Affinity maturation and class switching
Matthias Wabl* and Charles Steinbergt
Affinity maturation and class switching of antibodies are
temporally, but not mechanistically, related processes. The
basis of affinity maturation is the selection, in the germinal
centers, of antibodies that bind the antigen better. Early in an
immune response, the selection is from the primary repertoire;
later, it is from mutants generated by hypermutation at the
immunoglobulin loci. Recently, the door has been opened
for the study of the molecular mechanism of hypermutation,
which is expected to make a major contribution to general
biology. Class switching has been studied in the past for its
obvious clinical importance, but also at the basic level of DNA
recombination. Progress in understanding class switching has
been trailing the progress made in V(D)J recombination, but
new in vitro systems and gene-targeted mice are closing the
gap.
Addresses
*Department of Microbiology and Immunology, Universityof California,
San Francisco, CA 94143-0670, USA
tBasel Institute for Immunology, Postfach, CH-4005 Basel,
Switzerland; e-mail: mutator@itsa.ucsf.edu
Current Opinion in Immunology 1996, 8:89-92
© Current Biology Lid ISSN 0952-7915
Abbreviations
C constant
D diversity
J joining
H heavy
L light
RAG recombinationactivating gene
V variable
Introduction
"Vaccination is an astonishing thing. A little prick in the
skin and the immune system deals with amazing efficiency
with the corresponding microbe. Without vaccination,
however, the system responds poorly. Many have to die for
survivors to become immune. Clearly, the virgin immune
system does not aim at any particular microbes, but
stands in readiness to deal with the entire antigenic
universe. Microbes cannot mutate out of its range, but
they have their chance during the time it takes for the
immune response to get into focus," wrote Niels Jerne
[1]. Focusing the humoral immune response is achieved
by three processes: affinity maturation; class switching; and
memory formation. All three occur at about the same time
after B-lymphocyte activation.
Before concentrating on the advances reported in the past
year, let us briefly summarize the events in B-cell differen-
tiation, as generally viewed today. Immunoglobulin genes
are assembled in the bone marrow from gene segments:
the heavy (H) chain genes at the pro-B, and the light (L)
chain genes at the pre-B cell stage. When a functional
immunoglobulin molecule is formed and inserted into the
membrane, the pre-B cell becomes, by definition, a B cell,
which travels through blood and lymph to the spleen,
lymph nodes, and other sites in the periphery. When the
immature B cell meets a self-antigen, it is taken out of
the functional pool, either by clonal deletion (i.e. physical
destruction) or anergy (i.e. functional shut down), unless
the antigen receptor is 'edited' by replacing the L chain
and/or H chain genes. Mature B cells police the periphery,
and some of them come in contact with antigen. The
antigen-specific cells home into the germinal centers of
lymphoid follicles, where they proliferate, differentiate,
hypermutate and switch their immunoglobulin class. The
architecture of the germinal center provides an intricate
meeting place for the several cells involved in the immune
response, and this seems to provide the most effective
site for selecting mutant antibodies with higher affinities
[2]. T h e cells that accumulate mutations and acquire
higher affinity for antigen are destined to become memory
cells. During the primary response, no cells of clones
from this pool will differentiate into plasma cells [3].
Upon secondary stimulation by antigen, the memory cells
proliferate and develop into plasma cells, which now
secrete antibodies of higher affinity. Neither memory cells
nor plasma cells accumulate any more mutations.
In this review, we will concern ourselves with affinity
maturation and class switching. These are two quite
distinct processes, which will be described separately. At
the end, we will consider what, if anything, they have to
do with each other.
Affinity maturation
It seems self-evident that in any large repertoire not
aimed at a particular antigen, high-affinity antibodies
must be far outnumbered by low-affinity antibodies. After
immunization, especially after repeated immunizations,
the affinity of the antibodies in the serum increases dra-
matically with time: improvements of three to four orders
of magnitude are possible. This increase in the quality
of serum antibody, which focuses the immune response
to a particular antigen, is called affinity maturation. Early
in the response, affinity maturation has been attributed
to the selection among clones of the primary repertoire,
the repertoire generated by V(D)J rearrangement. As
antigen becomes limiting, clones with higher affinity will
have a selective advantage. But the primary repertoire
alone will rarely have the high affinities associated with
hyperimmunization. Later in the response, mutants with
even higher affinities will be progressively generated by
somatic hypermutation, and these will be selected.
90 Antigenrecognition
This increase cannot go on indefinitely. Increases in
affinity due to somatic hypermutation are usually less than
a 100-fold. In a landmark commentary, Foote and Eisen
[4 °.] have reminded us of the problems of multivalency
of the antigen, multivalency of the antibody and T-cell
help in affinity maturation. T h e y also made a case for
an 'affinity ceiling', by arguing that the highest affinity
(which is the ratio of on-rate to off-rate constants)
that can be selected in a T cell dependent response
is in the order of 1010M -I. Foote and Eisen reason
that, firstly, the maximum on-rate is not determined by
the closeness of fit to the antigen, but is limited by
the diffusion coefficients of antibody and antigen; and,
secondly, that the residence time of antigen required
for signal transduction and endocytosis will limit the
selectable off-rate. Thus, better affinities than 1010M -!
(106M -1 s-1 for the on-rate controlled by the diffusion
constant, divided by 10-4s -! for an effective off-rate) are
exhibited by overzealous fellows only. And if a particular
V region combination with an initial affinity near that
value starts to walk the 'rugged landscapes' [5] of affinities,
chances are that it will be downhill.
There are several exceptions to the 'standard' case
described above. T h e response to vesicular stomatitis virus
was reported to be of high affinity early on and to lack
affinity maturation [6]. Moreover, higher affinity antibodies
observed in secondary responses are often derived from
germline V gene segments that were not represented in
the primary response.
Hypermutation in the immunoglobulin genes is the
paradigm of a site-specific, stage-specific, and lineage-
specific mutator that generates point mutations, and
the process has become amenable to molecular analysis.
In the heavy chain genes, the segments encoding the
V region is the 'epicenter' [7] of mutation, with the
frequency decreasing progressively in both 5' and 3'
directions. T h e area of hypermutation of about 2 kb
includes the flanking regions. T h e 5' boundary near the
promoter region is distinct; the 3' boundary near the
enhancer region is more loosely defined [7]. Given this
distribution of mutations it was only natural that one
stared at the immunoglobulin V region sequences and
wondered what it is that attracts hypermutation. It should
be noted, however, that at the locus encoding ~. chains,
a segment of the major intron is as hypermutable as
the complementarity-determining regions [8]. Within the
hypermutable sequences, there are sites of even higher
intrinsic mutability than their neighbors, so-called 'hot
spots' [9-12]. Consensus sequence motifs can be found for
the hotspots [12,13,14"°,15]. But it came as a small surprise
that the actual V gene segment is not needed to trigger
the process. Neuberger, Milstein and colleagues [14 .°] re-
placed the V gene segment in a transgene by the bacterial
sequences gpt and neo r in a construct that contained the
other elements needed for h y p e r m u t a t i o n - - a promoter,
the major intron enhancer, and the 3' e n h a n c e r - - a n d the
bacterial sequences underwent hypermutation during an
immune response. This ought to put to rest speculation
that all hypermutation is really hyperconversion, for such
a process requires homologous donor sequences, and there
does not seem to be any such sequences in the mouse.
That is, neo r and ~0t have been probed for many years
in transfected mouse cells and transgenic mice, and no
cross-reacting sequences have ever been found.
Another advance of the last year was of a more technical
nature. Even though the final goal is description of
hypermutation in the whole animal, the study of the
mutator components in transgenic animals is cumbersome
and often involves a heroic effort. Thus, mutations at the
immunoglobulin loci have also been studied in lympho-
cyte lines. This approach will become especially important
in the search for trans-acting factors. Early studies with
endogenous genes in lines were recently extended by
transgenic approaches. T h e laboratory of Scharff and
our own laboratory described ~t gene constructs with
all the cis-acting elements necessary for hypermutation
comparable to the rate of the endogenous gene segments
encoding the V region [16-18].
A major finding from these in vitro studies was that the
mutator preferentially acts on G--C pairs [18], as it does
in frogs [19,20°]. What else do hypermutation in a mouse
cell line and hypermutation in a frog have in common?
Antigen-driven selection is non-existent in the former and
exceedingly weak in the latter. Thus, although it is by no
means the only possible interpretation, we think that the
mutator is targeted primarily to G - C base pairs, a bias that
is obliterated by antigen-driven selection. The validity of
this contention will be tested in the near future.
Although it is clear that the selection of higher-affinity
V region combinations is related to follicular dendritic
cells and the architecture of the germinal center, the
mechanism of antigen-driven selection does not yet seem
to be ripe for molecular analysis. Perhaps this situation
would be remedied if there were a system for generating
germinal centers from cells in vitro.
Class switching
The class of an antibody molecule and, hence, its biologi-
cal function, are dependent on the constant (C) region of
the H chain. For example, the E chain of IgE mediates
an allergic reaction, and the et chain in IgA directs
the molecule to various bodily secretions. By replacing
the CH segments of the primary response--usually I1
chain--class switching focuses the immune response
to various biological effects at various sites within the
organism. Because the V region is retained, the specificity
of the antibody remains the same, but the biological
consequences of mounting an immune response changes.
Class switching is being studied at two levels: induction
of the process by T-helper cells and interleukins; and the

generation of the switched genes by D N A recombination.
T h e regulation of class switching by interleukins is an
ever expanding field, and it is a subject with direct
clinical importance (for example, see [21]). Progress in
studying the mechanism of DNA recombination has been
slower. It has been known for five years that looping
out and deletion of DNA between so-called switch
regions is what generates immunoglobulin genes other
than g and 8. In rabbits [22] and in transgenic mice, the
switch recombination can also occur between homologs
and unrelated chromosomes, respectively. What seems
to be missing is the discovery of proteins analogous
to recombination activating genes RAG-1 and RAG-2,
which are the specificity determining factors in V(D)J
recombination, and of mutant mice analogous to scid.
A first step towards this direction was accomplished
by the laboratories of Maizels and Lieber [23,24"],
who developed extrachromosomal recombination assays
that closely recapitulate DNA deletional class switch
recombination. In these assays transcription stimulates the
switch sequence-dependent recombination, but only when
directed in the physiologic orientation. It is astonishing
that these assays work in B-cell lines, in which the
endogenous genes do not switch. This is reminiscent of
the fibroblasts transfected with RAG-1 and RAG-2 genes,
in which exogenous signal sequences, but not endogenous
V(D)J gene segments are joined.
It is well established that transcription of the so-called
germline transcripts is an important harbinger of the
switch recombination. These transcripts originate 5' to the
switch regions; the spliced products contain an I exon
joined to the C region. Their transcription is controlled
by interleukins. In fact, the weight of experiments on
switching currently is on the induction and function of
these transcripts. T h e y have been called 'sterile', because
to date no translation products have been reported. Thus,
the focus is on RNA. An attractive idea is that the RNA
would bind to the switch region and make it a target for
recombination, perhaps by the loss of superhelical turns
[25]. But if the correct splice site 3' of the I exon was
deleted in mice, there was no switch recombination [26"].
T h e straightforward interpretation is that the correctly
spliced transcript, and not just nuclear trancription of the
region, is needed, but it is also possible that only the
presence of the splicing machinery is required. Devoted
transcriptionists would argue that proteins analogous to
RAG-1 and RAG-2 are not going to be found, as the
specificity of the switch reaction lies exclusively in the
interleukin-dependent elements of the I region promoter.
Radbruch and colleagues [26"] suggest that the spliced
germline transcript is part of the switch recombinase.
Our laboratory found that the germline transcripts are
translatable and thus should not be called 'sterile' (J Bachl,
Affinity maturalion and class switching Wabl and Steinberg 91
M Wabl, unpublished data). It remains to be seen whether
this fact is biologically relevant. Clearly, the application of
recombination assays, as developed by Maizels and Lieber
[23,24"] by transfections, and by ourselves in a cell-free
system [27] will settle some of the issues discussed and
allow the identification of trans-acting factors.
Conclusions
Long ago, it was thought that IgM antibodies were never
somatically mutated. Once that myth was exploded, it
was apparent that affinity maturation and class switching
have little to do with each other in a mechanistic sense.
However, because IgM, with its ten identical combining
sites, is the first antibody to appear, cooperative binding
may to some extent counterbalance low affinity. This
compensation is lost upon class switching, but affinity
maturation comes to the rescue.
T h e immune system fights fire with fire; that is, it uses
the same tricks to fight microbes that the microbes use in
attempting to circumvent the immune system. Microbes
recombine their DNA. T and B cells recombine the genes
segments encoding the antigen receptor. Microbes mutate
at a rate of about 10-8 per base pair per cell generation. B
cells hypermutate their V exons at a rate that is some four
orders of magnitude greater. Microbes invade the organism
at different sites. B cells direct antibodies to different sites
by switching the antibody class.
After remaining a mystery for so many years, hyper-
mutation, the generator of the tighter-binding mutants
necessary for affinity maturation, is now accessible for
molecular analysis. With the coarse mapping of the cis
elements needed for hypermutation and the observation of
hot spots, the focus can shift to the trans-acting factors, (i.e.
to the mutator proteins). This will integrate the study of
hypermutation at the immunoglobulin locus into the larger
area of DNA repair. On the other hand, we are far from
understanding in molecular terms the process by which
those tighter-binding mutants are selected.
T h e area of H chain class switching is much more
advanced. T h e analysis of germline transcripts proceeds
apace, and systems to analyze the components of the
putative switch recombinase have been established. Soon
the first components will be isolated, and ultimately it
ought to be possible to direct the switch recombinase to a
particular switch region and bar it from another one.
Acknowledgements
This work was supported by National Institutes for Health grant IR01
GM37699 and by funds from the Markey Trust. The Basel Institute for
Immunology was founded and is sponsored by F Hoffmann-La Roche,
Basel, Switzerland.
92 Antigen recognition
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