 2001, Schuh et al., 2003). Since the

Discussion

Signals provided by the pre-BCR are part of an important checkpoint in early B cell development in order to control the outcome of rearrangement at the IgH locus. Cells expressing a functional μHC assemble with SLC the pre-BCR complex, which is then translocated to the cell surface from where it triggers intracellular signaling pathways leading to survival, proliferation and differentiation (Bradl et al., 2007, Hendriks and Middendorp, 2004, Vettermann et al., 2006). Pre-BCR-positive cells

Acknowledgements

We thank Edith Roth for excellent technical assistance. This work was supported in part by the project grant SFB466 and the research grant JA968/5 through FOR832 from the Deutsche Forschungsgemeinschaft (DFG) to H-MJ, intramural grants from the ELAN fond to WS, H-MJ and KH and the DFG Training Grant GK592 to KH.

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