(immunoglobulin genes/heavy chain class/fluorescence-activated cell sorting/somatic cell .... Cloned myeloma cell populations were stained with fluorescent.
Proc. Natl. Acad. Sci. USA
Vol. 77, No. 5, pp. 2909-2913, May 1980
Immunology
Isolation of variants of mouse myeloma X63 that express changed immunoglobulin class (immunoglobulin genes/heavy chain class/fluorescence-activated cell sorting/somatic cell genetics)
ANDREAS RADBRUCH, BERNHARD LIESEGANG, AND KLAUS RAJEWSKY Institute for Genetics, University of Cologne, Weyertal 121, D-5000 Kbln 41, West Germany
Communicated by Walter Gilbert, February 28,1980
ABSTRACT We have used fluorescence-activated cell sorting with class-specific antisera to isolate spontaneous variants in the expression of immunoglobulin heavy chain class from the mouse myeloma cell line X63 (IgGi, K). In the wild-type cell population, only one type of variants was found, namely, cells expressing IgG2b. From an IgG2b variant clone we isolated secondary variants that had either reverted to IgG1 expression or expressed IgG2a or IgG2a and IgG2b concomitantly. The variant heavy chains are of normal size. The variant immunoglobulins were characterized serologically, and all of them still expressed the wild-type idiotype. Wild-type and variant cell populations were screened for heavy chain class-switch variants by fluorescence microscopy. A variety of switch variants was found in addition to the ones isolated by cell sorting, and a clear pattern of class switching (y -_ y2b -_ 'y2a -_ a) with frequent reversion emerges from this analysis. Cells ex ressing new heavy chain classes occurred at frequencies ofabout 10-710-6/cell per generation, whereas revertants were as frequent as 10-6-10-5/cell per generation. A gene complex located on chromosome 12 of the mouse contains genes coding for the variable (V) (1), joining (J) (2), and constant (C) regions of immunoglobulin (Ig) heavy chains (3). The combination of genes from each group to form an active gene may contribute to the diversity of antibody-combining sites (4, 5) and to the range of antibody effector functions determined by the different classes of heavy chain constant regions (6). In the mouse, there are eight heavy chain classes (7, 8). During B-cell differentiation, different heavy chain classes can be expressed sequentially by the same cell [e.g., the 1u toy switch (9)] or simultaneously [e.g., ,u and 6 chains (8)]. In both cases the different immunoglobulins of a given B-cell clone bear similar or identical V regions. Transformed plasma cells, available as myelomas, serve as a model system to study this differentiation of heavy chain expression. From such cell lines variants of Ig expression have been isolated (10-14), and an example of class switching ("y2b -- y2a in the myeloma MPC 11) has been established. We report here the isolation of heavy chain class-switch variants from the mouse myeloma line X63.
MATERIALS AND METHODS Animals. The sources of mice, rabbits, and guinea pigs are listed in a previous paper (14). Myelomas and Myeloma Proteins. All myelomas were of BALB/c origin. The myeloma line MPC 11, secreting an IgG2b with K light chains (IgG2b, K), was a gift from S. Tonegawa (Basel, Switzerland). The myeloma cell line X63 (IgG1, K) was a gift from C. Milstein (Cambridge, England). These lines are adapted to growth in cell culture (13), as are the hybrid cell lines The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 2909
B1-8 (IgM, X) and S24 (IgG3, X; IgGl, K), which were a gift from M. Reth and T. Imanishi-Kari (K6ln, West Germany). The myelomas TEPC 183 (IgM, K), MOPC 104E (IgM, X), MOPC 315 (IgA, X2), MOPC 460 (IgA, K), MOPC 31C (IgGl, K), HOPC 1 (IgG2a, X), RPC 5 (IgG2a, K), MOPC 195 (IgG2b, K), and J 606 (IgG3, K) were obtained from Litton Bionetics (Kensington, MD), and MOPC 70A (IgGI, K) was from the Salk Institute (San Diego, CA). Maintenance of myeloma cells, cloning by limiting dilution, screening of clonal supernatants for class of secreted antibody by enzyme-linked immunosorbent assays, analysis of 14C-labeled secretion products in isoelectric focusing, and molecular weight determinations by NaDodSO4/polyacrylamide gel electrophoresis have been described (14, 15). Antisera. Immunoglobulin class and idiotype specific antisera were obtained from guinea pigs and rabbits as described (13). Antisera from goats were kindly provided by U. Wurzburg (Merck, Darmstadt, West Germany). For absorption of antisera and purification of specific antibodies, we prepared immunosorbents by coupling purified myeloma proteins to Sepharose 4B (Pharmacia) (16). Immunosorbents were eluted with 1 M glycine/HCI, pH 3.0 or 3.2. For selection of heavy chain class variants from X63 wildtype cells (IgGI), we combined guinea pig antisera against Ig from MOPC 104E, MOPC 315, J 606, MOPC 195, and HOPC 1 according to titer as determined in radioactive binding assays. This cocktail was designated C1 and absorbed on a X63 immunosorbent and, during the selection experiment, with X63 cells that had been enriched on the basis of an unspecific staining reaction with C1. C1 reacted equally well with IgM, IgG3, IgG2b, IgG2a, and IgA, but not with IgGl. For selection of secondary variants from the IgG2b variant clone X63.2b-7, we mixed another cocktail of guinea pig antisera against Ig from MOPC 104, MOPC 315, J 606, MOPC 31C, and HOPC 1. This cocktail, C2b, was absorbed on a MPC 11 sorbent and on MPC 11 and X63.2b-7 cells. C2b reacted with IgM, IgG3, IgG1, IgG2a, and IgA, but not with IgG2b. For the isolation of IgG2a variants from X63.2b-7, we absorbed a guinea pig antiserum against HOPC 1 with insolubilized MOPC 70A Ig and subsequently isolated anti-y2a antibodies on an RPC 5 immunosorbent. X63 idiotype specific antibodies were obtained from a rabbit serum against X63 Fab fragments, absorbed with mouse Ig and TEPC 183 Ig, and eluted from an X63 sorbent. We also used three guinea pig antisera against X63 Ig which we made specific for the X63 idiotype by absorption on MOPC 70A and MOPC 195 immunosorbents. Purified Ig class specific goat antibodies coupled with fluoresceine, rhodamine, or alkaline phosphatase were kindly donated by J. F. Kearney (Birmingham, AL) or prepared by absorption and elution on appropriate myeloma or hybrid cell line antibody sorbents. Fluoresceinated rabbit antisera against mouse Ig and against guinea pig Ig were obtained from Behringwerke (Marburg/Lahn, West Germany).
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Proc. Natl. Acad. Sci. USA 77 (1980)
Immunology: Radbruch et al.
Serology. The specificity of antisera and the class and idiotype of myeloma proteins were determined by enzyme-linked immunosorbent assays (15) and by radioimmunoassays (13). Immunofluorescence. Direct and indirect staining of cell surface Ig was done as described (13), the only modification being the use of Dulbecco's phosphate-buffered solution (17) instead of Dulbecco's modified Eagle's medium. Cell smears were prepared and stained for cytoplasmic Ig according to Kearney et al. (15). When cell smears were screened for class-switch variants, dead cells, as identified by phase-contrast microscopy, were excluded from the analysis. A small fraction of the cells that appear intact by phase-contrast microscopy exhibit very bright fluorescence which can be easily distinguished from that of normal myeloma cells. This fluorescence represents a technical artifact because the corresponding cells cannot be enriched by cell sorting. Consequently, cells of this type were also disregarded. Cell Sorting. We used the fluorescence-activated cell sorter FACS-1 (Becton, Dickinson, Mountain View, CA) at a flow rate of 2000-5000 cells per sec. Before sorting, cell suspensions were filtered through a nylon mesh (no. P30 Nytal, Schweiz. Seidengazefabrik AG, Thal, Switzerland). Other details of the sorting procedure have been described (13). Karyotyping. Metaphase plates were prepared as described (13). Slides with spread metaphase cells were dried for 1 week and stained for trypsin/Giemsa bands by a variation of the method of Seabright (18). In order to calculate the mean chromosome number of a cell line, we counted the chromosomes of 16-22 metaphase plates. RESULTS Variant cell lines Cloned myeloma cell populations were stained with fluorescent antisera specific for heavy chain classes other than that expressed by the wild-type cells and separated by fluorescenceactivated cell sorting. Because separated cell populations were enriched only 50-fold at most (13) for variants, we repeated sorting, growth, and resorting until variants were frequent enough for isolation by cloning. Four variant cell lines were isolated in this way and roughly characterized (Fig. 1). yI -_ 'y2b. The first selection aimed at the isolation of the most frequent heavy chain variant of X63 cells. X63 cells were subcloned twice and then selected with cocktail C1 (antibodies against ,u, oy2a, y2b, y3, and a). Variants were enriched by seven cycles of selecting the brightest 2.4-15% of the cells from 0.76-5.2 X 107 cells, one selection of 22% from 4.6 X 106, and
one selection of 20% from 1.5 X 105 cells. The resulting cells were analyzed for the class of cytoplasmic Ig. In about 105 cells we did not detect a single cell expressing ,u, zy3, y2a, or a determinants. However, 63% of the cells were positive for y2b only. The rest of the cells stained with anti-- 1 antibodies. The cells were cloned by limiting dilution, and clones secreting IgG2b were identified by enzyme-linked immunosorbent assays of culture supernatants. The isoelectric focusing banding patterns of the secreted proteins from 30 clones were identical. We chose one clone (X63.2b-7) for characterization and further variant selection experiments. "y2b -. 'y1. Cell line X63.2b-7 was subcloned twice and the subclone X63.2b-7.8.4 was chosen for further selection with cocktail C2b (antibodies against ,t, y1, y2a, 'y3, and a). In order to avoid any possibility of contamination of X63.2b-7 with the parental line, we banned all X63 wild-type cells from the tissue culture rooms before recloning X63.2b-7 for the second time. After three cycles of selection, separating 1.5%, 1.3%, and 58% from 1.5-3.5 X 107 cells, 20 out of 23 clones produced IgG1 only whereas the remaining three clones still expressed IgG2b alone. The secreted products of 16 of the revertant clones were compared by isoelectric focusing; they had banding patterns identical to that of X63 wild-type immunoglobulin (see Fig. 2A). Clone X63.Rl-18 was further characterized (see below). 'y2b -8y2a. Selection of X63.2b-7 cells with C2b had resulted in the isolation of revertants to IgGl expression. However, in the fluorescence microscope we could also detect y2a-positive cells in clone X63.2b-7.8.4, although at a much lower frequency than the revertant cells (see Table 1). The 'y2a variants were isolated by separating 1.5% of 1.5 X 107 cells with cocktail C2b and then further selecting with purified anti-y2a antibodies. Four times we separated between 0.6% and 6.3% from 0.43-3 X 107 cells. In the fifth sorting 10% of 2 X 103 cells were separated and directly cloned. From the resulting 112 clones, 13 produced IgG2a only, 44 expressed both y2a and oy2b determinants, and 55 produced IgG2b. We analyzed the isoelectric focusing patterns of secreted Ig from 6 clones of the first group and 16 of the second. The patterns were group specific (see Fig. 2). We chose X63.2a-93 as a representative of clones expressing y2a and y2b determinants simultaneously and X63.2a-25 of clones producing only IgG2a. A
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Immunology:
Radbruch et al. CO)
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Proc. Natl. Acad. Sci. USA 77 (1980)
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Comparison of variants The variant cell lines chosen for further analysis were propagated both in vitro and in vivo in BALB/c mice. The secreted Ig of each line was purified. We obtained a single protein from each variant except for X63.2a-93 (IgG2a-2b). This latter variant produced three different immunoglobulin species that could be separated by DE-52 chromatography (data not shown). The three proteins (931, 9311, and 9311I) were typed in radioactive-binding inhibition assays for heavy chain class. Protein 93I typed as an IgG2a, 93III as an IgG2b, and 93II carried both y2a and -y2b determinants. Fig. 2 shows the isoelectric focusing patterns of all variant myeloma proteins compared to the wild type. The banding patterns of IgG1 from wild-type X63 and X63.R1-18 are identical (Fig. 2A). The IgG2b proteins derived from X63.2b-7 and X63.2a-93 (Fig. 2B) have identical patterns, as have the IgG2a immunoglobulins produced by X63.2a-25 and X63.2a-93 (Fig. 2C). The IgG2a + 2b from X63.2a-93II shows a unique isoelectric focusing pattern (Fig. 2B). 100-
Inhibitor, pg FIG. 4. Serological analysis of idiotype of variant immunoglobulins in a radioactive binding inhibition assay. Binding of a guinea pig anti-X63 idiotype serum to 125I-labeled X63 IgG1 (ordinate) was inhibited by increasing amounts (abscissa) of X63.5.3.1 (IgG1, X), X63.R1-18 (IgG1, *), X63.2b-7 (IgG2b, 0), X63.2a-25 (IgG2a, *), X63.2a-93I (IgG2a, 3), X63.2a-931I (IgG2a + 2b), A), and X63.2a93111 (IgG2b, &) and by the myeloma proteins of MOPC 70A (IgGi, 0), RPC 5 (IgG2a, *), and MOPC 195 (IgG2b, 0).
FIG. 3. Analysis of size of variant immunoglobulins. The immunoglobulins were reduced by addition of 5% 2-mercaptoethanol to the sample buffer and subjected to electrophoresis on a 10% NaDodSO4polyacrylamide gel. Positions of heavy (H) and light (L) chains are marked. Marker proteins were bovine serum albumin (Mr 68,000), ovalbumin (Mr 46,000), and RNase (Mr 13,000).
The size of the variant heavy chains was determined by NaDodSO4/polyacrylamide gel electrophoresis of reduced immunoglobulins. The sizes of the variant X63 yI and y2a heavy chains were identical to that of wild-type X63 yyl chians (Fig. 3B), whereas X63 y2b chains appeared slightly larger and were similar in size to MOPC 195 y2b chains (Fig. 3). The X63.2a-93II Ig contained two heavy chains, one in the position of y2b and one in that of -y2a chains. We characterized the variant proteins serologically in radioimmunoassays. The analysis was similar to that previously reported for the 1B6 variant of MPC 11 (14), and the results are not documented here in detail. The 'y2b and 'yl variant proteins completely inhibited binding of an appropriate class specific antiserum to a '25I-labeled myeloma protein of the same class and at approximately the same concentration as the homologous myeloma protein itself. The two 'y2a variant proteins from X63.2a-25 and X63.2a-93 completely inhibited binding of a guinea pig anti-y2a serum to radiolabeled IgG2a and RPC 5 or HOPC 1, but they failed to inhibit binding of a goat anti-'y2a serum to IgG2a. The IgG2a of the variants may thus not express all determinants of normal y2a heavy chains. None of the variant immunoglobulins inhibited binding of antisera specific for the class of wild-type heavy chains to myeloma proteins of that class. The purified X63.2a-93II protein completely inhibited binding of anti-,y2a to IgG2a and anti-y2b to IgG2b, although in both cases comparable inhibition was achieved only at inhibitor concentrations higher than with homologous Ig. This result again suggests a hybrid composition of the X63.2a93II antibody, each molecule containing a -y2a and a y2b heavy chain (Fig. 2). The binding of four different anti-idiotypic sera to X63 wild-type protein or to the protein of the X63.2b-7 variant could be inhibited equally well by the wild-type protein or any of the variant proteins. An example of this is depicted in Fig. 4. This establishes that all our variants are derived from X63 and suggests that the variable region of the heavy chain is retained in the variant proteins without major change. The common origin of the X63 wild-type line and all variant cell lines was also established by karyotype examination. Marker chromosomes specific for the X63 myeloma line are present in all variant cell lines (data not shown). The karyotype analysis showed, in addition, that X63.2a-93 is unlikely to have arisen by spontaneous fusion because it has a mean chromosome number of 69.7 i 1.3 compared to 68.7 i 2 for X63.2b-7 and 68.2 ± 2.6 for X63.2a-25.
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Immunology: Radbruch et al.
Proc. Natl. Acad. Sci. USA 77 (1980)
Frequency of variants We screened defined numbers of cells in cytocentrifuge smears
longer express either y2a or y2b determinants. Here we could also detect cells expressing determinants of y2a or 'y2b and yyl simultaneously. Cells expressing determinants of two IgG classes were also found in X63.2b-7.8.4 and X63.2a-25.
with pairs of fluoresceine- and rhodamine-labeled class specific antisera, one directed towards the class produced by the majority of cells from that clone and the other directed against the variant class in question (Table 1). The frequency of variants per cell per generation was calculated on the basis of the number of variants per 105 or 106 cells, the generation time of X63.2b-7.8.4 cells, and the number of generations from cloning until the preparation of the slides. Our confidence in the fluorescence analysis is based on two points. First, many of the variant cells stain exclusively for the variant class in paired immunofluorescence. Second, variants of a particular type, as defined by fluorescence microscopy,
DISCUSSION The spontaneous class switches that occur in the X63 population have the following properties. (i) Class switches appear to occur in a distinct sequence-namely, from -y1 to 'y2b to -y2a to a. In many cases the switching cells lose the expression of a given class and achieve that of another; however, there are switch variants that coexpress two heavy chains of different classes (X63.2a-93) or at least antigenic determinants of two Ig classes (Table 1). (ii) In all cases, class-switch variants revert to wildtype class expression with a much higher frequency than that of their own occurrence (105-10-6 in contrast to 10-6-10-7 per cell per generation). Again, a state of two-class expression is often found in reverting cells (Table 1). (iii) The spontaneous class variants appear in general to be due to an exchange of the entire constant region of the heavy chain. The heavy chains of the variants described in this and a previous paper (14) have lost all constant-region antigenic determinants of the parental chains and, with one possible exception ('y2a), express the complete set of antigenic determinants of the new class. In addition, the chains are of correct size (Fig. 3). Determination of protein and mRNA sequences will establish definitively that switching involves the entire constant region of the heavy chain. (iv) The variable regions of wild-type and variant proteins are very similar if not identical, as shown by idiotypic analysis (Fig. 4). However, this point needs further study. In MPC 11, classswitch variants have been typed as idiotypically identical (12) or, as in our own experiments (14), crossreactive but nonidentical with the wild-type protein. We know that the sequences of amino acids 21 -60 of the heavy chain of our MPC 11 variant and the amino-terminal 45 residues of X63.2a-25 and X63.2b-7 heavy chains are identical to those of the corresponding wildtype proteins (R. Dildrop, T. Geske, J. Bovens, and K. Beyreu-
could invariably be enriched and isolated when the attempt was made, even when the variants were present in the wild-type population in low frequency (see frames in Table 1). Cell sorting was accompanied by enrichment of microscopically detectable variants until these cells comprised a large fraction of the total cell population and could be established as true variants by cloning and subsequent analysis of the clones (see above). In addition, we never found enrichment for variants of a type that we had not already detected microscopically in the wild-type population. In none of the cell lines examined could we detect cells expressing IgM, IgG3, or IgA, except for X63.2a-25, where we detected two cells expressing IgA. Variants expressing IgG2b in X63 wild-type cells and variants expressing IgG2a in X63.2b-7.8.4 cells occurred at frequencies of 2.3 X O-7 and 1.6 X 10-7, respectively. In all variant cell lines we could detect revertants at higher frequencies (Table 1). In -y2a variants we tried to find clones that would not give rise to any revertants. However, examination of 100 subelones of X63.2a-25 (IgG2a, K) always revealed y2b revertants at frequencies of 10-5-10-6 (data not shown). In some of the subelones we could also detect 'yl revertants at a frequency of about 10-6. The double producer X63.2a-93 has a high frequency of segregants that no Cellline X63.5.3.1 X63.R1-18
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5/105 0/105 + 0/106 7/106 (1.2 X 10-5) (2.9 x 10-7) X63.2b-7.8t 23/2 x 105 0/105 + (2.6 X 10-6) X63.2b-7.8.4t 0/106 0/106 [24/105 + 3/105 x 105 19/8 x 105 05/8 0/106 (5.6 x 10-6)l (7.4 X 10-7) 5.9 x 10-7) 1.6 x 10-7) X63.2b-7.9.5 13/105 + 10/105 (3.6 X 10-6) (2.8 X 10-6) X63.2a-25.19.1.4§ 0/106 0/106 31/105 1 11/105 + 105/105 48/105 2/106 (1.6 X 10-5) (1 x 10-5) (5.2 x 10-5) (2.6 X 10-5) (1.6 x 10-7) X63.2a-93.1.41 10/105 13/105 + 32/105 66/105 (1 x 10-5) (1.3 X 10-5) (3.2 X 10-5) (6 X 10-5) Cytocentrifuge smears of 105 cells per slide were incubated with antisera against wild-type Ig class and variant Ig class each tagged either with rhodamine or fluorescein. Values are given as the number of cells expressing the class indicated at the top of the table per total number of cells screened. Values in parentheses are the frequency of variants per cell per generation, calculated on the basis of the generation time of X63.2b-7.8.4 (20 hr), the number of generations until preparation of the slide, and the number of variants per total cell number screened. The frequencies of variants that have been isolated (Fig. 1) appear in frames. * Wild-type heavy chain class. t Subclone of X63.2b-7. t Subclone of X63.2b-7.8. § X63.2a-25 subcloned three times. X63.2a-93 subcloned twice. 1 We cannot exclude that these cells express y2a or y2b determinants in addition.
Immunology:
Radbruch et al.
ther, personal communication). It will be of particular interest to compare the primary structures of wild-type and variant heavy chains at the V-J boundary. The molecular mechanism by which class switching in myeloma cells is brought about cannot be deduced from the present analysis. However, a few points that are relevant to the problem can already be made: X63 cells possess three to four copies of chromosome number 12 (unpublished results). If more than one of these chromosomes synthesize X63 heavy chain mRNA, the switching event would have to involve more than one chromosome in variants that express the new Ig class only [X63 cells are in principle permissive for the coexpression of different Ig classes (see ref. 19 and the double producers described in this paper)]. Concomitant constant-region gene deletion on homologous chromosomes, discussed further below, could account for this, but is incompatible with reversion. We, therefore, consider it likely that only one heavy chain locus is expressed in X63 cells, and we would like to think that class switching reflects the expression of genes in that locus. The order of the class switches, yl y2b - y2a - a, must be based on a specific mechanism operating at the level of mRNA or DNA. A gene deletion model has been proposed on the basis of DNA hybridization data (20). In this model, the V gene expressed by the cell lies in front of the C gene cluster on one of the two homologous chromosomes and is transcribed with the C gene next to it. Switching involves sequential deletion of C genes on the same chromosome. The recent data of Rabbitts et al. (21) suggest that C gene deletion occurs on both homologous chromosomes. The order of C genes emerging from the work of Honjo and Kataoka (20) and of Rabbitts et al. (21) agrees with the order of "forward" class switches observed in the present and in earlier (12, 14) experiments. However, the finding both of variants expressing heavy chains of different class simultaneously and of revertants (Fig. 1 and Table 1) cannot be easily understood on the basis of constant-region gene deletion. With respect to the double producer line X63.2a-93, one might invoke chromosome duplication in combination with deletion on one of the duplicated chromosomes. It is difficult to see how such a mechanism could account for the many cases of two-class expression in the microscopic analysis (Table 1). We do not yet know, however, whether the double staining cells generally express two different heavy chains such as in X63.2a-93 or hybrid heavy chains such as those found in mutagenized MPC 11 cells (22). Reversion, furthermore, is a difficult problem for a deletion model, in particular because the reversion frequency is 1-2 orders of magnitude higher than that of forward switching. This difference argues against the idea that both the reversion and the "forward" switching are due to somatic recombination between homologous chromosomes. In the myeloma cell, class switches cannot be obligatorily accompanied by the loss of all copies of the previously expressed constant region gene. It is significant in this context that we have found revertants in each of 100 clones of X63.2a-25. Reversion but not two-chain production could be due to inversion of constant region genes or their excision and propagation as extrachromosomal elements. However, it might well be that some or all of the class switches observed in this study reflect regulation at the RNA level. Although there is evidence that RNA splicing is not responsible for heavy chain class determination in certain transformed cell lines (23, 24), those data appear insufficient to make a general case. The concomitant expression of heavy chains of different class but identical variable regions is presumed to occur in normal B-cell development, at least for ju and a chains (8). In addition,
Proc. Natl. Acad. Sci. USA 77 (1980)
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"forward" switches from At to -y and a are a characteristic feature of B-cell maturation (9). Thus, the spontaneous class switches in myeloma cells may be based on a mechanism similar to that operating in B-cell ontogeny, although switches between IgG subclasses in B cells have not so far been reported. Finally, we note that the present experiments demonstrate a way in which groups of antibodies can be obtained that share a given variable region but differ in their heavy chain class. We are in the process of constructing, by hybridoma variant selection, such groups of antibodies with known antigen- or idiotypebinding properties in order to study the role of antibody classes in the regulation of the immune system. For example, our technique permits the conversion of a noncomplement-fixing antibody into a complement-fixing species. This might be of some practical use in experimental and clinical work. We thank Drs. S. Tonegawa, C. Milstein, M. Potter, T. Imanishi-Kari, and M. Reth for gifts of myeloma cells, Drs. J. F. Kearney, U. Wtirzburg, and R. Grutzmann for gifts of reagents, and Dr. R. S. Jack for helpful discussions in the initial phase of this work. We thank Ms. G. Giels for excellent technical assistance and Mr. W. Santins for help with the cell sorter. This work was supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 74.
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