Stochastic pairing of Ig heavy and light chains frequently generates B cell antigen receptors that are subject to editing in vivo

Tatiana Novobrantseva1,3, Shengli Xu2, Joy En-Lin Tan2, Mitsuo Maruyama1,4, Stephan Schwers1,5, Roberta Pelanda1,6 and Kong-Peng Lam1,2

1 Institute for Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany
2 Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
3 Present address: Biogen Idec, MA 02142, USA
4 Present address: National Institute for Longevity Sciences, 474-8522 Aichi, Japan
5 Present address: Bayer Healthcare AG, 51368 Leverkusen, Germany
6 Present address: National Jewish Medical and Research Center, Denver, CO 80206, USA

Correspondence to: K.-P. Lam; E-mail: mcblamkp{at}imcb.nus.edu.sg


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We examined the generation and selection of the B cell antibody repertoire through crossing of mice bearing distinct Ig heavy (H) and light (L) chain rearranged variable region transgenes. Ig gene knock-in and transgenic mice whose H and L chains pair to form a non-autoreactive, functional B cell antigen receptor (BCR) have significantly reduced pre-B cells in the bone marrow as their B cell progenitors rapidly differentiate into surface IgM+ B cells. The presence of a pre-B cell compartment in these Ig transgenic mice, however, indicates the induction of receptor editing. Here, 18 distinct combinations of H and L chains were generated that we showed could pair in vitro to form BCRs of unknown specificities. Of these, nine induced receptor editing in vivo as evidenced by the presence of pre-B cells and endogenous L chain rearrangements in mice bearing these H and L chain transgenes. These data thus suggest that about half of the emerging antibody repertoire is negatively selected during B lymphopoiesis due to the likely encoding of autoreactive or non-functional BCRs.

Keywords: antibody repertoire, autoimmunity, B cell development, gene targeting, receptor editing


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During development, B lymphocytes rearrange the various gene segments termed variable (V), D and J for the Ig heavy (H) chain locus and V and J for the light (L) chain {kappa} or {lambda} locus (1). This combinatorial gene rearrangement process makes available an enormous number of H and L chain V regions from a limited number of germ line gene segments for the formation of the B cell antibody repertoire. Since each antibody molecule is made up of H and L chains that together determine its specificity, the random association of the H and L chains further contributes to the diversity of the antigen-binding sites that are being generated (2).

The fully assembled Ig is expressed on the surface of a B lymphocyte as a B cell antigen receptor (BCR) (3) and each B cell expresses a BCR of a distinct specificity (2). The random processes of Ig gene rearrangement and H and L chain association inevitably generate BCRs recognizing self-antigens. Studies with transgenic mice that express autoreactive antibodies indicate that two basic mechanisms of negative selection of autospecific antibodies operate to maintain tolerance in the B cell lineage (4). The first mechanism is receptor editing in which autoreactive B cells in the bone marrow attempt to re-express an innocent BCR through secondary Ig gene rearrangements, mainly at the L chain loci (57). If the cells fail in this process, they are clonally deleted by apoptosis or rendered unresponsive (810).

A possible second role for receptor editing emerged from experiments in which hemi- and homozygous mice expressing 3-83 Ig transgene reacting against MHC class I molecules were compared: receptor editing might serve the purpose of substituting functionally incompetent BCRs (11, 12).

An analysis of the Ig{kappa} loci in {lambda}+ IgM-expressing B cells in mice indicated that 47% of the recombining sequence-inactivated V{kappa}J{kappa} joints were in-frame, suggesting that BCR editing occurs frequently during normal B cell development (13). Recently, an Ig{kappa} allele that harbors a human C{kappa} gene segment allowing direct quantification of cells losing the initial L chain gene rearrangement in the periphery was combined with several transgenic H chains in the mouse (14). Out of five H and L chain combinations studied, three were found to induce editing (14, Table 2B), in good agreement with the 47% estimate described above (13).


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Table 2. Pre-B cell compartment in various H and L chain transgenic mice

 
We have previously shown that the bone marrow pre-B cell compartment is the site of receptor editing in mice bearing autoantibody transgenes (15). Pre-B cell compartment is virtually absent in transgenic mice whose H and L chain pair to form a non-autoreactive, functional BCR (15), probably reflecting the rapid differentiation of B cell progenitors into immature IgM+ B cells. This phenomenon provides us with a means to evaluate whether the association of a given H and L chain generates an innocent or an autoreactive/non-functional BCR in vivo. Thus, another approach to estimate the fraction of the BCRs that is edited during B cell development is to randomly inter-breed a number of Ig transgenic mice bearing distinct H and L chain V region genes and to determine the size of pre-B cell compartment. Random combination of H and L chains would mimic the stochastic pairing of Ig H and L chains that occurs during normal B cell development. Here, we have produced 18 strains of Ig H chain knock-in and L chain knock-in or transgenic mice bearing BCRs of unknown specificities to assess the fraction of the BCRs that will be expressed on peripheral B cells in vivo.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
The generation of the B1-8f (16), glD42i (17), T15i (18), 3-83Hi (15) and VHPEf (19) H chain insertion mice and the 3-83{kappa}i (20) and D23{kappa}i (21) L chain insertion mice has been described previously. The generation of the VH12 knock-in allele (Lam and Rajewsky, unpublished data) mice will be described elsewhere. The V{kappa}4 conventional transgenic mouse strain (22) was obtained from Stephen Clarke (University of North Carolina, Chapel Hill). Ig knock-in/transgenic mice generated in this study were heterozygous at the H and L chain loci with the exception of mice bearing the T15 H chain which were homozygous for the T15 insertion. Mice were of mixed genetic background and analyzed between 6 and 10 weeks of age.

Flow cytometry
Single-cell suspensions were obtained from the spleen and bone marrow of mice as described (23) and stained with fluorochrome (FITC, PE or CyChrome) or biotin-conjugated antibodies for flow-cytometric analyses on a FACScan or FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA). Biotin-conjugated mAbs were revealed with streptavidin–CyChrome. The following mAbs were used in this study: R33-24-12 (anti-IgM), R33-18-10 (anti-{kappa}), LS136 (anti-{lambda}1), Ac146 (anti-VHB1-8), S7 (anti-CD43) and RA3-6B2 (anti-B220).

Transfection of P3X63Ag8.653 cells
The 3-83{kappa} and D23{kappa} (21) VJs were cloned into the expression plasmids pSVEneo and pE{kappa}A20/44 (24), respectively. The V{kappa}4 construct (22) was obtained from Stephen Clarke (Chapel Hill) and used without any modification. The B1-8, VH12, VHPE, 3-83H and glD42 VDJs were cloned separately into the plasmid pEVHC{gamma}1 (25) that expressed these H chains as secreted {gamma}1 proteins. Linearized plasmids bearing H and L chain genes were co-transfected in various combinations into the P3X63Ag8.653 myeloma cells (26) by electroporation at 250 V, 950 µF. Selection for cells that had incorporated both the H and L chain-containing plasmids was carried out in the presence of 0.25 µg mycophenolic acid ml–1 and 500 µg G418 ml–1. In the transient transfection studies, H chain-encoding plasmids were electroporated into P3X63 cells that had already incorporated a D23{kappa} L chain construct (21). Transfected cells were not sub-cloned prior to the assay for the presence of secreted antibodies.

ELISA
To test for the association of H and L chains in vitro, ELISA plates were coated overnight at 4°C with 5 µg/ml of anti-mouse {gamma}1 (A85-1; rat IgG1, {kappa}) antibodies. Plates were subsequently washed three times with 0.5% Tween-20 in PBS and blocked with 1% BSA in PBS for 2 h at room temperature. Thereafter, serial dilutions of control mouse {gamma}1 antibody (MOPC-21; IgG1, {kappa}) and supernatants from stably or transiently transfected cell clones were added to the wells and incubated for 2 h or overnight. After washing, HRP-coupled anti-mouse {kappa} (R8-140, rat IgG1, {kappa}) antibody was used for detection. Plates were developed with ImmunoPure TMB substrate (Pierce, Rockford, IL, USA) and read in an ELISA reader at 450 nm. Antibodies used as standard, capture and detection in the ELISA were purchased from BD Pharmingen (San Diego, CA, USA).

Southern blot analysis of transgenic splenic B cells
Splenic B cells were obtained by positive selection using MACS (Miltenyi, Bergisch Gladbach, Germany) with anti-B220 or anti-IgM mAb-coupled magnetic beads. The purity of B cells obtained was >90% as assessed by anti-B220 and anti-IgM mAb staining in FACS analysis. Genomic DNA was prepared, digested with SacI and fractionated on a 1% agarose gel. Analysis of the wild-type {kappa} locus was performed using the EcoRI–EcoRI probe as shown in Fig. 2 and by Pelanda et al. (15).



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Fig. 2. Pairing of H and L chains in vitro. ELISA of culture supernatants obtained from P3X63 cells co-transfected with the various H and (A) V{kappa}4, (B) 3-83{kappa} or (C) D23{kappa} L chains. The graphs show the data obtained from representative clones that were transfected with individual combinations of H and L chains. The MOPC-21 ({gamma}, {kappa}) mAb (at 120, 60, 30, 10 and 4 ng ml–1) was used as the positive control, whereas culture supernatants from non-transfectants or single H or L chain transfectants were used as the negative control in the ELISA. Transfected cells were not sub-cloned prior to the assay.

 
Stromal cell cultures
Bone marrow cells were seeded onto a layer of mitomycin C-inactivated ST2 stromal cells and cultured in DMEM with 15% FCS and 50 ng ml–1 of IL-7 (Pharmingen) for 5 days. Subsequently, the non-adherent cells were harvested and re-plated onto a fresh layer of ST2 cells in the absence of IL-7. After a further 4 days of culture, the non-adherent cells were analyzed by flow cytometry.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Ig knock-in and transgenic mice expressing H and L chains that form an innocent BCR virtually lack pre-B cells in the bone marrow
We have previously shown that Ig knock-in mice bearing a targeted VDJ rearrangement derived from a B1-8 antibody and a 3-83{kappa} L chain have almost undetectable levels of pre-B cells in the bone marrow and produce an essentially monoclonal population of peripheral B cells in which these H and L chains are expressed (15). This suggests that B cell precursors that express pre-rearranged H and L chains can rapidly differentiate into IgM+ B cells. The B1-8 H and 3-83{kappa} L chains encode an antibody of unknown but apparently innocuous specificity. In contrast, mice expressing 3-83H and 3-83{kappa} L chains that form a BCR that recognizes MHC class I molecules have a normal-size pre-B cell compartment in the bone marrow where editing of the autoreactive BCR occurs (15).

To test if validity of this observation goes beyond one specific BCR, we analyzed another mouse strain that harbors targeted rearranged H and L chain transgenes: VH12 H and V{kappa}4 L chains. In contrast to the B1-8 H and 3-83{kappa} L chain combination that encodes an antibody of unknown specificity, the VH12 and V{kappa}4 chains together encode an antibody that recognizes phosphatidyl choline (22). B cells that express this specificity are positively selected and maintained in the peripheral immune system as B1 cells (22). As shown in Fig. 1(A), flow-cytometric analyses revealed the presence of a population of B220+ IgM pre-B cells in the bone marrow of either the wild-type, VH12 insertion or V{kappa}4 transgenic mice. However, this population of pre-B cells was 6- to 10-fold reduced in VH12/V{kappa}4 transgenic mice compared with the control single-Ig transgenic or wild-type animals. This is consistent with the initial observation involving the B1-8H/3-83{kappa} mice (15). Thus, transgenic B cell precursors that express functional H and L chain combinations can indeed bypass the pre-B cell stage of development and directly differentiate into immature IgM+ B cells. This phenomenon, i.e. the lack of a normal-size pre-B cell compartment in the bone marrow of Ig transgenic mice, thus provides us with a means to determine whether a particular H and L chain combination encodes a functional BCR that is not subject to receptor editing and promotes further B cell development.



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Fig. 1. Bone marrow B cell development in tg mice bearing distinct H and L chain V region. (A) Lack of pre-B cells in the bone marrow of mice bearing VH12 and V{kappa}4 transgenes. FACS plots depicting the B220 versus IgM profiles of total (upper panel) and B220 versus CD43 profiles of IgM-negative (lower panel) bone marrow cells from wild-type, V{kappa}4tg, VH12f/+ and VH12f/+, V{kappa}4tg mice are shown. Cells were stained with FITC-anti-IgM, PE-anti-B220 and bio-anti-CD43 mAbs. (B) FACS plots depicting the B220 versus IgM profiles of bone marrow cells derived from B1-8f/+, V{kappa}4tg; 3-83Hi/+, V{kappa}4tg and glD42i/+, V{kappa}4tg; VHPE/+, V{kappa}4tg and T15i/+ with T15i/T15i, V{kappa}4tg mice. Cells were stained with FITC-anti-IgM and PE-anti-B220 mAbs. Numbers indicate the percentage of total lymphocytes. (C) IgM-negative bone marrow cells from Ig-insertion mice bearing distinct H chain V region and 3-83{kappa} L chain genes. FITC-anti-IgM, PE-anti-B220 and biotin-anti-CD43 mAbs were used for the staining. Numbers indicate the percentage of IgM-negative lymphocytes. Mice bearing the H and L chain combinations 3-83Hi/3-83{kappa} and VHPE/3-83{kappa} are not shown. (D) Bone marrow B cell development in various Ig H, D23{kappa} insertion mice. IgM-negative bone marrow cells stained with PE-anti-IgM, CyChrome-anti-B220 and FITC-anti-CD43 mAbs are shown. Numbers indicate the percentage of IgM-negative lymphocytes.

 
BCRs encoded by some H and L chain combinations do not promote B cell development in vivo
To determine the fraction of H and L chain combinations that would promote B cell development in vivo, we inter-bred various gene-targeted H and L chain transgenic mice that were generated in the laboratory. We surmised that the random crossing of the various H and L chain transgenic mice would mimic the random pairing of the Ig H and L chains that occurs during the generation of the B cell antibody repertoire in vivo. It is worth noticing that all Ig chains used in the study were selected to the periphery by murine immune system.

The H chain insertion mice used in this study are B1-8f (16), VH12f (27), VHPEf (19), glD42Hi (17), T15i (18) and 3-83Hi (15); the L chain transgenic mice used are 3-83{kappa}i (20) and D23{kappa}i (21). A V{kappa}4 conventional transgenic mouse (22) is also included in this study. A list of these transgenic mice with the description of the individual H and L chains and the specificities of the BCRs they are derived from is shown in Table 1. Mice used in the present study were heterozygous at the H and L chain loci with the exception of the T15i mice in which the H chain was bred to homozygosity because of the relative instability of the targeted T15 allele (18). In total, 18 strains of H and L chain double-transgenic mice bearing BCRs of mostly unknown specificities were generated.


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Table 1. Origin and specificity of VHDHJH and V{kappa}J{kappa} in tg mice

 
The various H chain insertion mice were first bred with the V{kappa}4 transgenic (tg) mice. As a result, mice bearing B1-8/V{kappa}4, 3-83H/V{kappa}4, VHPE/V{kappa}4 and T15/V{kappa}4 (Fig. 1B) have reduced numbers of pre-B cells in their bone marrow. The B220+ IgM pre-B cells in these mice are reduced ~10- and 5-fold compared with mice bearing an H or the V{kappa}4 chain alone, respectively. This is similar to the situation found in VH12/V{kappa}4 mice whose H and L chain encode a BCR of a known specificity that can promote B cell development in vivo. In contrast, mice bearing glD42H/V{kappa}4 (Fig. 1B) possess a pre-B cell compartment in the bone marrow that is comparable in size to that of the control mice that harbor only the glD42 H chain. Thus, this latter H and L chain combination apparently was edited during B lymphopoiesis in vivo.

Analyses of the crosses of the various H chain transgenic mice with the 3-83{kappa} L chain insertion mice are depicted in Fig. 1(C) where the IgM cells in the bone marrow are gated for the expression of B220 and CD43 to phenotypically identify pre-B cells. Similar to the B1-8/3-83{kappa} mice that we had analyzed previously (15) and re-capitulated here, mice bearing glD42H/3-83{kappa} also have a significantly reduced population of B220+ CD43 IgM pre-B cells in their bone marrow. The pre-B cells in glD42H/3-83{kappa} mice were reduced ~5-fold compared with either glD42H or 3-83{kappa}i mice. However, normal numbers of pre-B cells are present in mice bearing VH12/3-83{kappa}, T15/3-83{kappa} or VHPE/3-83{kappa} (data not shown), similar to the situation in mice bearing the autoreactive 3-83H/3-83{kappa} (15). Thus, the BCRs encoded by these latter H and L chain combinations are apparently not readily selected into the periphery in vivo.

Finally, the various H chain insertion mice were crossed with the D23{kappa} L chain insertion mice. The D23{kappa} L chain is derived from a ‘natural’ polyreactive antibody (28). Flow-cytometric analyses depicted in Fig. 1(D) indicated that mice bearing 3-83H/D23{kappa}, glD42H/D23{kappa}, VHPE/D23{kappa} or T15/D23{kappa} also possess a normal-size population of B220+ CD43 pre-B cells in their bone marrow, in contrast to mice that express B1-8/D23{kappa} or VH12/D23{kappa}. Thus, the pairings of the various H chains with the D23{kappa} L chains again generate some BCRs that induce editing in vivo.

A summary of the various crosses of the H and L chain transgenic mice and an indication of the presence or absence of a normal-sized pre-B cell compartment in the bone marrow of these mice are depicted in Table 2(A). Of the 18 BCRs of mostly unknown specificities that are generated through the random pairing of H and L chains brought about by the random crossing of Ig transgenic mice, nine seem to be unable to drive B lymphopoiesis in vivo as evidenced by the presence of a normal-sized pre-B cell compartment in mice bearing these H and L chains.

The inability of certain H and L chains to promote B cell development in vivo is not due to their inability to pair with each other
The inability of certain H and L chain combinations to promote B cell development in vivo may be due to their inability to pair with each other to form a BCR. Alternatively and more likely, the association of certain H and L chains may generate a BCR of autoreactive specificity or a functionally incompetent receptor (11, 12) and cells bearing these BCRs are counter-selected as was shown previously for mice bearing 3-83H/3-83{kappa} (15). To distinguish between pairing and autoreactivity/signaling incompetence, we performed transfection studies to test the association of these H and L chains in cultured cells.

The various H and L chains were cloned into C{gamma}1 and C{kappa} expression vectors, respectively, and co-transfected into the Ig-less P3X63Ag8.653 plasmacytoma cells. Supernatants from clones that had been transiently transfected or that had stably integrated the co-transfected DNA constructs were assayed in a sandwich ELISA using the anti-C{gamma}1 antibody as capture and anti-{kappa} antibody as detection reagents.

In the first set of transfection studies as depicted in Fig. 2(A), the glD42H chain was co-transfected with the V{kappa}4 L chain to determine pairing of these two chains in vitro. Supernatant from a cell clone that had been transfected with this H and L chain combination shows a titration pattern in ELISA that is indistinguishable from supernatants obtained from the clones transfected with H and L chains encoding BCRs that did not induce editing in vivo, namely VH12/V{kappa}4, 3-83H/V{kappa}4 (Fig. 1) and VHPE/V{kappa}4 (data not shown). Thus, the ELISA data indicate that glD42 H and V{kappa}4 L chains could associate with each other to form an intact antibody.

Similarly, H and L chain combinations such as T15/3-83{kappa}, VH12/3-83{kappa} and VHPE/3-83{kappa} that were replaced during B maturation in vivo could also pair in vitro as shown by cell transfection studies and ELISA (Fig. 2B). The 3-83H/3-83{kappa} pair that is known to encode a functional BCR with specificity for an MHC class I antigen (13) and the glD42/3-83{kappa} combination that could drive B lymphopoiesis in vivo (Fig. 1C) served as positive controls in this second series of transfection studies.

Finally, the various H chains were transiently transfected into P3X63 cells that had already stably integrated a D23{kappa} L chain gene. Again, the ELISA data suggest that the H and L chain combinations such as 3-83H/D23{kappa}, glD42H/D23{kappa} and VHPE/D23{kappa} in which the inserted D23{kappa} chain is replaced in peripheral B cells (data not shown) could associate to form intact antibodies in vitro (Fig. 2C).

Taken together, these data indicate that all H and L chains employed in this study can pair with each other at least in the form of secreted antibody, in agreement with previous studies suggesting that virtually all randomly chosen H/L chains (2931) associate. Secreted antibody production suggests that the H and L chains would also be capable of forming BCR complexes on the B cell surface. Thus, the inability of certain H and L chain combinations to promote B cell development in vivo is likely not due to their inability to pair.

Induction of endogenous {kappa} L chain rearrangements in Ig H and L chain transgenic mice with a pre-B cell compartment
The presence of pre-B cells in transgenic mice whose H and L chains are able to pair in vitro suggests that editing of the BCR occurs in vivo. To confirm that receptor editing indeed takes place, we examined the wild-type {kappa} allele in mice bearing the various H and 3-83{kappa} L chain transgenes using a strategy that we had previously employed for the analysis of the autoreactive 3-83H/3-83{kappa} mice (15). The configurations of the wild-type and targeted {kappa} alleles are shown in Fig. 3(A). Rearrangement of the upstream V{kappa} genes to the downstream J{kappa} elements on the wild-type {kappa} locus results in the elimination of the gene segment and the disappearance of the SacI–SacI band as detected by the indicated probe in a Southern blot analysis. As seen in Fig. 3B, Southern blot analysis indicated the absence of or severe reduction in the intensity of the germ line {kappa} band in DNA isolated from purified splenic B cells of VH12f/+, 3-83{kappa}i (lane 3); T15i/T15i, 3-83{kappa}i/+ (lane 4) and VHPEf/+, 3-83{kappa}i/+ (lanes 5 and 6) mice that all have a normal-sized pre-B cell compartment in their bone marrow. This suggests the frequent occurrence of V{kappa}J{kappa} rearrangements at the wild-type {kappa} locus of these mice. The same analysis performed for the VHPEf/D23{kappa}i, glD42/D23{kappa}i and VH12/D23{kappa}i combinations gave similar results (data not shown). We assume that the remaining three combinations that induce a large pre-B cell compartment in vivo behave alike. In contrast, splenic B cells from control B1-8f/+, 3-83{kappa}i/+ mice (lane 1) that express both the transgenic H and L chain in vivo and lack a pre-B cell compartment largely retained the wild-type {kappa} locus in the germ line configuration, as was also shown elsewhere (15). Thus, the presence of a pre-B cell compartment in Ig H and L chain transgenic mice indeed demonstrates the occurrence of receptor editing in vivo.



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Fig. 3. Receptor editing occurs in B cells bearing certain H and 3-83{kappa} L chain transgenes. (A) Configuration of the wild-type (WT) {kappa} and 3-83{kappa}i targeted alleles with the indicated SacI restriction enzyme site. (B) DNA from purified splenic B cells (upper panel) and liver (lower panel) obtained from B1-8f/+, 3-83{kappa}/+ (lane 1); B1-8f/+, 3-83{kappa}/3-83{kappa} (lane 2); VH12f/+, 3-83{kappa}/+ (lane 3); T15i/T15i, 3-83{kappa}/+ (lane 4) and VHPEf/+, 3-83{kappa}/+ (lanes 5 and 6) mice were analyzed by Southern blotting for the configuration of the WT {kappa} locus. Two mice of the VHPEf/+, 3-83{kappa}i/+ genotype were included to demonstrate the variability between individual animals. DNA in lanes 1 and 6 comes from animals that represented several samples, whereas lane 5 shows an animal with the least amount of editing for this genotype. Lane 2 is the control for the targeted {kappa} allele. In the lower panel, liver sample from only one VHPEf/+, 3-83{kappa}/+ mouse was used.

 
BCRs that do not promote B lymphopoiesis in vivo can drive B cell development in cell culture
The early stages of B cell development can be examined in vitro in a stromal cell culture system (32). Recently, the ST2 stromal cell culture system was used to study the differentiation of autoreactive B cells bearing the 3-83H/3-83{kappa} antibody that recognizes H-2Kk and 2-Kb but not H-2Kd. In vivo the 3-83 B cells edit their BCRs even on the H-2Kd genetic background; however, the in vitro-generated B cells express the transgenic H and L chains (11). It was speculated that editing in H-2Kd mice is caused by a weakly cross-reacting antigen that is unrelated to MHC class I (11). The ST2 stromal system might lack this so far unidentified autoantigen.

In the present experiments, we examined whether certain H and L chain combinations that could not give rise to mature B cells with the specificity imposed by the transgenes in vivo could do so in vitro. Due to the lack of anti-idiotypic antibodies that could recognize the different H and L chain combinations and the difficulty in obtaining large numbers of surface IgM+ B cells as required for Southern blot analysis, we instead measured the proportion of B220+CD43IgM pre-B cells in the stromal cell cultures, assuming that in the case of particular H and L chain combinations that are unable to drive B cell maturation, the cells will be trapped at the pre-B cell stage.

We restricted our experiments to mice that harbor the B1-8, VH12 and T15 H chains in combination with the 3-83{kappa} L chain. As can be seen in Fig. 4, mice transgenic for either B1-8 H or 3-83{kappa} L chain generated a population of B220+CD43IgM pre-B cells in the stromal cell cultures. However, there are significantly less pre-B cells in the bone marrow stromal cell cultures of mice bearing both the B1-8H and the 3-83{kappa} chains. This is in agreement with the in vivo data shown in Fig. 1(C). The larger proportion of the pre-B cells present in the in vitro culture compared with the in vivo situation could be due to less-efficient B cell differentiation in vitro. In bone marrow cultures of mice transgenic for VH12/3-83{kappa} and T15/3-83{kappa}, the population of pre-B cells is also drastically reduced compared with WT or single insertion mice (Fig. 4; and data not shown). Taken together, these data indicate that in contrast to the situation in vivo, the tested H and L chain combinations could drive B cell differentiation in vitro and are compatible with the view that the developmental block occurring in vivo results from the engagement of the BCR by self-antigens, which may not be present in the cell culture system.



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Fig. 4. Stromal cell cultures of bone marrow B cells derived from mice transgenic for certain H and 3-83{kappa} L chains. Figure shows the B220 versus CD43 profiles of the surface IgM-negative cells present in the stromal cell cultures. Numbers indicate the percentage of pro-B and pre-B cells as a fraction of total cultured cells recovered that are gated to exclude the ST2 stromal cells.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BCR replacement by ongoing V(D)J recombination in newly arising B cells is a major mechanism of tolerance induction, antibody diversification and quality control in the B cell compartment [for review see Nemazee and Weigert (33)]. Examining V{kappa}J{kappa} joints in {lambda}+ mouse B cells and finding almost half of them to be in-frame, Retter and Nemazee concluded that receptor editing is a frequent event in normal B cell development (13). Interestingly, 55 to 75% of all antibodies emerging in human bone marrow, as well as ~40% of new emigrant B cells are self-reactive, as found in a recent study by Nussenzweig and colleagues (34). In a previously described approach, the same group addressed this problem by determining the fraction of murine B cells in which a V{kappa}J{kappa} joint knocked into the Ig{kappa} locus was replaced during B cell development by a different VLJL rearrangement (14). The fraction of cells which had undergone this type of receptor editing was in the order of 25% for the various transgenic V{kappa}J{kappa} joints.

It would be interesting to compare the fraction of cells in which receptor editing was successful with the fraction of newly arising cells which attempt to edit their receptors. This would yield information on the efficiency of cellular rescue by BCR replacement, which is limited by the ‘success’ rate of individual replacement reactions and their average frequency over the window of available developmental time. In an attempt to approach this difficult problem, we have made use of a variety of H and L chain transgenic mice, a majority having individual VHDHJH or V{kappa}J{kappa} joints knocked into the physiological position in the Ig H or Ig{kappa} locus, respectively. Most of these gene rearrangements had originally been isolated from hybridomas secreting antibodies of known specificity (Table 1). In their particular combination in those hybridomas, they had apparently encoded antibody specificities which had made it into the peripheral B cell compartment. By assorting them in mice in different combinations and determining the fraction of combinations in which receptor editing is induced in these animals, we hoped to get an impression of how frequently random combinations of V{kappa}J{kappa} and VHDHJH joints encode antibody specificities which are subject to receptor editing in vivo.

There are obvious limitations to this approach. Most importantly and quite apart from quantitative considerations, the gene rearrangements used are derived from pre-selected antibodies, some of which display some degree of autoreactivity and/or have undergone affinity maturation in germinal centers. Their random combination may lead to a pattern of receptor editing differing from that resulting from the combination of germ line-encoded antibody V regions expressed in newly arising B cells in vivo. However, we have no reason to think that such differences would be of major concern. These chains are stably expressed in the majority of the peripheral B cells of single-transgenic mice, as far as this has been analyzed (1521). Furthermore, data presented in this paper (Fig. 2) and elsewhere (13, 30, 31) indicate that most, if not all, of the transgenic H and L chains can associate with each other, thus ruling out another major concern of the experimental approach adopted.

In agreement with previous work, we find two classes of BCRs in our analysis, those mediating ‘accelerated’ B cell development as characterized by a strongly reduced pre-B cell compartment [the site of receptor editing; reviewed in Nemazee and Weigert (33)] and stable expression of the transgenic BCR in most peripheral B cells and others which are edited at the pre-B/immature B cell stage. In quantitative terms, these two classes of BCRs seem to be of equal size. If we add to our list five similar ‘random’ combinations of transgenic H and L chains from an earlier study (14, Table 2B), there are 12 BCRs that are edited and 11 that are not.

With the reservations pointed out above, this result indicates that a major fraction of the BCRs generated by random H and L chain combination is subject to receptor editing and that this process may rescue roughly one half of the corresponding cells (14). Our data lead to the conclusion that about half of the newly arising cells will induce receptor editing, differentiating this data from the previous studies that quantified the number of cells in the periphery that has been derived through receptor editing (e.g. 13, 14, 33, 34). Combining our data with those from previous studies, one can conclude that descendant(s) of most cells initiating editing in the bone marrow will make it to the periphery.

In conclusion, the present data suggest that a large fraction of BCRs generated in newly arising B cells might not be selected into the peripheral B cell repertoire in vivo without initiating a process of BCR replacement.


    Acknowledgements
 
We are extremely grateful to Klaus Rajewsky, who encouraged this project to materialize and critically read the manuscript. We thank Stephen Clarke for the gift of the V{kappa}4 transgenic mouse strain and A.-T. Tan and C. Koenigs for expert technical help. This work was supported by research grants from the Volkswagen Foundation, the Deutsche Forschungsgemeinschaft through SFB 243, the Human Frontier Science Program, the Koerber Foundation, the Land Nordrhein-Westfalen to Klaus Rajewsky and the Biomedical Research Council of the Singapore Agency for Science, Technology and Research to K.-P.L.


    Abbreviations
 
BCR   B cell antigen receptor
H   heavy
L   light
V   variable

    Notes
 
Transmitting editor: D. Tarlinton

Received 13 August 2004, accepted 9 January 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Tonegawa, S. 1983. Somatic generation of antibody diversity. Nature 302:575.[ISI][Medline]
  2. Rajewsky, K. 1996. Clonal selection and learning in the antibody system. Nature 381:751.[CrossRef][ISI][Medline]
  3. Reth, M. 1992. Antigen receptors on B lymphocytes. Annu. Rev. Immunol. 10:97.[CrossRef][ISI][Medline]
  4. Melamed, D., Benschop, R. J., Cambier, J. C. and Nemazee, D. 1998. Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection. Cell 92:173.[ISI][Medline]
  5. Gay, D., Saunders, T., Camper, S. and Weigert, M. 1993. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177:999.[Abstract/Free Full Text]
  6. Tiegs, S. L., Russell, D. M. and Nemazee, D. 1993. Receptor-editing in self-reactive bone marrow B cells. J. Exp. Med. 177:1009.[Abstract/Free Full Text]
  7. Radic, M. Z., Erikson, J., Litwin, S. and Weigert, M. 1993. B lymphocytes may escape tolerance by revising their antigen receptors. J. Exp. Med. 177:1165.[Abstract/Free Full Text]
  8. Hartley, S. B., Crosbie, J., Brink, R., Kantor, A. B., Basten, A. and Goodnow, C. C. 1991. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature 353:765.[CrossRef][ISI][Medline]
  9. Nemazee, D. A. and Burki, K. 1989. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337:562.[CrossRef][ISI][Medline]
  10. Goodnow, C. C., Crosbie, J., Jorgensen, H., Brink, R. A. and Basten, A. 1989. Induction of self-tolerance in mature peripheral B lymphocytes. Nature 342:385.[CrossRef][ISI][Medline]
  11. Braun, U., Rajewsky, K. and Pelanda, R. 2000. Different sensitivity to receptor editing of B cells from mice hemizygous or homozygous for targeted Ig transgenes. Proc. Natl Acad. Sci. USA 97:7429.[Abstract/Free Full Text]
  12. Kouskoff, V., Lacaud, G., Pape, K., Retter, M. and Nemazee, D. 2000. B cell receptor expression level determines the fate of developing B lymphocytes: receptor editing versus selection. Proc. Natl Acad. Sci. USA 97:7435.[Abstract/Free Full Text]
  13. Retter, M. W. and Nemazee, D. 1998. Receptor editing occurs frequently during normal B cell development. J. Exp. Med. 188:1231.[Abstract/Free Full Text]
  14. Casellas, R., Shih, T. A., Kleinewietfeld, M. et al. 2001. Contribution of receptor editing to the antibody repertoire. Science 291:1541.[Abstract/Free Full Text]
  15. Pelanda, R., Schwers, S., Sonoda, E., Torres, R. M., Nemazee, D. and Rajewsky, K. 1997. Receptor editing in a transgenic mouse model: site, efficiency, and role in B cell tolerance and antibody diversification. Immunity 7:765.[ISI][Medline]
  16. Lam, K.-P., Kuhn, R. and Rajewsky, K. 1997. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene-targeting results in rapid cell death. Cell 90:1073.[ISI][Medline]
  17. Pewzner-Jung, Y., Friedmann, D., Sonoda, E., Jung, S., Rajewsky, K. and Eilat, D. 1998. B cell deletion, anergy, and receptor editing in "knock in" mice targeted with a germline-encoded or somatically mutated anti-DNA heavy chain. J. Immunol. 161:4634.[Abstract/Free Full Text]
  18. Taki, S., Meiering, M. and Rajewsky, K. 1993. Targeted insertion of a variable region gene into the immunoglobulin heavy chain locus. Science 262:1268.[ISI][Medline]
  19. Maruyama, M., Lam, K. P. and Rajewsky, K. 2000. Memory B-cell persistence is independent of persisting immunizing antigen. Nature 407:636.[CrossRef][ISI][Medline]
  20. Pelanda, R., Schaal, S., Torres, R. M. and Rajewsky, K. 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 5:229.[ISI][Medline]
  21. Novobrantseva, T. 2000. I. Rearrangement and Expression of Immunoglobulin Kappa and Lambda Light Chain Genes Can Precede Heavy Chain Expression during Murine B Cell Development. II. B Cells Expressing a Natural Autoreactive Immunoglobulin Receptor In Vivo, pp. 56–68. PhD Thesis, University of Cologne, Cologne, Germany.
  22. Arnold, L. W., Pennell, C. A., McCray, S. K. and Clarke, S. H. 1994. Development of B-1 cells: segregation of phosphatidyl choline-specific B cells to the B-1 population occurs after immunoglobulin gene expression. J. Exp. Med. 179:1585.[Abstract/Free Full Text]
  23. Lam, K.-P. and Stall, A. M. 1994. Major histocompatibility complex class II expression distinguishes two distinct B cell developmental pathways during ontogeny. J. Exp. Med. 180:507.[Abstract/Free Full Text]
  24. Kocks, C. and Rajewsky, K. 1988. Stepwise intraclonal maturation of antibody affinity through somatic hypermutation. Proc. Natl Acad. Sci. USA 85:8206.[Abstract]
  25. Simon, T. and Rajewsky, K. 1988. ‘Enhancer-constitutive’ vectors for the expression of recombinant antibodies. Nucleic Acids Res. 161:354.
  26. Kearney, J. F., Radbruch, A., Liesegang, B. and Rajewsky, K. 1979. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J. Immunol. 123:1548.[Abstract]
  27. Lam, K. P. and Rajewsky, K. 1999. B cell antigen receptor specificity and surface density together determine B-1 versus B-2 cell development. J. Exp. Med. 190:471.[Abstract/Free Full Text]
  28. Baccala, R., Quang, T. V., Gilbert, M., Ternynck, T. and Avrameas, S. 1989. Two murine natural polyreactive autoantibodies are encoded by nonmutated germ-line genes. Proc. Natl Acad. Sci. USA 86:4624.[Abstract]
  29. De Lau, W. B., Heije, K., Neefjes, J. J., Oosterwegel, M., Rozemuller, E. and Bast, B. J. 1991. Absence of preferential homologous H/L chain association in hybrid hybridomas. J. Immunol. 146:906.[Abstract/Free Full Text]
  30. Hamel, P. A., Klein, M. H., Smith-Gill, S. J. and Dorington, K. J. 1987. Relative noncovalent association constant between immunoglobulin H and L chains is unrelated to their expression or antigen-binding activity. J. Immunol. 139:3012.[Abstract/Free Full Text]
  31. Kaushik, A., Schulze, D. H., Bonilla, F. A., Bona, C. and Kelsoe, G. 1990. Stochastic pairing of heavychain and kappa lightchain variable gene families occurs in polyclonally activated B cells. Proc. Natl Acad. Sci. USA 87:4932.[Abstract/Free Full Text]
  32. Rolink, A. G., Winkler, T., Melchers, F. and Andersson, J. 2000. Precursor B cell receptor-dependent B cell proliferation and differentiation does not require the bone marrow or fetal liver environment. J. Exp. Med. 191:23.[Abstract/Free Full Text]
  33. Nemazee, D. and Weigert, M. 2000. Revising B cell receptors. J. Exp. Med. 191:1813.[Abstract/Free Full Text]
  34. Wardemann, H., Yurasov, S., Schaefer, A., Young, J. W., Meffre, E. and Nussenzweig, M. C. 2003. Predominant autoantibody production by early human B cell precursors. Science 301:1374.[Abstract/Free Full Text]
  35. Reth, M., Hammerling, G. J., and Rajewsky, K. 1978. Analysis of the repertoire of anti-NP antibodies in C57BL/6 mice by cell fusion. I. Characterization of antibody families in the primary and hyperimmune response. Eur J Immunol. 8:393–400.[ISI][Medline]
  36. Eilat, D., Hochberg, M., Pumphrey, J., and Rudikoff, S. 1984. Monoclonal antibodies to DNA and RNA from NZB/NZW F1 mice: antigenic specificities and NH2 terminal amino acid sequences. J Immunol. 133:489–94.[Abstract/Free Full Text]
  37. Nemazee, D. A., and Burki, K. 1989. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature. 337:562–566.[CrossRef][ISI][Medline]
  38. Sonoda, E., Pewzner-Jung, Y., Schwers, S., Taki, S., Jung, S., Eilat, D., and Rajewsky, K. 1997. B cell development under the condition of allelic inclusion. Immunity 6:225–233.[ISI][Medline]




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