Early function of Pax5 (BSAP) before the pre-B cell receptor stage of B lymphopoiesis

Abstract

The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin mu (Igmu) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell stage. The pre-BCR contains, in addition to Igmu, the surrogate light chains lambda5 and VpreB and the signal transducing proteins Igalpha and Igbeta. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5 gene, which codes for the B cell-specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Igalpha) gene and VH-to-DHJH recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes approximately 10- and approximately 50-fold, respectively. Here we demonstrate that complementation of these deficiencies in pre-BCR components by expression of functionally rearranged Ig mu and chimeric Igmu-Igbeta transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (-/-) mice in contrast to RAG2 (-/-) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igmu transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.

Figure 1

Expression of a rearranged Igμ transgene fails to advance B cell development in Pax5 mutant mice. Bone marrow cells from 8–11-d-old mice of the indicated genotype were analyzed by flow cytometry using an FITC-conjugated anti-B220 antibody (RA3-6B2) in combination with biotinylated anti-CD25 (7D4), anti-CD43 (S7), anti-CD2 (RM2-5), or anti–c-kit (ACK4) antibodies. The biotin-conjugated antibodies were revealed by incubation with PE-coupled streptavidin. The percentage of B220⁺ cells is indicated in each quadrant. The number of B220⁺ cells was consistently lower in the bone marrow of Pax5 mutant mice compared with RAG2 mutant mice, which may reflect the poorer health of Pax5-deficient mice (25), the ultrasensitivity of Pax5 (−/−) pre-BI cells to apoptotic signals (Nutt, S.L., data not shown), or blockage at different stages of pro-B cell development in the two mutant mice (see Discussion).

Figure 2

Igμ and bcl-2 transgene expression in pre-BI cells of Pax5- deficient bone marrow. (A) Intracellular Igμ staining. Bone marrow cells of Pax5 mutant mice containing or lacking the Igμ transgene were analyzed by intracellular staining for expression of the Igμ protein (see Materials and Methods). Pre-BI cell were identified by staining with APC-coupled anti–c-kit (ACK45) and PE-conjugated anti-B220 (RA3-6B2) antibodies. The presence of the intracellular Igμ protein within the c-kit⁺ B220⁺ cell population was detected with an FITC-conjugated anti-μ antibody (R6-60.2) and is displayed to the right. (B) Expression of a bcl-2 transgene fails to rescue the early B cell developmental block in Pax5-deficient mice. The transgene of the Eμ-bcl-2-36 strain (29, 30) was crossed into the Pax5 mutant background, and the bone marrow of 10–14-d-old Pax5 (−/−) mice was analyzed by flow cytometry using FITC-conjugated anti-B220 (RA3-6B2) and biotinylated anti-CD43 (S7) antibodies (revealed by PE-streptavidin). The percentage of B220⁺ cells is indicated in each quadrant. 10 mice of each genotype were analyzed, indicating that the CD43⁺ B220⁺ cell population corresponded on average to 5.4% [Pax5 (−/−)], 7.1% [Pax5 (−/−), bcl-2 transgene], 7.2% [Pax5 (+/+)], and 10.8% [Pax5 (+/+), bcl-2 transgene] of the total bone marrow cells (data not shown).

Figure 3

Expression of the Igμ-Igβ transgene is not sufficient to rescue the early developmental arrest in Pax5 mutant mice. (A) Flow cytometric analysis. Bone marrow cells of Pax5 mutant mice containing or lacking the Igμ-Igβ transgene (16) were analyzed by flow cytometry as described in the legend to Fig. 1. (B) Expression of the Igμ–Igβ fusion protein. Bone marrow pre-BI cells from mice of the indicated genotypes were enriched by short-term culturing in IL-7 containing medium and then analyzed for Igμ–Igβ protein expression by immunoblotting (see Materials and Methods). The faster migrating polypeptide corresponds in size to the unglycosylated form of the Igμ-Igβ fusion protein. The size of marker proteins (given in kD) is indicated to the left.

Figure 4

The Pax5 function is required for normal signaling responses of the pre-BCR. (A) Constitutive expression of the pre-BCR on Pax5-deficient pre-BI cells carrying the Igμ transgene. Pre-BI cells from Pax5 mutant mice were grown for 3 wk on stromal ST2 cells in the presence of IL-7 followed by flow cytometric analysis with biotinylated anti-λ5 (LM34), anti-μ (M41.42), and anti–pre-BCR (SL156) antibodies. Incubation with PE-coupled streptavidin was used to visualize the biotinylated antibodies. Unstained control cells are indicated by a line. Note that the cell surface expression of λ5 in the absence of Igμ is in agreement with the finding that the surrogate light chains λ5 and VpreB are expressed in association with an unidentified 130 kD glycoprotein on the surface of pre-BI cells before any productive V(D)J rearrangement (44). (B) Expression of the TdT gene in transgenic, Pax5-deficient pre-BI cells. Total RNA (10 μg) isolated from cultured pre-BI cells was simultaneously analyzed for TdT and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression by RNase protection assay. Only the relevant parts of the autoradiograph containing the RNase-protected fragments are shown. Pre-BI cells derived from Pax5 (−/−) bone marrow lacking (lane 2) or containing the Igμ transgene (lane 3) were compared with pre-BI cells established from the fetal liver of a control embryo (lane 1). Note that the TdT gene is not expressed in fetal B lymphopoiesis (38).