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212 Part III Immunologic Basis of Hematology

IMMUNOGLOBULIN GENE REARRANGEMENT AND is that functional Ig rearrangements are rare, so the chance that two
functional rearrangements will occur in an individual cell is extremely
EXPRESSION low. An increasingly accepted, second model of allelic exclusion is
that the expression of heavy chain protein from a successfully rear-
The defining feature of a B lymphocyte is its expression of cell surface ranged allele inhibits rearrangements at the other heavy chain allele.
Ig, which is formed by two heavy chains and two light chains, each
of which is encoded by multiple gene segments. The process of Ig
gene rearrangement occurs in a step-wise manner as murine and Light Chain Gene Rearrangement
human B cells mature through the cellular stages of development
just described. 8,9 Ig light chain protein can be encoded by the kappa (κ) or lambda
(λ) genes. Greater than 90% of murine B cells express κ protein.
However, the proportions of human κ and λ proteins are more
Heavy Chain Gene Rearrangement equivalent, with approximately 60% of human B cells expressing κ
light chain protein.
The initial Ig rearrangement events during B-cell development occur The human κ gene is located on chromosome 2 and includes
at the heavy chain locus. The Ig heavy chain locus includes multiple around 40 Vκ region genes, clustered in up to seven families, five
variable (V), diversity (D), joining (J), and constant (C) region gene functional Jκ region genes, and one Cκ region gene. The human
segments that are separated from one another by introns. The genes λ locus is located on human chromosome 22. Approximately 30
that encode Ig heavy chain protein are located on human chromo- human Vλ genes exist and are grouped into 10 families. There are
some 14 (Fig. 20.2). The V region genes are located at the 5′ end of seven human Cλ genes, four of which are functional and three of
the Ig heavy chain locus, and each consists of approximately 300 base which are pseudogenes. Each Cλ gene is located 3′ of a respective Jλ
pairs. These genes, which are separated by short intron sequences, are gene. Light chain genes do not include D region loci.
organized into seven families based on sequence homology. There are The initial event in light chain recombination involves the joining
about 25 human D region genes located 3′ to the V region. These of a V region to a J region. The VJ complex remains separated
also are grouped into families, and at least 10 have been described. from the light chain C region by an intron, the entire complex is
Downstream of the D region are six human J region genes. Finally, transcribed, and further splicing of the intron results in formation of
10 C region genes representing alternative Ig isotypes are arranged a mature V–J–C transcript. Light chain rearrangements in mice occur
in tandem. initially at the kappa locus. If rearrangements at the first κ allele are
The transcription of the unrearranged heavy chain locus occurs unsuccessful, attempts are made to rearrange the second κ gene. If
prior to actual Ig gene recombination. This results in the production this fails, the λ locus is used.
of developmentally regulated transcripts of unrearranged Ig genes,
referred to as germline or sterile transcripts. Multiple species of sterile
transcripts have been described, and some could conceivably encode The Ig Recombinatorial Machinery
proteins. A mechanistic link between transcription and Ig gene rear-
rangement has been hypothesized. For example, transcription might The process of Ig heavy and light chain gene rearrangement is
make unrearranged Ig genes accessible to both RNA polymerase dependent on enzymes that delete intronic sequences and join coding
and V(D)J recombinase, the germline transcripts could function in segments of DNA. The enzymes that mediate these functions act
the rearrangement reaction, or transcription could alter structural through recognition of recombination signal sequences that are
characteristics of DNA, making the recombination signal sequences, located 3′ of each heavy chain V region exon, 5′ of each heavy
described later, better targets for recombination. chain J segment, and 5′ and 3′ of each heavy chain D region gene.
The initial event during heavy chain gene rearrangement occurs Fig. 20.2 shows the association of these recognition sequences with
as early as the CLP stage and juxtaposes a D region segment to a J the various heavy chain exons. Each recombination signal sequence
segment. Although in theory any D region gene can join with equal consists of conserved heptamer and nonamer sequences separated by
frequency to any J region gene, there may be preferential utilization nonconserved DNA segments of 12 or 23 base pairs. During Ig gene
of selected D and J region genes at various times during fetal and recombination, these recognition sequences form loops of DNA,
adult B-cell development. The next recombination event involves the which in turn bring the coding exons in apposition to one another.
rearrangement of a V region gene to the D–J complex, and this occurs These noncoding loops are subsequently deleted and degraded.
at the pro-B-cell stage of development. Evidence suggests that biased The expression of two highly conserved proteins, referred to as
usage of J proximal V genes occurs in the newly generated repertoire recombinase-activating genes-1 (RAG-1) and RAG-2, is required for
10
of neonatal mice and humans. The heavy chain C region remains heavy and light chain gene recombination. Mice and humans in
separated from the rearranged VDJ complex by an intron, and this whom Rag genes are not expressed do not generate B or T cells. Results
entire sequence is transcribed. RNA processing subsequently leads from cell-free systems that measure V(D)J recombination indicate
to deletion of the intron between the VDJ complex and the most that RAG proteins are involved in cleavage of DNA at recombination
proximal C region genes. After translation, µ heavy chain protein is signal sequences and the subsequent joining of coding sequences to
expressed in the cytoplasm of pre-B cells (Fig. 20.2). one another. In addition to the RAG proteins, general DNA repair
The E2A-encoded transcription factors are particularly important enzymes, those encoded by the Ku complex of genes in particular, also
for Ig gene recombination and mediate their effects via binding to play a critical role in Ig heavy chain gene recombination.
specific promoter sequences located 5′ of each heavy chain V region
and one or more heavy chain enhancer regions located 3′ of the J
region genes and downstream from the CH region genes (see Fig. Generation and Selection of the Primary B-Cell
20.2). Before Ig gene rearrangement, E12 and E47 proteins may be Repertoire
in an inactive state owing to their heterodimeric association with
another protein known as Id. In this configuration, DNA binding For the organism to mount an effective humoral immune response,
by E12 and E47 does not occur. Thus, successful transition from the an array of Igs with unique antigen-binding specificities, together
pro-B- to pre-B-cell stage is dependent on cessation of Id expression. referred to as the Ig repertoire, must be generated. Several mechanisms
This conclusion is consistent with the fact that mice expressing an Id have evolved to ensure that this occurs.
transgene have a complete block in B-cell differentiation. First, heavy and light chain proteins can be encoded by multiple
Each pro-B cell has two Ig heavy chain genes, but only one of germline V, J, and, in the case of the heavy chain, D region genes,
these encodes heavy chain protein in any given cell. This phenom- and the combinatorial diversity among them is enormous. Second,
enon is known as allelic exclusion. One theory for how this occurs nucleotides not encoded in the germline can be added to D–J and