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
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Abstract |
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Keywords: antibody repertoire, autoimmunity, B cell development, gene targeting, receptor editing
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Introduction |
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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 loci in
+ IgM-expressing B cells in mice indicated that 47% of the recombining sequence-inactivated V
J
joints were in-frame, suggesting that BCR editing occurs frequently during normal B cell development (13). Recently, an Ig
allele that harbors a human C
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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Methods |
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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 streptavidinCyChrome. The following mAbs were used in this study: R33-24-12 (anti-IgM), R33-18-10 (anti-), LS136 (anti-
1), Ac146 (anti-VHB1-8), S7 (anti-CD43) and RA3-6B2 (anti-B220).
Transfection of P3X63Ag8.653 cells
The 3-83 and D23
(21) VJs were cloned into the expression plasmids pSVEneo and pE
A20/44 (24), respectively. The V
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
1 (25) that expressed these H chains as secreted
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 ml1 and 500 µg G418 ml1. In the transient transfection studies, H chain-encoding plasmids were electroporated into P3X63 cells that had already incorporated a D23
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 1 (A85-1; rat IgG1,
) 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
1 antibody (MOPC-21; IgG1,
) 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
(R8-140, rat IgG1,
) 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 locus was performed using the EcoRIEcoRI probe as shown in Fig. 2 and by Pelanda et al. (15).
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Results |
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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 V4 L chains. In contrast to the B1-8 H and 3-83
L chain combination that encodes an antibody of unknown specificity, the VH12 and V
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
4 transgenic mice. However, this population of pre-B cells was 6- to 10-fold reduced in VH12/V
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
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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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 3-83i (20) and D23
i (21). A V
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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Analyses of the crosses of the various H chain transgenic mice with the 3-83 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
mice that we had analyzed previously (15) and re-capitulated here, mice bearing glD42H/3-83
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
mice were reduced
5-fold compared with either glD42H or 3-83
i mice. However, normal numbers of pre-B cells are present in mice bearing VH12/3-83
, T15/3-83
or VHPE/3-83
(data not shown), similar to the situation in mice bearing the autoreactive 3-83H/3-83
(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 L chain insertion mice. The D23
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
, glD42H/D23
, VHPE/D23
or T15/D23
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
or VH12/D23
. Thus, the pairings of the various H chains with the D23
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 (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 C1 and C
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
1 antibody as capture and anti-
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 V4 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
4, 3-83H/V
4 (Fig. 1) and VHPE/V
4 (data not shown). Thus, the ELISA data indicate that glD42 H and V
4 L chains could associate with each other to form an intact antibody.
Similarly, H and L chain combinations such as T15/3-83, VH12/3-83
and VHPE/3-83
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
pair that is known to encode a functional BCR with specificity for an MHC class I antigen (13) and the glD42/3-83
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 L chain gene. Again, the ELISA data suggest that the H and L chain combinations such as 3-83H/D23
, glD42H/D23
and VHPE/D23
in which the inserted D23
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 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 allele in mice bearing the various H and 3-83
L chain transgenes using a strategy that we had previously employed for the analysis of the autoreactive 3-83H/3-83
mice (15). The configurations of the wild-type and targeted
alleles are shown in Fig. 3(A). Rearrangement of the upstream V
genes to the downstream J
elements on the wild-type
locus results in the elimination of the gene segment and the disappearance of the SacISacI 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
band in DNA isolated from purified splenic B cells of VH12f/+, 3-83
i (lane 3); T15i/T15i, 3-83
i/+ (lane 4) and VHPEf/+, 3-83
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
J
rearrangements at the wild-type
locus of these mice. The same analysis performed for the VHPEf/D23
i, glD42/D23
i and VH12/D23
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
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
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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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 L chain. As can be seen in Fig. 4, mice transgenic for either B1-8 H or 3-83
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
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
and T15/3-83
, 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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Discussion |
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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 VJ
joints knocked into the physiological position in the Ig H or Ig
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
J
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.
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Acknowledgements |
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Abbreviations |
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BCR | B cell antigen receptor |
H | heavy |
L | light |
V | variable |
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Notes |
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Received 13 August 2004, accepted 9 January 2005.
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References |
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