Immunoglobulin Class Switching

Jürgen Schmitz, Miltenyi Biotec, Bergisch Gladbach, Germany

Andreas Radbruch, Deutsches Rheuma-Forschengszentrum Berlin, Berlin, Germany

Copyright © 1998 Elsevier Ltd. All Rights Reserved.

The class of an antibody determines its functional properties and thus the kind of humoral immune response against a particular antigen. In 1964, Nos-sal and collaborators observed for the first time that individual B cells can switch the class of the immunoglobulin (Ig) they produce, while retaining the antigenic specificity. The molecular basis for this process, termed isotype or class switching, is a recombination between highly recombinogenic DNA sequences, so-called switch (S) regions. The switch recombination machinery is induced in naive and memory B cells upon activation by antigen or mitogen and targeted to distinct S regions by cytokines. These cytokines target recombination to particular S regions by inducing transcription of the respective S regions prior to recombination and this transcription is somehow functionally linked to the recombination event.

Molecular basis of immunoglobulin class switching

Mice have antibodies of the classes IgM, IgD, IgG3, IgGl, IgG2b, IgG2a, IgE and IgA, and humans of the classes IgM, IgD, IgG3, IgGl, IgG2, IgG4, IgE, IgAl and IgA2. Antibody classes differ in the Ig heavy chain constant region gene (CH gene) that is expressed. The CH genes are clustered at the 3' end of the Ig heavy chain variable region genes (VH genes). The entire murine CH gene locus spans approximately 200 kb with eight CH genes that are arranged in the order 5' C^-Q-C^-C^l -C72b-C72a-Ce-Ca 3'. The human CH gene locus extends over more than 300 kb with nine functional genes and two pseudogenes (i|>) that are ordered 5' C(i-CR-C73-Cyl-4»£-Cal-4vC^2-C74-Ce-C„2 3'.

Coexpression of C^ and C8 with the same VDJ gene on mature naive IgM"" IgD+ B cells is due to differential termination of transcription and differential splicing of transcripts encompassing both constant region genes.

The molecular basis of antibody class switching to the expression of C7, Ce, and C„ genes in activated B cells is a recombination which positions the new CH gene 3' next to the VDJ gene. The apparent sites of Ig class switch recombination are located within the S regions, highly repetitive DNA sequences which are present 5' of each Cn gene, except C8.

All murine and most humans S regions are sequenced at least partially. They are 1-10 kb in length, highly repetitive and GC-rich. Murine and human S^ are almost homogeneously composed of the two pentamer sequences GAGCT and GGGGT and the heptamer sequence (C/T)AGGTTG. All other S regions also contain multiple copies of the pentameric sequences. AH murine S regions except S^ are composed of tandem repeats that vary both in sequence and in length, with 49 bp repeats for S71, S73 and S72b, 52 bp repeats for S72a, 80 bp repeats for S„ and 40 bp repeats for St. Both human and murine S^ are more homologous to Sf and S„ than to the S7 regions, which have considerable homology among each other. The homology of murine S„ regions to S^ decreases with the distance on the chromosome (S73 > S71 > S72b > S72a). The S regions are sufficiently conserved between human and mouse to allow human S regions to be used as substrate for switch recombination in murine cells. The S^, S6 and S„ regions are more homologous between the two species than the S7 regions. The length of the S regions is subject to considerable allelic variation (length polymorphism) indicating that there is no functional requirement for a particular size of a given S region.

S regions are preferred targets not only for the switch recombination machinery but also for other bacterial and eukaryotic recombination systems. The extraordinary recombinogenic potential might be due, in part, to the formation of unusual DNA structures promoted by the repetitive character, the high degree of homology and the high GC consent of S regions.

The primary mechanism for switching that relocates a new CH gene next to the VDJ gene is an intrachromosomal recombination between a donor and an acceptor S region, with looping out and deletion of the intervening DNA as a circle, that can be isolated from switched cells. In rare cases the deleted DNA is reintegrated in inverted form either back into the CH gene locus or even into other regions of the genome.

More than 150 DNA sequences at S region recombination sites have been analyzed. Within S regions, switch recombination breakpoints are evenly distributed, without apparent preference of a particular area within the S region and without specific signal sequences present at or juxtaposed to all switch recombination sites. Some recombination sites reveal a few base pairs of homology between donor and acceptor S region. Analyzing switch recombination sites is difficult and results have to be taken with care due to the possibility that during amplification and cloning of recombined switch sequences, additional recombinations might take place in bacteria used for cloning and the sites identified may be not the sites of the primary switch recombination event in B cells.

Mitotic switch recombination by unequal crossing-over between sister chromatids may occur occasionally. It results in the asymmetric segregation of Cn genes, giving rise to one daughter cell with a deletion of CM genes and another with a duplication.

Switch recombinations normally occur in cis, but S regions can also be recombined in trans. This has been demonstrated in several B cell lines, for normal B cells of transgenic mice with an additional Sp region integrated outside the CH gene locus, and at a surprisingly high frequency for rabbit B cells. Transchromosomal recombination is also responsible for some c-rayc gene translocations, in which only one of the recombination partners is an S region.

Coexpression of IgM along with IgG, IgA or IgE on the surface, with all Cu genes still in germline configuration, has been reported in both normal and transformed B cells. However, in most cases it has not been ruled out that Ig of one of the classes had been passively picked up, e.g. by class-specific Fc receptors. If cells expressing IgM together with other, non-IgD isotypes exist, they might do so either by differential processing of long transcripts spanning the VDJ, C^, G,, Cu and Ce genes or by trans-splicing of the VDJ exon from primary VDJ-C^ transcripts to Cy, Cu or Ce exons from Cy, Ca or Cf switch transcripts (see below). Both models have been proposed, but so far clear evidence showing that either or both of them are of biological relevance has not yet been provided. Instead, it has been shown that surface IgAl-bearing B lymphocytes from human peripheral blood, i.e. IgAl+ memory B cells, express only IgAl and have already performed switch recombination, as seen in IgA-secreting plasma cells. This leaves little room for a physiological role of Ig class switching without switch recombination, except for IgD expression.

Induction of switch recombination

The switch recombination machinery can be induced in naive IgM- and IgD-expressing B cells and in memory B cells in vitro, by cross-linking surface Ig with mimics of T cell-independent type 2 (TI-2

antigens) such as anti-Ig antibody conjugated to dex-tran, by B cell mitogens (TI-1 antigens) such as bacterial lipopolysaccharide (LPS), or by activated T cells. Triggering of CD40 on B cells by its hgand (CD40L) on activated T cells, appears to play a crucial role for switch induction in the process of T cell-dependent B cell activation. Point mutations or deletions in the gene encoding the CD40L are responsible for the X-linked hyper-IgM syndrome, which is characterized by dramatically reduced levels of serum Ig of classes other than IgM, reflecting a severe defect in T cell-dependent immune responses and Ig class switching. Similarly, CD40- and CD40L-deficient mice show normal IgM but no detectable IgG, IgE and IgA antibody responses to T cell-dependent antigens, while antibody responses to type II T cell-independent antigens are not affected.

Within a period of about 2 days after onset of B cell stimulation, activated B cells begin to undergo class switching at high frequency. Frequencies of switched cells can be as high as 10% per generation. Switch recombination is not necessarily a one-hit event in individual cells. Usually activated B cells undergo switch recombination not only on the expressed but also on the allelically excluded IgH allele.

Intra-S region recombinations can also occur in switching cells, especially involving sequences in the area of the SM pentameric repeats and 5' thereof, which are frequently deleted on the chromosome after the switch re combination between two different S regions has taken place. Intra-S region recombinations are also observed on S regions of non-switched excluded IgH alleles, in particular on SM, and on downstream S regions, the latter in the case of S^-inactivated cells. Secondary or even tertiary switches involving two or more S regions downstream of S^ on the chromosome can also occur, allowing B cells to switch sequentially to different isotypes. This seems to be the dominant although not obligatory pathway for switching to IgE, which often occurs via IgGl in mice or IgG4 in humans.

All switch recombinations inducing B cell differentiation signals also induce DNA synthesis and proliferation in resting B cells, but it is not clear whether replication of DNA is required for switch recombination. Several groups have reported evidence that DNA synthesis is associated with switch recombination since inhibition of DNA synthesis in B cells also inhibits switching. Small mutations (substitutions, insertions and deletions), indicative of error-prone DNA synthesis, have been identified near the sites of switch recombination.

In hybrid cells of class switch-performing activated B cell blasts and immortalized plasma cells, switch recombination is arrested indicating that the switch recombination machinery may be actively turned off by an inhibitor in the plasma cell stage.

Directing switch recombination to distinct switch regions

The choice of the S regions involved in class switch recombination in individual cells is not random. This first became evident from the observation that switch recombinations involving S regions of the same class of Ig occur on both IgH alleles of individual switched cells. Since those B cells could hardly have been selected for a particular switch recombination on a non-expressed IgH allele, this analysis has shown that activated B cells expressing particular isotypes are committed to switch to particular classes of Ig.

When and how is this commitment induced? It is clear that, depending on the mode of activation of the B cell and dependent on cytokines produced by T cells or other cells, including natural killer (NK) cells, mast cells, basophils and macrophages, distinct S regions are targeted for recombination in activated B cells. In the mouse, three cytokines, interleukin 4 (IL-4), interferon y (IFNy), and transforming growth factor (3 (TGF0), have been shown to play pivotal roles in this process; others may be identified in the future. IL-4 targets the S71 and, at higher concentrations, also the Sf region for switch recombination, IFNy targets the S73 and S72a regions for switch recombination, and TGF(3 targets the S72b and S„ regions for switching. Thus, each of the three cytokines regulates two of the six non-IgM, non-IgD murine Ig isotypes in a positive manner. Furthermore, in the mouse, IFNy suppresses IL-4-induced switching to IgGl and IgE, while IL-4 suppresses IFNy-induced switching to IgG3 and IgG2b. Neither IL-4 nor IFNy are obligatory for switch induction. IFNy receptor-deficient mice can still produce IgG3 and IgG2b, and IL-4-deficient mice can still produce IgGl, albeit in reduced amounts. For human B cells, less is known about the signals that induce or suppress certain S regions for recombination, except that both IL-4 and IL-13 target the S^4 and Se regions for switch recombination, probably by signaling via a shared receptor component of the IL-4 and IL-13 receptors.

At the molecular level, targeting of certain S regions for switch recombination is strictly correlated with induction of transcription of those S regions. This has been well established for cell lines which are stably committed to switch to particular Ig isotypes, as well as for murine and human B cells stimulated by switch-targeting cytokines. Human and murine S region transcripts (switch transcripts)

initiate at multiple initiation sites of a promoter, the IH promoter, located upstream of the respective S region, proceed through the S region and terminate at the normal polyadenylation sites downstream of the respective CH gene. The primary transcripts are processed into mature switch transcripts and transported into the cytosol. The processed switch transcripts, also called 'sterile' or 'germline' transcripts, differ from the productive VDJ Cn mRNAs by an exon, the IH exon, encoded just 5' of the S region that, instead of the VDJ exon, is spliced to the first CH exon. Although transcription from the VH promoter proceeds through the S^ region after VDJ recombination, in addition switch transcripts containing an exon initiate from the Iu promoter. After switch recombination, transcription from this promoter proceeds through the hybrid S region and hybrid switch transcripts containing the l,t exon and the exons of the switched Q ( gene are generated. Transcription of hybrid S regions might be required for sequential switching to downstream isotypes. Due to stop codons in the IH exons, switch transcripts are not translated into proteins (i.e. they are sterile). At most, short sequences may be translated into peptides.

The functional dependency of switch recombination on S region transcription has been effectively-demonstrated by targeted deletion or replacement in murine genomes of the LI and I72b exons together with their cytokine-responsive promoters, which results in an almost complete and selective deficiency in germline transcription of the adjacent S regions as well as switch recombination to S7I and S72b, respectively. Deletion of the JH gene segments and the promoter leads to a drastic reduction of switch recombination involving S^.

Along with transcription of S regions, cytokines such as IL-4 have also been shown to induce selective hypomethylation of previously hypermethylated CpG dinucleotides and to induce DNAase 1 hypersensitive sites at the regions of initiation of germline transcription. In general, induction of demethylation and of DNAase I hypersensitivity has been shown to reflect binding of inducible transcription factors to promoter and/or enhancer DNA sequences. Identification and characterization of the cytokine-respon-sive transcription elements that control S region transcription and cloning of the cytokine-inducible nuclear transcription factors that bind to these elements is just in its infancy. At present most information is available on the murine If promoter. A minimal 179 bp Ie promoter sequence was shown to impart IL-4-inducible and IFNy-suppressive transcription to a heterologous reporter gene. Using elec-trophoretic mobility shift assays (EMSA), several nuclear protein complexes have been demonstrated to bind to this region, but only two of the proteins involved have been cloned so far, STAT6 (STF-IL4) and BSAP. STAT6 is a member of the signal transducer and activator of transcription family of proteins that is activated by tyrosine phosphorylation in response to IL-4. STAT6-null mice are deficient in class switching to IgE. BSAP, also referred to as sa-BP or NF-sp-Bl, is an early B cell lineage-specific activator protein which binds to an indispensable DNA element of the Ie promoter, but also to several other regions within the murine IgH locus, including sites 5' of the S„ and S72a region, repetitive sequence motifs of the S(l region and sequences within the Ig 3' C„ enhancer.

Molecular components of the switch recombinase

So far, efforts to identify components of the switch recombinase have focused on S region-binding proteins. A few such proteins such as LR1 and NFkB or SNUP, SNAP and SNIP have been cloned, but so far no functional role in switch recombination could be attributed to any of them unequivocally (Figure 1). An interesting observation is that transcription of

(A) Induction of S region transcription

jti Su Cil lv3 S'fl

l^i Cn primary switch transcripts mature switch Iranscripts

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