© Rockefeller University Press,
0022-1007/2003/12/1699 $5.00
The Journal of Experimental Medicine, Volume 198, Number 11, 1699-1706
Identification of a Pre-BCR Lacking Surrogate Light Chain
Yu-wen Su,
Alexandra Flemming,
Thomas Wossning,
Elias Hobeika,
Michael Reth and
Hassan Jumaa
Institute for Biology III, Albert-Ludwigs University of Freiburg, and Max Planck Institute for Immunobiology, 79108 Freiburg, Germany
Address correspondence to Hassan Jumaa, Max Planck Institute for Immunobiology, Stuebeweg 51, D-79108 Freiburg, Germany. Phone: 49-761-5108437; Fax: 49-761-5108423; email: jumaa{at}immunbio.mpg.de
 |
Abstract
|
---|
SLP-65-/- pre-B cells show a high proliferation rate in vitro. We have shown previously that
5 expression and consequently a conventional pre-B cell receptor (pre-BCR) are essential for this proliferation. Here, we show that pre-B cells express a novel receptor complex that contains a µ heavy chain (µHC) but lacks any surrogate (SL) or conventional light chain (LC). This SL-deficient pre-BCR (SL-pre-BCR) requires Ig-
for expression on the cell surface. Anti-µ treatment of pre-B cells expressing the SL-pre-BCR induces tyrosine phosphorylation of substrate proteins and a strong calcium (Ca2+) release. Further, the expression of the SL-pre-BCR is associated with a high differentiation rate toward
LC-positive cells. Given that B cell development is only partially blocked and allelic exclusion is unaffected in SL-deficient mice, we propose that the SL-pre-BCR is involved in these processes and therefore shares important functions with the conventional pre-BCR.
Key Words: B cell development adaptor signaling proliferation receptor
 |
Introduction
|
---|
The development of B cells is a highly regulated process that can be divided into distinct stages according to the expression of various surface markers and the recombination status of the heavy chain (HC) and light chain (LC) genes (1, 2). The recombination of the HC locus is initiated in pro-B cells (c-kit+B220+CD43+), which represent the earliest distinguishable B cell population (3). Productive recombination of the HC locus results in µHC expression, which pairs with the SL chain and the signaling components Ig-
/Ig-ß to form the pre-BCR on the cell surface (4, 5). The SL chain consists of the noncovalently associated VpreB and
5 polypeptides that have sequence and presumed structural homology to the Ig V- and C-type domains, respectively (6). Successful LC recombination and the subsequent association between HC and LC proteins lead to the expression of the B cell receptor (BCR) complex on the cell surface and the production of immature B cells that leave the BM and continue differentiation to become mature B cells (7, 8). Binding of the BCR to its specific ligand is an essential requirement for the selection and activation of immature and mature B cells, respectively (9). Although it is still unclear whether ligand binding is required for proper pre-BCR function, recent results indicate that the pre-BCR can react with ligand molecules on stromal cells (1012). Whereas the biological role of this pre-BCR ligand interaction is not clear at present, it is established that pre-BCR expression is crucial for pre-B cell proliferation and differentiation and therefore represents a key checkpoint in B cell development (1315). For instance, B cell development is completely blocked at the pro-B cell stage in mice deficient for Ig-
or Ig-ß (16, 17). In µMT mice that lack the transmembrane region of the µ chain, a complete block of B cell development and a loss of allelic exclusion were reported (18, 19). Mice deficient for the SL component
5, VpreB, or both showed an incomplete block of B cell development but intact allelic exclusion (2022). Since the conventional pre-BCR cannot be expressed in both the µMT mice and the SL-deficient mice, it was suggested that prematurely expressed LCs allow the expression of the µHC on the surface of SL-deficient pre-B cells (23).
Pre-BCR engagement results in activation of protein tyrosine kinases (PTKs) of the Src, Syk, and Tec families that mediate the phosphorylation of substrate proteins (24). One major substrate of Syk is the adaptor protein SLP-65, which is also known as BLNK or BASH (2527). Phosphorylated SLP-65 is a central modulator of Ca2+ response that couples Syk and the Tec family kinase Btk to phospholipase C (PLC)-
2 (2830). After phosphorylation by Syk and Btk, PLC-
2 is activated and generates the second messenger inositol 1,4,5-trisphosphate which leads to Ca2+ release from intracellular stores (31). Mice deficient for SLP-65 show a partial block of B cell development at the pre-B cell stage and, due to a defect of receptor down-regulation pre-B cells from these mice express large amounts of the pre-BCR on their surface (3234). Here, we exploit the increase of µHC expression on the surface of SLP-65/
5 double mutant (SLP-65/
5-/-) pre-B cells to characterize a novel pre-BCR complex that is expressed without any surrogate or conventional LCs.
 |
Materials and Methods
|
---|
Mice.
Ig-
-/-,
5-/-, and SLP-65-/- mice were generated as described previously (17, 20, 32). Single deficient mice were crossed to generate the SLP-65/
5-/- and SLP-65/Ig-
-/- mice. All animal experiments were performed in compliance with guidelines of the German law and the MPI for Immunobiology.
Cell Purification, Cell Culture, and Cell Lines.
Cell suspension was prepared from the murine BM and cultured in Iscove's medium containing 10% FCS (Vitromex), 100 U/ml penicillin, 100 U/ml streptomycin (GIBCO BRL), 5 x 10-5 M 2-ME, and IL-7 as described previously (35). The SLP-65/
5-/- pre-B cell line mcR2 was established by culturing BM cells in IL-7supplemented medium for extended times (>6 mo). The pre-B cell lines Dec and Oct were derived from SLP-65-/- mice and the pro-B cell line from SLP-65/Ig
-/- mice.
Flow Cytometry.
Aliquots of single cell (106) suspensions from cell culture was stained for FACS® analysis (FACSCalibur; Becton Dickinson) using FITC, cy5 or biotinanti-IgM (µ chain specific; Southern Biotechnology and Dianova), biotinanti-IgMa (Becton Dickinson), FITC or biotinanti-
(Southern Biotechnology), FITCanti-
(Southern Biotechnology), and streptavidin-cy5 (Dianova). Antimouse surrogate LC (SL-156), anti-VpreB (Vp245), and anti-
5 (LM34) rat mAbs were gifts from A.G. Rolink (University of Basel, Basel, Switzerland).
Retroviral Constructs and Transduction.
Retroviral transductions were performed as described previously (36). pMOWS-
5 vector encoding murine
5 was used for transfection performed in the PhoenixTM retroviral producer cell line using GeneJuiceTM (Novagen) according to manufacturer's instructions. For transduction, pre-B cells derived from the BM of SLP-65/
5-/- mice, which were cultured for 23 d were mixed with viral supernatants and centrifuged at 1,800 rpm at 37°C for 3 h. Transduction efficiency, measured 1 d later, was between 1 and 10%.
Southern Blotting.
Genomic DNA was digested with EcoRI at 37°C overnight. 10 µg of DNA was loaded, separated in 0.8% TAE-agarose gel, and blotted to Nylon membrane (PerkinElmer). The membrane was hybridized overnight with a P32-labeled probe corresponding to genomic DNA sequences downstream of JH4 at the µ locus as described previously (34). The results were visualized by the development with HyperfilmTM MP high performance autoradiography film (Amersham Biosciences).
In Vitro Differentiation Assay.
BM-derived cells from SLP-65-/- and SLP-65/
5-/- mice were cultured for 1 wk with IL-7 and then divided into two pools, one cultured continuously with IL-7 and the other without IL-7 for 2 d. The differentiated B cells were analyzed by FACS® using anti-µHC and anti-
LC stain.
Surface Biotinylation, Immune Purification, and Western Blot Analysis.
SLP-65/Ig-
-/- pre-B cells, SLP-65/
5-/- pre-B cells (mcR2), SLP-65-/- (Dec) pre-B cells, J558LµM3 (surface µHC and
LC positive), and WEHI-231 (surface µHC and kLC positive) were used in biotinylation experiments of surface cellular proteins. Cells (2 x 107) were washed twice with ice cold PBS. The cell pellets were resuspended in EZ-LinkTM Sulfo-NHS-Biotin (Pierce Chemical Co.) at 1 µg/ml in PBS and incubated on ice for 30 min. After PBS washing, the cell pellets were lysed in ice cold digitonin lysis buffer (50 mM Tris-HCl, pH 7.4; 0.5% digitonin [Sigma-Aldrich], 137.5 mM NaCl, 1% glycerol, 1 mM Na-orthovanadate, 0.5 mM EDTA, pH 8, and protease inhibitor cocktail [Sigma-Aldrich]) and kept on ice for 15 min. After 15 min centrifugation at 14,000 rpm and 4°C, the supernatant was collected. µHC and µHC-associated proteins were immune purified from the supernatant by incubation with goat antimouse IgM (µ chain specific; Southern Biotechnology) plus protein G sepharose beads (Amersham Biosciences) at 4°C overnight. Purified proteins were washed with PBS or lysis buffer, subjected to 12% SDS-PAGE, and blotted to ImmobilonTM PVDF transfer membrane (Millipore). The biotinylated proteins were revealed by streptavidin-HRPO (Pierce Chemical Co.) following ECL developmental systemTM (Amersham Biosciences) according to manufacturer's instructions. µHC,
LC, and
LC were detected with the same antibodies used for FACS® analysis. Ig-
was detected with rabbit polyclonal antibody (a gift from J.C. Cambier, Integrated Department of Immunology, Jewish Medical and Research Center, and University of Colorado Health Sciences Center, Denver, CO).
Cell Stimulation.
Cell pellets were incubated with 20 µg/ml of goat antimouse IgM or goat antimouse kappa (Southern Biotechnology) at 37°C for 2 min. Stimulation was stopped by the addition of ice cold PBS, and after centrifugation the cell pellets were lysed in ice cold lysis buffer (50 mM Tris-HCl, pH 7,4); 1% n-octyl-ß-D-glucopyranosid [Applichem], 137.5 mM NaCl, 1% glycerol, 1 mM Na-orthovanadate, 0.5 mM EDTA, pH 8, supplemented with protease inhibitor cocktail as described above). Tyrosine-phosphorylated cellular proteins were immune purified from the total cellular lysates by incubation with antiphosphotyrosine antibody 4G10 (Upstate Biotechnology) at 4°C overnight. The purified proteins were subjected to 10% SDS-PAGE, revealed by antiphosphotyrosine antibody (4G10, Upstate Biotechnology) followed by HRPO-labeled antimouse antibody (Pierce Chemical Co.) and developed as described above.
Ca2+ Mobilization.
Cells (5 x 106) resuspended in Iscove's medium containing 1% FCS were incubated with 5 µg/ml of Indo-1 a.m. (Molecular Probes) and 0.5 µg/ml of pluronic F-127 (Molecular Probes) at 37°C for 45 min. The cell pellets were then resuspended in Iscove's medium (containing 1% FCS) and kept on ice. Ca2+ response was induced by the addition of goat antimouse IgM (Southern Biotechnology) at a final concentration of 20 µg/ml or antimouse kappa (Southern Biotechnology) at the same concentration as a control.
 |
Results
|
---|
SLP-65/
5-/- Pre-B Cells Express a Novel µ-Containing Receptor.
BM-derived SLP-65-/- pre-B cell cultures repeatedly contained cell populations that were positive for µHC but negative for conventional LCs and the antibody SL-156 recognizing
5 in association with µ (not depicted). We postulated that µHC might be expressed on the surface of pre-B cells in the absence of
5. To test this hypothesis, we analyzed in parallel BM-derived pre-B cells from WT, SLP-65-/-,
5-/-, and SLP-65/
5-/- littermates by FACS®. At day 4 of in vitro culture, the cells were stained with SL-156, anti-
/
, and anti-µHC. No evident pre-BCR expression (µHC+/SL-156+) was detected in the WT pre-B cell culture, and most µHC+ WT cells were also
/
+ (Fig. 1
A, top). However, the SLP-65-/- pre-B cell culture contained a clear µHC+/SL-156+ pre-B cell population (74%) in contrast to the WT,
5-/-, and SLP-65/
5-/- cultures (Fig. 1 A, left). In addition,
20% of the SLP-65-/- pre-B cells were µHC+/SL-156-/
-/
-, indicating that they express the SL-pre-BCR that lacks a conventional or a surrogate LC (Fig. 1 A, second left and right panels). The µHC+/SL-156- cells in the pre-B cell cultures from
5-/- and SLP-65/
5-/- mice consisted of two distinct populations. A minor population that expressed the µHC as a part of the BCR together with a conventional LC (
or
) and a major population expressing the µHC as a part of the SL-pre-BCR. The ratio of the SL-pre-BCRexpressing cells and its amount on each cell are increased in SLP-65/
5-/- compared with
5-/- pre-B cell cultures (Fig. 1 A, bottom four panels).
To confirm that the µHC of the SL-pre-BCR is expressed on the surface as a subunit of a receptor complex, we generated mice deficient for SLP-65 and the signal transduction subunit Ig-
(SLP-65/Ig-
-/- mice). BM-derived pre-B cells from these mice showed no µHC expression on the cell surface (Fig. 1 B). To show that the SL-pre-BCR can also be detected in cells that have not been cultured in vitro, we analyzed freshly isolated BM cells from SLP-65/
5-/- and SLP-65/Ig-
-/- mice by FACS®. These experiments showed that a distinct B cell fraction (6%) in the BM of SLP-65/
5-/- but not of SLP-65/Ig-
-/- mice expressed the SL-pre-BCR (Fig. 1 C).
The SL component VpreB might associate with µHC in the absence of
5 and allow surface expression of an incomplete pre-BCR (37). To exclude this possibility, we performed FACS® analysis with anti-VpreB and show that VpreB is not associated with µHC in the SL-pre-BCR on the surface of SLP-65/
5-/- pre-B cells (Fig. 2
A, second panel). However, surface VpreB expression was easily detected when
5 was retrovirally transduced into SLP-65/
5-/- pre-B cells, and the ratio of the transduced cells increased from 9% at day 4 to 46% at day 18 posttransduction (Fig. 2 A, third panel and not depicted). Further, we analyzed the Ig HC rearrangement status in genomic DNA from WT, SLP-65-/-, and SLP-65/
5-/- pre-B cell cultures. This analysis showed that similar to WT and SLP-65-/- the DNA from SLP-65/
5-/- pre-B cells contained several submolar JH-rearranged bands, indicating that the cells were polyclonal and excluding the possibility that few clones with abnormal µH chains gave rise to surface µHC expression (Fig. 2 B). In summary, SLP-65/
5-/- and
5-/- pre-B cells express a SL-pre-BCR that cannot provide the cells with a strong selective advantage to dominate the culture, since a considerable proportion of these pre-B cells is µHC- in contrast to SLP-65-/- pre-B cells that are mostly µHC+ (Fig. 1 A). Further, the reconstitution of pre-BCR (i.e.,
5) expression in SLP-65/
5-/- pre-B cells resulted in the proliferative selection of µHC+ cells (Fig. 2 A).
The SL-pre-BCR Is Associated with Enhanced Differentiation In Vitro.
We further tested the ability of SLP-65/
5-/- pre-B cells to differentiate in vitro. 2 d after withdrawal of IL-7 to induce differentiation, we observed
LC recombination and the appearance of similar amounts of BCR+ cells in SLP-65-/- and SLP-65/
5-/- cultures (Fig. 3
, panels 12). To show that pre-B cells with a surface SL-pre-BCR are not blocked in development, we compared the in vitro differentiation of sorted µHC+ pre-B cells from SLP-65/
5-/- and SLP-65-/- cultures. After 2 d of IL-7 removal, 30 and 19% of the SLP-65/
5-/- and SLP-65-/- pre-B cells differentiated to BCR+ cells, respectively (Fig. 3, panels 34). In contrast, the SLP-65/
5-/- pre-B cells that did not express an SL-pre-BCR showed a poor differentiation capacity (Fig. 3, panel 5). This indicates that the SL-pre-BCR is capable of inducing differentiation and that the µHC of this receptor can pair with
LC. Sorted µHC- pre-B cells from SLP-65-/- cultures did not grow in culture and therefore were not analyzed.

View larger version (31K):
[in this window]
[in a new window]
|
Figure 3. The SL-pre-BCR is associated with enhanced differentiation. BM-derived pre-B cells from a 1-wk culture were incubated for 2 d in the absence or presence of IL-7 (top and bottom, respectively). Unsorted and µHC+ (µ+) and µHC- (µ-) sorted cells were analyzed in parallel. Sorted µHC- SLP-65-/- pre-B cells showed poor survival and therefore were not included. µHC versus LC FACS® profiles of the indicated genotypes are shown. Numbers refer to the percentage of cells in each quadrant.
| |
Pre-B Cell Lines Expressing the SL-pre-BCR.
Prolonged in vitro culture of BM-derived SLP-65/
5-/- pre-B cells allowed the generation of cell lines that express the SL-pre-BCR. Analysis of five independent lines showed a similar pattern of surface marker expression compared with short-term cultures and were CD19+/CD43+/CD25-/µHC+/VpreB-/
5- (Fig. 4
A and not depicted). Interestingly, the DS-1 antibody, which is specific for the IgHCa allotype present in SLP-65/
5-/- mice, did not detect the µHC on these cell lines (mcR2) and on cells from short-term culture (Fig. 4 B, top, and not depicted). Given that the DS-1 antibody detects only µHC associated with an SL or conventional LC but not free µHC (38), we conclude that the SL-pre-BCR contains a µHC without any light chain. To prove that the µHC of the SLP-65/
5-/- cells is recognizable by DS-1 after association with a light chain, IL-7 was removed for 2 d from the culture to induce
LC recombination. Indeed, all SLP-65/
5-/- cells with a surface
LC were readily detected with DS-1 (Fig. 4 B, bottom). Similar to the results of sorted µHC+ cells (Fig. 3), the SLP-65/
5-/- pre-B cells showed a higher differentiation capacity compared with SLP-65-/- pre-B cells. We used the SLP-65/
5-/- pre-B cell lines to further characterize the SL-pre-BCR. Western blot analysis revealed that the SLP-65/
5-/- pre-B cells mainly contain a complete µHC and not the truncated Dµ protein (Fig. 4 D). Biotinylation of surface proteins followed by anti-µ immune purification and streptavidin Western blot analysis confirmed the FACS® data that µHC is expressed on the surface of SLP-65/
5-/- pre-B cells (Fig. 4 C, top). Further, the µHC of both the pre-BCR and the SL-pre-BCR was associated with Ig-
, whereas VpreB was only detected in the context of the pre-BCR (Fig. 4 C, bottom). In these surface biotinylation experiments, no µHC-associated proteins were detected that might correspond to a light chain or similar molecules (not depicted).

View larger version (41K):
[in this window]
[in a new window]
|
Figure 4. Pre-B cell lines that express the SL-pre-BCR. (A) FACS® analysis of the SLP-65-/- (Dec) or SLP-65/ 5-/- (mcR2) pre-B cell lines stained for conventional pre-BCR (µ versus VpreB, top; µ versus 5, bottom). (B) FACS® analysis of Dec and mcR2 pre-B cells cultured with IL-7 (top) or without IL-7 for 2 d (middle and bottom). Cells were stained with antibody DS-1 for the µHCa allotype that detects conventional pre-BCR (Dec) but not the SL-pre-BCR (mcR2) (µ versus µa, top). After IL-7 removal, differentiation and LC expression enable the recognition of the µHCa allotype in the mcR2 cells (middle and bottom). Numbers refer to the percentage of cells in the gate. (C) Western blot analysis of protein complexes obtained with anti-µ immune purification. The surface cellular proteins of the indicated cell lines were biotinylated before cell lysis. The purified protein complexes were separated by SDS gel electrophoresis. After blotting, the membrane was incubated with streptavidin-HRPO (tol) and subsequently with antibodies recognizing µHC, LC, LC, VpreB, or Ig- . The pro-B cell line was established from BM cells of an SLP-65/Ig- -/- mouse by extended culture in IL-7. The cell lines J558LµM3 and WEHI-231 were used as controls for cells expressing a BCR with LC and LC, respectively. (D) Western blot analysis of total cellular lysates for µHC expression in the indicated cell lines.
| |
Engagement of the SL-pre-BCR Induces a Massive Ca2+ Response.
To analyze the signaling competence of the SL-pre-BCR, we compared the phosphorylation of PTK substrate proteins after anti-µ treatment of SLP-65-/- and SLP-65/
5-/- short-term cultures and pre-B cell lines. In all cell types, anti-µ treatment induced tyrosine phosphorylation of similar substrate proteins including PLC-
2, Syk, Ig-
, and the adaptor protein LAT (linker for activation of T cells) (Fig. 5
A). This confirms our recent results showing that LAT is expressed in pre-B cells and phosphorylated upon pre-BCR engagement (35). The induction of substrate tyrosine phosphorylation was lower in the short-term cultures compared with the cell lines (Fig. 5 A, lanes 16 and 712). Further, the SLP-65/
5-/- pre-B cells showed the highest induction of tyrosine phosphorylation (Fig. 5 A, lane 11). Compared with the short-term culture of SLP-65-/- pre-B cells and the SLP-65-/- pre-B cell line Dec, anti-µ treatment of the SLP-65/
5-/- short-term culture and the pre-B cell line mcR2 induced a strong Ca2+ response (Fig. 5 B). Treatment with anti-
as a control induced neither phosphorylation nor Ca2+ release (Fig. 5, A and B, middle). These results demonstrate a profound difference between the SL-pre-BCR and the conventional pre-BCR in the induction of Ca2+ release.

View larger version (38K):
[in this window]
[in a new window]
|
Figure 5. Engagement of the SL-pre-BCR induces PTK substrate phosphorylation and a massive Ca2+ release. (A) Antiphosphotyrosine Western blot analysis of proteins purified by antiphosphotyrosine precipitation. BM-derived cells from SLP-65-/- and SLP-65/ 5-/- mice cultured for 1 wk (lanes 16), pre-B cell lines Dec (lane 79) and mcR2 (lanes 1012) were left unstimulated (lanes 1, 4, 7, and 10), stimulated with 20 µg/ml anti-µ (lanes 2, 5, 8 and 11) or 20 µg/ml anti-kappa (lanes 3, 6, 9 and 12) at 37°C for 2 min. For all lanes, 1 x 107 cell equivalents were loaded. (B) BM-derived pre-B cells and cell lines (described in A) were loaded with Indo-1 and treated with anti-µ (left and right lanes) to induce Ca2+ response. Antikappa treatment was used as a negative control (middle lane).
| |
 |
Discussion
|
---|
Our experiments demonstrate that the µHC forms an Ig-
associated and signaling-competent complex on the surface of pre-B cells that lack any SL or conventional light chains. These cells can be detected in the BM of SLP-65/
5-/- mice and can be grown in vitro in IL-7supplemented medium. The SL-pre-BCR on the surface of SLP-65/
5-/- pre-B cells contains a complete µHC of the same molecular weight as the µHC from control cell lines, so that it is unlikely that deletions within the variable region or the CH1 domain of µHC lead to SL-pre-BCR expression in the absence of SL or conventional light chains. Previous reports showed that some human µH chains with special VH genes could be expressed on the cell surface in the absence of SL or conventional LC (39). These aberrant µH chains induced apoptosis and were only found in pro-B cells, but not in preB or mature B cells, indicating that pro-B cells with this µHC are negatively selected. For several reasons, we assume that a functional rather than an aberrant µHC is expressed in the SLP-65/
5-/- pre-B cells. First, SLP-65/
5-/- pre-B cells are polyclonal, arguing against the use of highly selected VH genes able to generate µHC with LC-independent expression on the cell surface. Second, no increased apoptosis was detected when µHC+ SLP-65/
5-/- pre-B cells were sorted and analyzed for differentiation. Third, the µHC+ SLP-65/
5-/- pre-B cells differentiated readily to immature B cells, whereas µHC- SLP-65/
5-/- cells showed a poor differentiation capacity, demonstrating that the µH chains do not block but rather promote development. It is unlikely that the µH chains of the sorted µHC+ SLP-65/
5-/- pre-B cells are replaced during differentiation, since this would suggest that replacement in the µHC+ cells is more frequent than de novo recombination in µHC- cells.
We reported previously that SLP-65-/- pre-B cells show an increased proliferation capacity that can lead to pre-B cells tumors (33, 34). Since the SLP-65/
5-/- pre-B cells showed a decreased proliferation capacity compared with SLP-65-/- pre-B cells, we concluded that the high proliferation rate of SLP-65-/- pre-B cells in vitro required the expression of the conventional pre-BCR (33). In the present study, we show that reconstitution of
5 expression in SLP-65/
5-/- pre-B cells lead to the enrichment of cells with a conventional pre-BCR. Thus, the SL-pre-BCR on SLP-65/
5-/- pre-B cells does not seem to induce cell proliferation. In full agreement with this, no pre-B cell tumors were observed in SLP-65/
5-/- mice (>500).
We have shown recently that the adaptor protein LAT is involved in pre-BCR signaling and in the rescue of SLP-65-/- pre-B cells (35). SLP-65/
5-/- pre-B cells express high amounts of LAT, which explains their capability to mobilize Ca2+ in the absence of SLP-65. However, it is currently unclear why receptor engagement induces a strong Ca2+ response in SLP-65/
5-/- pre-B cells compared with SLP-65-/- pre-B cells.
In a recent study, Ohnishi and Melchers (40) demonstrated that mutations in the non-Ig portion of
5 lead to increased pre-BCR accumulation on the cell surface, suggesting that
5 controls the internalization of the pre-BCR. Further, they showed that the same
5 mutations abolished the constitutive, cell autonomous signaling capacity of the pre-BCR. However, the signaling capacity of these mutant pre-BCR molecules was restored after cross-linking with anti-µ, suggesting that
5 is involved in the cross-linking of the pre-BCR on the surface of pre-B cells (40). These results indicate that, in the uninduced situation, the SL-pre-BCR may be less efficiently cross-linked and internalized than the conventional pre-BCR and that cross-linking of the pre-BCR is required for the proliferation but not the differentiation of pre-B cells.
However, in BM-derived pre-B cells from WT mice we could not detect conventional pre-BCR or SL-pre-BCRexpressing cells, and most µHC+ cells in these cultures were also
LC+, indicating that they were immature B cells. We assume that defective receptor internalization due to a deficiency in SLP-65,
5, or both simply allows the detection rather than the formation of both the conventional pre-BCR and the SL-pre-BCR.
Although the exact role and significance of the SL-pre-BCR are not fully clear, this receptor may explain previous results. For instance, B cell development is only partially blocked in
5-/- mice, which is in contrast to the complete developmental block in µMT mice lacking the transmembrane region of µHC (18). In both cases, no expression of a conventional pre-BCR and a block in differentiation are expected. However, the
5-/- pre-B cells bypass this block, indicating that the µHC forms alternative complexes in the absence of an SL. According to previous reports, prematurely expressed conventional light chains could pair with µHC and compensate the loss of the SL chain in
5-/- pre-B cells (23). However, this rescue of SL deficiency required an early onset of
LC expression, which might be unlikely because kLC expression is largely restricted to cells at later stages of B cell development (23). We propose that in
5-/- pre-B cells, an SL-pre-BCR may induce differentiation of these cells and explain the difference between the
5-/- and µMT mice. In addition, the fact that the µHC forms an SL-pre-BCR on pre-B cells may also explain why allelic exclusion still operates in SL chain deficient but not in µMT mice (19, 22).
Normally, a µHC unpaired with an SL or a conventional LC is retained in the ER by the chaperone BiP (41). BiP binds to the unpaired CH1 domain of µHC and retains it in the ER until an SL or a conventional LC displaces BiP and allows the transport of the completely assembled receptor to the cell surface. Our experiments with surface biotinylation did not detect additional proteins that might associate with µHC and allow its dissociation from BiP. The staining experiments with the µHC-allotypespecific antibody (DS-1), which recognizes µHCa only when it is associated with an SL or a conventional LC, suggest that µHC is expressed on the surface without associated proteins. It is currently unclear how the µHC can circumvent the BiP retention control in SLP-65/
5-/- pre-B cells. A better understanding of the SL-pre-BCR should help to clarify the requirements for surface µHC expression, allelic exclusion, pre-B cell proliferation, and differentiation.
 |
Acknowledgments
|
---|
We thank Peter J. Nielsen for discussions; Andreas Wuerch and Hubertus Kohler for Ca2+ measurement; and Cathrin Eschbach and Ingrid Fidler for technical help.
This work was supported by a grant from the Ministry of Science, Research and Arts of Baden-Württemberg to M. Reth and by the Deutsche Forschungsgemeinschaft (SFB 620).
Submitted: August 20, 2003
Accepted: October 15, 2003
 |
Footnotes
|
---|
Y.-w. Su and A. Flemming contributed equally to this work.
Abbreviations used in this paper: BCR, B cell receptor; HC, heavy chain; LAT, linker for activation of T cells; LC, light chain; PLC, phospholipase C; PTK, protein tyrosine kinase; SL, surrogate light chain.
 |
References
|
---|
- Osmond, D.G., A. Rolink, and F. Melchers. 1998. Murine B lymphopoiesis: towards a unified model. Immunol. Today. 19:6568.[CrossRef][Medline]
- Hardy, R.R., C.E. Carmack, S.A. Shinton, J.D. Kemp, and K. Hayakawa. 1991. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:12131225.[Abstract]
- Benschop, R.J., and J.C. Cambier. 1999. B cell development: signal transduction by antigen receptors and their surrogates. Curr. Opin. Immunol. 11:143151.[CrossRef][Medline]
- Tenboekel, E., F. Melchers, and A.G. Rolink. 1998. Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression. Immunity. 8:199207.[Medline]
- Karasuyama, H., A. Rolink, Y. Shinkai, F. Young, F.W. Alt, and F. Melchers. 1994. The expression of Vpre-B/lambda 5 surrogate light chain in early bone marrow precursor B cells of normal and B cell-deficient mutant mice. Cell. 77:133143.[Medline]
- Burrows, P.D., R.P. Stephan, Y.H. Wang, K. Lassoued, Z.X. Zhang, and M.D. Cooper. 2002. The transient expression of pre-B cell receptors governs B cell development. Semin. Immunol. 14:343349.[CrossRef][Medline]
- Rajewsky, K. 1996. Clonal selection and learning in the antibody system. Nature. 381:751758.[CrossRef][Medline]
- Rolink, A.G., F. Melchers, and J. Andersson. 1999. The transition from immature to mature B cells. Curr. Top. Microbiol. Immunol. 246:3943.[Medline]
- Pike, K.A., and M.J.H. Ratcliffe. 2002. Cell surface immunoglobulin receptors in B cell development. Semin. Immunol. 14:351358.[CrossRef][Medline]
- Gauthier, L., B. Rossi, F. Roux, E. Termine, and C. Schiff. 2002. Galectin-1 is a stromal cell ligand of the pre-B cell receptor (BCR) implicated in synapse formation between pre-B and stromal cells and in pre-BCR triggering. Proc. Natl. Acad. Sci. USA. 99:1301413019.[Abstract/Free Full Text]
- Bradl, H., and H.M. Jack. 2001. Surrogate light chain-mediated interaction of a soluble pre-B cell receptor with adherent cell lines. J. Immunol. 167:64036411.[Abstract/Free Full Text]
- Rolink, A.G., T. Winkler, F. Melchers, and J. Andersson. 2000. Precursor B cell receptordependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment. J. Exp. Med. 191:2331.[Abstract/Free Full Text]
- Martensson, I.L., A. Rolink, F. Melchers, C. Mundt, S. Licence, and T. Shimizu. 2002. The pre-B cell receptor and its role in proliferation and Ig heavy chain allelic exclusion. Semin. Immunol. 14:335342.[CrossRef][Medline]
- Conley, M.E., J. Rohrer, L. Rapalus, E.C. Boylin, and Y. Minegishi. 2000. Defects in early B-cell development: comparing the consequences of abnormalities in pre-BCR signaling in the human and the mouse. Immunol. Rev. 178:7590.[CrossRef][Medline]
- Melchers, F., E. ten Boekel, T. Seidl, X.C. Kong, T. Yamagami, K. Onishi, T. Shimizu, A.G. Rolink, and J. Andersson. 2000. Repertoire selection by pre-B-cell receptors and B-cell receptors, and genetic control of B-cell development from immature to mature B cells. Immunol. Rev. 175:3346.[CrossRef][Medline]
- Gong, S., and M.C. Nussenzweig. 1996. Regulation of an early developmental checkpoint in the B cell pathway by Ig beta. Science. 272:411414.[Abstract]
- Pelanda, R., U. Braun, E. Hobeika, M.C. Nussenzweig, and M. Reth. 2002. B cell progenitors are arrested in maturation but have intact VDJ recombination in the absence of Ig-alpha and Ig-beta. J. Immunol. 169:865872.[Abstract/Free Full Text]
- Kitamura, D., J. Roes, R. Kuhn, and K. Rajewsky. 1991. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature. 350:423426.[CrossRef][Medline]
- Kitamura, D., and K. Rajewsky. 1992. Targeted disruption of mu chain membrane exon causes loss of heavy-chain allelic exclusion. Nature. 356:154156.[CrossRef][Medline]
- Kitamura, D., A. Kudo, S. Schaal, W. Muller, F. Melchers, and K. Rajewsky. 1992. A critical role of lambda 5 protein in B cell development. Cell. 69:823831.[Medline]
- Mundt, C., S. Licence, T. Shimizu, F. Melchers, and I.L. Martensson. 2001. Loss of precursor B cell expansion but not allelic exclusion in VpreB1/VpreB2 double-deficient mice. J. Exp. Med. 193:435445.[Abstract/Free Full Text]
- Shimizu, T., C. Mundt, S. Licence, F. Melchers, and I.L. Martensson. 2002. VpreB1/VpreB2/lambda 5 triple-deficient mice show impaired B cell development but functional allelic exclusion of the IgH locus. J. Immunol. 168:62866293.[Abstract/Free Full Text]
- Pelanda, R., S. Schaal, R.M. Torres, and K. Rajewsky. 1996. A prematurely expressed Ig-kappa transgene, but not a V-kappa-J-kappa gene segment targeted into the Ig-kappa locus, can rescue B cell development in lambda-5-deficient mice. Immunity. 5:229239.[Medline]
- Tamir, I., and J.C. Cambier. 1998. Antigen receptor signalingintegration of protein tyrosine kinase functions. Oncogene. 17:13531364.[CrossRef][Medline]
- Wienands, J., J. Schweikert, B. Wollscheid, H. Jumaa, P.J. Nielsen, and M. Reth. 1998. SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation. J. Exp. Med. 188:791795.[Abstract/Free Full Text]
- Fu, C., C.W. Turck, T. Kurosaki, and A.C. Chan. 1998. BLNKa central linker protein in B cell activation. Immunity. 9:93103.[Medline]
- Goitsuka, R., Y. Fujimura, H. Mamada, A. Umeda, T. Morimura, K. Uetsuka, K. Doi, S. Tsuji, and D. Kitamura. 1998. BASH, a novel signaling molecule preferentially expressed in B cells of the bursa of Fabricius. J. Immunol. 161:58045808.[Abstract/Free Full Text]
- Ishiai, M., M. Kurosaki, R. Pappu, K. Okawa, I. Ronko, C. Fu, M. Shibata, A. Iwamatsu, A.C. Chan, and T. Kurosaki. 1999. BLNK required for coupling Syk to PLC gamma 2 and Rac1-JNK in B cells. Immunity. 10:117125.[Medline]
- Su, Y.W., Y. Zhang, J. Schweikert, G.A. Koretzky, M. Reth, and J. Wienands. 1999. Interaction of SLP adaptors with the SH2 domain of Tec family kinases. Eur. J. Immunol. 29:37023711.[CrossRef][Medline]
- Hashimoto, S., A. Iwamatsu, M. Ishiai, K. Okawa, T. Yamadori, M. Matsushita, Y. Baba, T. Kishimoto, T. Kurosaki, and S. Tsukada. 1999. Identification of the SH2 domain binding protein of Bruton's tyrosine kinase as BLNKfunctional significance of Btk-SH2 domain in B-cell antigen receptor-coupled calcium signaling. Blood. 94:23572364.[Abstract/Free Full Text]
- Kurosaki, T., A. Maeda, M. Ishiai, A. Hashimoto, K. Inabe, and M. Takata. 2000. Regulation of the phospholipase C-gamma 2 pathway in B cells. Immunol. Rev. 176:1929.[CrossRef][Medline]
- Jumaa, H., B. Wollscheid, M. Mitterer, J. Wienands, M. Reth, and P.J. Nielsen. 1999. Abnormal development and function of B lymphocytes in mice deficient for the signaling adaptor protein SLP-65. Immunity. 11:547554.[Medline]
- Flemming, A., T. Brummer, M. Reth, and H. Jumaa. 2003. The adaptor protein SLP-65 acts as a tumor suppressor that limits pre-B cell expansion. Nat. Immunol. 4:3843.[CrossRef][Medline]
- Jumaa, H., L. Bossaller, K. Portugal, B. Storch, M. Lotz, A. Flemming, M. Schrappe, V. Postila, P. Riikonen, J. Pelkonen, et al. 2003. Deficiency of the adaptor SLP-65 in pre-B-cell acute lymphoblastic leukaemia. Nature. 423:452456.[CrossRef][Medline]
- Su, Y.-w., and H. Jumaa. 2003. LAT links the pre-BCR to calcium signaling. Immunity. 19:295305.[Medline]
- Ketteler, R., S. Glaser, O. Sandra, U.M. Martens, and U. Klingmuller. 2002. Enhanced transgene expression in primitive hematopoietic progenitor cells and embryonic stem cells efficiently transduced by optimized retroviral hybrid vectors. Gene Ther. 9:477487.[CrossRef][Medline]
- Seidl, T., A. Rolink, and F. Melchers. 2001. The VpreB protein of the surrogate light-chain can pair with some mu heavy-chains in the absence of the lambda 5 protein. Eur. J. Immunol. 31:19992006.[CrossRef][Medline]
- Stall, A.M. 1996. Mouse immuoglobulin allotypes. Weir's Handbook of Experimental Immunology. 5th edition. L.A. Herzenberg, D.M. Weir, L.A. Herzenberg, and C. Blackwell, editors. Blackwell Science Publishers, Boston, MA. 116.
- Minegishi, Y., and M.E. Conley. 2001. Negative selection at the pre-BCR checkpoint elicited by human mu heavy chains with unusual CDR3 regions. Immunity. 14:631641.[CrossRef][Medline]
- Ohnishi, K., and F. Melchers. 2003. The nonimmunoglobulin portion of
5 mediates cell-autonomous pre-B cell receptor signaling. Nat. Immunol. 4:849856.[CrossRef][Medline]
- Haas, I.G., and M. Wabl. 1983. Immunoglobulin heavy chain binding protein. Nature. 306:387389.[Medline]