In T-dependent responses, some of the progeny of activated IgM- and IgD-expressing B cells undergo heavy-chain class (isotype) switching and produce antibodies with heavy chains of different classes, such as γ, α, and ε (Fig. 1). Class switching is initiated in B cells in extrafollicular foci, driven by extra follicular helper T cells, and the process continues to occur for some Ig classes in germinal centers, driven by Tfh cells in the light zone. The capacity of B cells to produce different antibody classes provides a remarkable plasticity in humoral immune responses by generating antibodies that perform distinct effector functions and are involved in defense against different types of infectious agents. B cells change the classes of the antibodies they produce by changing the constant regions of the heavy chains, but the specificity of the antibodies (which is determined by the variable regions) remains unaltered. The molecular mechanisms responsible for the change in heavy-chain constant regions are described below.
Fig1. Immunoglobulin (Ig) heavy-chain isotype switching. B cells activated by helper T-cell signals (CD40 ligand [CD40L], cytokines) undergo switching to different Ig isotypes, which mediate distinct effector functions. Selected examples of switched isotypes are shown. All isotypes are capable of neutralizing microbes and toxins. APRIL, A proliferation-inducing ligand; BAFF, B cell–activating factor; IL, interleukin; TGF-β, transforming growth factor–β.
Class switching in response to different types of pathogens is regulated by cytokines produced by the helper T cells that are activated by these pathogens.
• The humoral response to many helminthic parasites is dominated by IgE antibodies, which participate in mast cell mediated elimination of helminths; IgE antibodies also mediate immediate hypersensitivity (allergic) reactions including anaphylaxis. Some activated extrafollicular helper cells (not Th2 cells) that only make IL-4 can drive the class switch response to IgE, even in the absence of germinal centers. Specialized BCL-6 and GATA-3 expressing Tfh cells that secrete both IL-4 and IL-13 in the light zone of the germinal center are required for the generation of high-affinity IgE.
• Switching from IgM to IgG is a prominent aspect of T-dependent antibody responses against many bacteria and viruses. IgG antibodies are also transferred through the placenta to protect newborns, and they have longer half-lives in the blood than other Ig classes, so the production of IgG con tributes in many ways to the protective capacity of humoral immunity. In mice, switching to IgG sub classes is induced by the cytokine IFN-γ, which may be produced by extrafollicular T cells or Tfh cells activated by these microbes. However, there is no evidence that class switching to IgG in humans involves IFN-γ. The details of which cytokines secreted by human helper T cells drive switching differentially to IgG1 and IgG3, remain to be established. IL-10 in combination with Il-4 contribute to class switching to IgG4. Human IgG2 antibodies are mainly directed against carbohydrates and it is presumed that class switching to IgG2 might be T-independent though, as for other human IgG iso types, the contributions of specific cytokines to IgG2 class switching are not known.
• In addition, B cells in different anatomic sites switch to different Ig classes, in part because of the cytokines produced at these sites. Specifically, B cells in lymphoid organs draining mucosal tissues switch to IgA, which is the antibody class that is most efficiently transported through epithelia into mucosal secretions, where it prevents microbes from entering through the epithelia. Switching to IgA is stimulated by transforming growth factor–β (TGF-β), which is produced by many cell types in mucosal and other tissues. Cytokines of the TNF family, BAFF and APRIL, also stimulate switching to IgA. Because these latter cytokines can be produced by myeloid cells and some stromal cells, they may facilitate some IgA class switching in the absence of T-cell help. Some individuals who inherit mutant versions of the TACI gene, which encodes a receptor for these cytokines, have a selective deficiency of IgA production.
The molecular mechanism of class switching, a process called switch recombination, involves the Ig heavy-chain DNA in B cells being cut and recombined such that a previously formed VDJ exon (that encodes the V domain) is placed adjacent to a downstream C region, and the intervening DNA is deleted (Fig. 2). These DNA recombination events involve nucleotide sequences called switch regions, which are located in the introns between the J and C segments at the 5′ ends of each CH locus, other than for the δ gene. Switch regions are 1 to 10 kilobases long, contain numerous tandem repeats of GC-rich DNA sequences, and are found upstream of every heavy-chain gene. Upstream of each switch region is a small exon called the I exon (for initiator of transcription) preceded by an I region promoter. Signals from cytokines induce transcription from a particular I region promoter reading through the I exon, switch region, and adjacent CH exons. These transcripts are known as germline transcripts. They are not translated into proteins but are required for class switching to proceed. Germline transcripts are found at both the µ locus and the downstream heavy chain locus to which an activated B cell is being induced to switch. At each participating switch region, the germline transcript facilitates the generation of double-stranded DNA breaks, as described later. The DNA break in the upstream (µ) switch region is joined to the break in the downstream-selected switch region. As a result, the rearranged VDJ exon just upstream of the µ switch region in the IgM-producing B cell recombines with the Ig heavy-chain gene located immediately after the transcriptionally active downstream switch region.
Fig2. Mechanisms of Ig heavy-chain class switching. When antigen-activated B cells encounter helper T-cell signals (CD40 ligand [CD40L] and, in this example, interleukin-4), the B cells undergo switching to Ig classes other than immunoglobulin M (IgM) (in this example, IgE). These stimuli initiate germline transcrip tion through the Iε-Sε-Cε locus and the proximal CH genes are deleted, leading to recombination of the VDJ exon upstream of the µ locus with the Cε gene. Switch regions are indicated by circles labeled Sµ, Sγ, and Sε. Iµ, Iγ, and Iε represent the initiation sites for germline transcription. (Note that there are multiple Cγ genes located between Cδ and Cε and Cα genes downstream of Cε, but these are not shown.)
Cytokines determine which CH region will undergo germline transcription. For instance, IL-4 induces germline transcription through the Iε-Sε-Cε locus (see Fig.2). This leads first to the production of germline ε transcripts in an IgM-expressing B cell and then to recombination of the Sµ switch region with the Sε switch region. The intervening DNA is lost and the VDJ exon is thus brought adjacent to Cε. The end result is the production of IgE with the same V domain as that of the original IgM produced by that B cell.
The key enzyme required for class switching (and somatic hypermutation, described later) is AID. AID is a cytidine deaminase that removes an amino group from cytosines in single-stranded DNA templates, converting cytosine (C) residues to deaminated uracil (U) residues (Fig.3). How AID is targeted to switch regions is poorly understood. This enzyme has a propensity for certain GC-containing tetranucleotide motifs. Switch regions are rich in these motifs, and cytokine-induced transcription through these regions (see below) makes them accessible to AID. However, similar motifs are present throughout the genome, and the propensity of AID to target switch regions can be partially explained by the fact that these GC-rich regions contribute to increased stalling of RNA polymerase II, which, when stalled, efficiently recruits AID. (Stalling refers to the RNA polymerase complex stopping for a while at a particular site on the DNA, which provides an opportunity for the previously moving complex to recruit other repair factors and enzymes.) Switch region transcripts tend to form stable DNA-RNA hybrids involving the template strand of DNA, thus freeing up the nontemplate strand, which forms an open single-stranded DNA loop called an R-loop. The generation of single-stranded DNA by R-loop formation is critical because AID can target only single-stranded DNA. The R-loop is therefore a region where a large number of C residues in the switch DNA sequence are converted to U residues by AID. An enzyme called uracil N-glycosylase (UNG) removes the U residues, leaving abasicsites. The APE1 endonuclease cleaves these abasicsites, generating a nick at each position. While R-loops facilitate the formation of discontinuities in the nontemplate strand of DNA, a break in double-stranded DNA requires that nicks also be generated on the opposite template strand of DNA. The GC-rich switch region RNA that remains tightly bound to the template strand DNA is degraded by a protein complex called the RNA exosome, thus exposing C residues transiently on the template strand and allowing AID, UNG, and APE1 to generate some nicks on this strand as well. Nicks that are generated on both strands contribute to double stranded breaks both in the Sµ “donor” switch region and in the downstream “acceptor” switch region that is involved in a particular class switch event. The double-stranded breaks in the two switch regions are joined together (ligated) by use of the machinery involved in double-stranded break repair by nonhomologous end joining. In this process, the DNA between the two switch regions is deleted, and the net result is that the original rearranged V region DNA is fused to a new constant region.
Fig3. Mechanism by which activation-induced deaminase generates double-stranded breaks at switch regions. Germline transcripts form DNA-RNA hybrids in the switch region and activation-induced cytidine deaminase (AID) deaminates C residues to generate U residues in single stranded DNA. Uracil N-glycosylase (UNG) removes U residues to generate abasicsites where the APE1 endonuclease creates nicks that lead to a double-stranded break. While this figure only illustrates the generation of a double-stranded break in the μ switch region, a similar double-stranded break occurs around the same time in the switch region for a downstream isotype, thus facilitating switch recombination and isotype switching.
