Immunoglobulin class switching

Cell, Vol. 36, 801-603,

April 1964, Copyright

0 1984 by MIT

lmmunoglobulin

Class Switching

Akira Shimiru and Tasuku Honjo Department of Genetics Osaka University, Medical School Kita-ku, Osaka 530, Japan

The enormous diversity of immunoglobulins (lg) relies upon two features: a large number of variable (V) regions bind to specific antigens; and they associate with different constant (C) regions that exert a variety of effector functions such as complement fixation. The V region repertoire is shared by the lg classes and subclasses (IgM, IgD, IgG,, IgGna, lgGPb, IgGa, IgA, and IgE) which are determined by one of eight C,, region genes in mouse (C,, C,, C,,, CyEa, CyLb, C+, C,, and C,, respectively). Somatic amplification of both types of lg diversity is mediated by DNA rearrangements of the lg genes. Multiple germ-line DNA segments (K-JL or VH-DH-JH) are recombined to form a complete V gene. Thus the antigen specificity of each lymphocyte is determined by random association of two or three germ-line DNA segments. The VH-0-J” recombination seems to be a key step for differentiation between T and 6 lymphocytes because it is the first DNA rearrangement in the B cell lineage. Common precursors to T and B lymphocytes may not be committed to differentiate to either T or B cells until either the B cell receptor (i.e. lg) gene or the T cell receptor gene is rearranged successfully. The rearrangements of the T and B cell receptor genes may compete in determining the lineage of lymphocytes. B lymphocytes that have completed the correct VH-DJ,, and VL-JL recombinations produce IgM because the C, gene is the closest to the Ja segments. The progeny of a single lymphocyte retain essentially the same VH region sequences, except for base replacements due to somatic mutation. However, the lg produced among the progeny often changes from IgM to another class. Paradoxically, the constant region (C,) of the lg is replaced by another CH region while the variable region (V,) remains constant, This phenomenon, known as class switching, is mediated by another DNA rearrangement, called S-S recombination, The initial molecular genetic analyses of myeloma DNAs producing different lg classes revealed that specific CH genes are deleted, depending on the CH genes expressed (Honjo and Kataoka, PNAS 75, 2140-2144, 1978). We proposed a model that explains the class switch phenomenon by supposing that the expressed VH gene recombines with a different Ca gene and the intervening DNA segment is deleted. We also proposed an order for the CH genes on the basis of the CH gene deletion profile in various myelomas. Structural comparison of the germ-line and expressed y (or a) genes has shown that after switching a complete VH gene is directly linked to the C, (or C,) gene (Davis et al., Nature 283, 733-739, 1980; Kataoka et al., PNAS 77, 919-923, 1980; Maki et al., PNAS 77, 2138-2142, 1980). Recombination takes place in an intervening sequence

that lies between the VH and C,, genes. The nucleotide sequences of the regions containing frequent recombination sites are comprised of tandem repeats of short unit sequences that share dispersed TGAGC and TGGGG (Nikaido et al., JBC 257, 7322-7329, 1982; Kataoka et al., Cell 23, 357-368, 1981). This stretch is termed the S region. Isolation and characterization of partially overlapping DNA segments encompassing the whole C,, gene cluster of mouse (Shimizu et al., Cell 28, 499-506, 1982) has demonstrated that the order of CH genes is 5’-J”(6.5 kb)-Q-(4.5 kb&(55 kb)-C,s-(34 kb)-C&,-(21 kb)-C&l5 kb)-C+,-( 14 kb)-C-(12 kb)-C--3’, in agreement with our earlier proposal. An S region was identified at the 5’flanking region of each CH gene except for the Ca gene. The organization of the human CH gene family, which maps to band q32 (14q32) of chromosome 14, is similar to mouse; but unique to humans are the presence of several pseudogenes and a large duplicated set of C,-C,C, genes. The human CH gene order is 5’-J+,-(8 kb)-C,-(5 kb)-Ca-(unknown distance)-C+-(26 kb)-C,,-( 19 kb)-Cn-( 13 kb)-C&unknown distance)C,,-( 18 kb)-Cd-(23 kb)-C,,-( 10 kb)&-3’ (Flanagan and Rabbitts, Nature 300, 709-713, 1982). The CrZ gene is a truncated pseudogene that has two large deletions including the CH1 and CH2 exons, and the CL gene is a processed gene located on chromosome 9 (Max et al., Cell 29, 691-699, 1982; Hisajima et al., PNAS 80, 2995-2999, 1983; Ueda et al., EMBO J. 1, 1539-1544,1982). The pseudo-C, gene, the chromosome location of which is unknown, has lost the S region (Takahashi et al., Cell 29, 671-679, 1982). There are two views on the structure of the S regions. Kataoka et al. (1981, op. cit.) assumed that the S-S recombination might be a sort of homologous recombination recognizing scattered short homology unit sequences. Davis et al. (Science 209, 1360-1365, 1981) proposed that class-specific recombinases might be responsible for S-S recombination. The nucleotide sequences surrounding the recombination sites usually contain TGGG and TGAG (Nikaido et al., op. cit.). Attempts to analyze molecular mechanisms of S-S recombination in vitro were recently reported (Kataoka et al., PNAS 80, 2666-2670, 1983). They have constructed two recombinant X phages that carry different inserts, S,-C, and S,-C,. Each phage has different genetic markers on right and left arms. Host bacteria were chosen so that only hybrid phages produced by recombination within inserted DNA can grow. Using this system Kataoka et al. (1983, op. cit.) found that an E. coli extract can catalyze the recombination of two phages predominantly within the S regions. The nucleotide sequences of the actual recombination sites are similar to those of myeloma cells (table). The recombination sequences are also reminiscent of Chi sequence that stimulates generalized recombination (Kenter and Birshtein, Nature 293, 402-404, 1981). It is not clear whether the S region sequence was recognized by the Chi-stimulated bacterial recombination system. It is also

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possible that the S region is a target for homologous recombination. In either case, the S region was shown to serve as an efficient recombination site even in bacteria. DNA fragments with nucleotide sequence almost identical to the mouse S, region have been cloned from Drosophila DNA (Sakoyama et al., Nucl. Acids Res. 70, 4203-4214, 1982). These findings suggest that prototypes of the eucaryote recombination system might be found in lower eucaryotes and possibly in procaryotes. It may be reasonable to assume that the lg gene system has borrowed, rather than invented, the recombinational mechanism from an unknown precedent. Two molecular mechanisms have been proposed for SS recombination. The looping-out model is the simplest to explain deletion and cis recombination (Honjo and Kataoka, op. cit.). The unexpected finding of an S, segment sandwiched between the S, and S,, regions flanking an expressed yl gene led to the proposal of a sister chromatid exchange model (Obata et al., PNAS 78, 24372441, 1981). Multiple recombination between sister chromatids can easily reverse the linear order of the S regions in the chromosome. Since the transcriptional orientation of all the CH genes is the same as that of the JH segments, the inversion model as proposed for DH-JH recombination (Alt et al., PNAS 78, 4118-4122, 1982) is unlikely. Deletion of CH genes has been found universally in plasmacytomas and is considered to be a general principle of class switching in the plasma cell (Cory et al., Nature 285, 450-456, 1980; Yaoita et al., Nature 286, 850-853, 1980; Rabbitts et al., Nature 283, 351-356, 1980). More recently, Radbuch and Sablitzky (EMBO J. 2, 1929-1935, 1983) have demonstrated that the C, gene deletion takes place in IgGa-producing spleen cells (plasmablasts) stimulated with lipopolysaccharide (LPS) and collected by a fluorescence-activated cell sorter. Populations of surface IgG3+ (slgG3+) cells that were surface IgM- (slgM-), cultured for 6 days with LPS, contained only about half as many C, genes as spleen and liver cells. C, deletion is nearly complete in populations of slgM3+/slgM- cells from 9 day old cultures, indicating that the C, gene was deleted in most IgG3+/lgM- cells from both chromosomes. Unexpectedly, slgM+ cells from 8 day culture also contained an average of only one copy of the C, gene per cell. It remains to be seen whether C, deletion took place only in inactive chromosomes or in both chromosomes of a half-population of slgM’ cells. In the latter case, the slgM must have derived from long-lived r-chain mRNA.

Similarity of Nucleotide Common

pentamers

Sequences

Surrounding

of S regions

Recombination

Siies

TGAGC, TGGGG

Frequent tetramers surrounding S-S recombination sites in myeloma cells

TGAG. TGGG

Sequences surrounding S region recombination sites in E. coli

TGAG, TGGG, TGGT

Chi sequence

GCTGGTGG

The presence of more than one lg class on the B lymphocyte surface has been difficult to explain by the deletion model. The most frequent double-bearers carry F and 6 chains with the same VH region. Since the C, gene exits very close to the C, gene, these genes could be cotranscribed and expressed by an RNA processing mechanism (Liu et al., Science 209, 1348-1353, 1980). Studies with p + b coexpressing lymphocytes, lymphomas, and hybridomas have shown that the C, and Ca genes are not rearranged in these cells, and the same VH region is found in both p and 6 mRNAs (Maki et al., Cell 24, 353365, 1981; Moore et al., PNAS 78, 1800-1804, 1981; Knapp et al., PNAS 79, 2996-3000, 1982). These results support the model that the ~1and 6 mRNAs are proccessed from a large precursor RNA by differential RNA splicing. Yaoita et al. (Nature 297,697-699,1982) collected more than 80% pure p+ Z+ cells by a cell sorter from spleens of nematode-infected SJA/S mice, in which T cell defects cause accumulation of pee+e+ B lymphocytes without secretion of IgE into sera (Hirano et al., PNAS 80, 3435-3438, 1983). As these cells do not have Fc, receptors, both p and t chains are produced in these cells de novo. They found that all CH genes are in the germ-line context, although JH loci are rearranged. This result indicates that S-S recombination is not obligatory for the surface t-chain production. They proposed a two-step model for class switching, involving first the initiation of a large RNA transcript (about 180 kb for C,) encompassing multiple Cr., genes and its differential RNA splicing, then a second step including deletion of the intervening DNA segment by SS recombination. T cell help may exert its effects at the second step, in which T cells recognize the surface lg and stimulate proliferation and S-S recombination selectively in B cells bearing a particular isotype. A subclone of an Abelson-virus-transformed cell line (18-81 A-2) has been shown to switch from p to y2b producer without deletion of the C, gene (Alt et al., Nature 269, 325-331, 1982). This observation is consistent with the above two-step model, but p mRNA was no longer found. There may be an intermediate stage between the two steps, in which p mRNA synthesis is terminated. Quantitative estimation of the C, gene in this cell line would strengthen this observation because a recent report (Burrows et al., Nature 306, 243-246, 1983) indicates the deletion of the C, gene in a y2b-chainproducing pre-B cell line derived from the 18-81A line. Further support for the presence of a large multi-C,, gene transcript was provided by a recent study on IgA-producing TEPC 15 myeloma cell line (Woloschak and Liarakos, J. Cell. Biochem., in press). Total cellular poly(A)+ RNA, but not cytoplasmic poly(A)+ RNA, contained 11.5 and 2.3 kb RNA species hybridizing to C,, C,, and C,,, probes. Unfortunately, neither C, nor C&, sequences were translated. Although it is not proved that 11.5 kb RNA is a single species containing C,, Cynb, and C, sequences, the results clearly demonstrate that multiple CH genes are transcribed in TEPC 15. Since the chromosome of TEPC 15 rearranged

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for the expression of the (Y chain has the C, and Cypb sequences deleted, transcription of the C, and C+, genes must have taken place at the other “inactive” chromosome, which retains all the CH genes. If the 11.5 kb RNA carries both C, and C+ sequences, it could be an intermediate in splicing the primary transcript of more than 100 kb (which remains to be identified). Alternatively, 11.5 kb mRNA may correspond to a collection of aberrantly transcribed C,, C+, and C, sequences. They have also identified similar high molecular weight RNA species in mouse spleen and Peyer’s patch. Primer extension experiments with total cellular poly(A)+ RNA of Peyer’s patch demonstrated that a C, primer was able to prime for the synthesis of CyPb sequences while a C, primer was not. Taking all the recent observations together, we would like to elaborate the two-step model for class switching (Yaoita et al., op. cit.). We define five different biochemical stages of B cells (figure). Stage I represents 6 cells producing only IgM and expressing mostly on their surface. Stages II and III include B cells carrying two isotypes on their surface, IgM-IgD and IgM-IgG (IgE or IgA), respectively. These two stages differ from each other in the range of transcription and pair of spliced exons. B cells of stage IV express only IgG (IgE or IgA) on their surface. The final stage (V) of B cell maturation corresponds to a plasma cell, which secretes a large amount of IgG (IgE or IgA). The expression of the membrane-bound or secreted form of lg is not an absolute difference and is defined for each stage by the major species produced. All of the above stages are known to exist in vivo. Steps A and B involve initiation of longer transcripts and differential splicing. Stage II may not be obligatory and can be skipped (step A’). Theoretically, steps A, B, and A’ are reversible. Step C involves S-S recombination, resulting in irreversible deletion of C,, genes. Step D augments efficiency of lg gene transcription. T cells may take regulatory

recombination

of tmnrcrtpticm

splidng

effects at step C and probably at step D as discussed above. However, T-cell-independent antigens or polyclonal B cell stimulators like LPS stimulate rapid proliferation of B cells and promote S-S recombination (step C) without T cell help as described above (Radbugh and Sablitzsky, op. cit.). Several lines of evidence suggest that certain V,, regions are associated with specific CH isotypes. A possible explanation for this phenomenon is a predetermined VH-CH linkage at DNA level. Abelson-virus-transformed preS cell lines were shown to switch from p to y2b with deletion of the C, gene (Akira et al., Cell 34, 545-556, 1983; Burrows et al., op. cit.). The results provide an example of predetermined isotype expression before VL gene rearrangement. Nonetheless, it is hard to imagine how the product of V-D-J recombination determines the pair of S-S recombination It remains to be seen whether S-S recombination in pre-B cells also takes place under physiological conditions We prefer another explanation-that a V,, region associated with a Cn region of a proper effector function is selected at step C or D. Finally, a recent report that UV illumination of Epstein-Barr-virus-transformed B cell lines (IgM producers) results in class switching (Rosen and Klein, Nature 306, 189-190, 1983) provides an exciting opportunity to study molecular mechanism of class switching.