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214 Part III Immunologic Basis of Hematology

VDJ junctions by a nuclear enzyme known as terminal deoxynucleotidyl the µ heavy chain via a carboxyl-terminal (C-terminal) cysteine. One
transferase (TdT). Two splice variants of TdT, encoded by a single role of the surrogate light chains is to select heavy chains that will
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gene, have been identified. The short (509-amino acid) variant cata- ultimately be capable of pairing with conventional light chains. If
lyzes the addition of nontemplated nucleotides at coding joints and this does not occur, then these cells will likely be deleted.
the long (529-amino acid) form is a 3′–5′ exonuclease that catalyzes The µ heavy chain–surrogate light chain complex is additionally
the deletion of nucleotides at coding joints. Third, DNA joints that associated with two transmembrane proteins, Igα (CD79a) and
form during recombination are often imprecise and can occur at any Igβ (CD79b), and the entire complex is referred to as the pre-BCR
of several nucleotides in the germline. Although out-of-frame joints (Fig. 20.3). The intracellular tails of both Igα and Igβ contain
that cannot be transcribed may result, this junctional diversity has immunoreceptor tyrosine activation motifs (ITAMs) critical to the
the potential to generate different amino acid sequences, resulting in signaling function of the pre-BCR (Fig. 20.3, upper panel). Lipid
added diversity of the Ig repertoire. Finally, somatic mutation of V rafts that contain mediators of intracellular signaling such as Lyn
region genes can occur, usually in secondary lymphoid tissues. This are constitutively associated with the pre-BCR in human pre-B cells.
latter process, which results in an increased affinity of the antibody Cross-linking of the pre-BCR leads to an increase in Lyn kinase activ-
for antigen, is discussed in more detail in the section on secondary ity, phosphorylation of the Igβ chain, and recruitment and activation
B-cell development. within the pre-BCR complex of additional signaling intermediates,
Because the process of Ig gene recombination is random, some including spleen tyrosine kinase (Syk), B-cell linker protein (BLNK),
B cells that are self-reactive may be produced. Several mechanisms phosphatidylinositol 3-kinase (PI3K), Bruton’s tyrosine kinase (Btk),
have been proposed to account for the fate of such self-reactive VAV, and phospholipase C-γ (PLCγ2). These events lead to calcium
cells. In some cases, the presence of self-antigen may not activate flux and activation of signaling cascades within the pre-B cell.
self-reactive B cells. This scenario may result from weak B-cell affinity These signaling pathways are crucial in developing pre-B cells.
for the antigen or the autoantigen may be present at an extremely One of the best examples of this requirement is the prototypical
low concentration. In other instances, interaction of antigen with the humoral immunodeficiency, X-linked agammaglobulinemia (XLA).
autoreactive B cell may result in anergy. The level of membrane Ig on XLA results from mutations within the gene segments that encode
such anergic B cells may be reduced up to 20-fold, the cell’s ability to the nonreceptor tyrosine kinase, Btk. In males who express a defective
proliferate may be impaired, and differentiation into Ig-secreting cells Btk protein, pre-B-cell clonal expansion is markedly depressed, and
may be blocked. Finally, self-reactive B cells may be clonally deleted. there is an almost complete loss of immature B cells in the BM and in
Clonal deletion may result from cytolysis by other cells, such as secondary lymphoid organs. As a result, affected males develop recur-
BM macrophages, or autoreactive B cells may undergo a physiologic rent bacterial infections early in life because of a profound decrease
change resulting in cell death after receptor engagement. 11 in circulating Ig. A nearly identical clinical phenotype also has been
The recognition of self-antigen by a B cell may not necessarily observed in persons with mutations in additional components of
result in anergy or deletion but instead may lead to receptor editing. the pre-BCR signaling complex, including the µ heavy chain, λ5,
In this process, which represents the most common mechanism for Igα, Igβ, the key B-cell adaptor protein BLNK, and the lipid kinase
negative selection, rearranged κ light chain alleles can be replaced PLCγ2.
by secondary rearrangements of upstream Vκ genes to downstream, How signaling through the pre-BCR is initiated is unclear. It has
unrearranged Jκ segments. These secondary rearrangements, which been suggested that this occurs by binding of the extracellular portion
may delete the primary VκJκ complex or separate it from Cκ by of the pre-BCR to an environmental ligand. The identification of
inversion, are possible because of the continual presence of unrear- such ligands has been difficult, although galectin-1 may function in
ranged Vκ regions upstream of the joined VκJκ coding segments. this capacity. It has also been proposed that constitutive signaling
Finally, unsuccessful rearrangements of the initial κ light chain allele occurs after pre-BCR surface expression. Recent structural studies
are followed by rearrangements of the second allele, increasing the suggest that the pre-BCR constitutively assembles as an oligomer,
likelihood of generating a less self-reactive B-cell receptor (BCR). providing a potential mechanism for this behavior. 13,14

THE PRO-B- AND PRE-B-CELL CHECKPOINTS The Pre-B- to B-Cell Transition

B-cell progenitors progress through two critical checkpoints as they Pre-BCR-expressing cells exhibit two distinct functional responses.
mature into B lymphocytes. The first occurs at the pro-B- to pre-B- Initially they undergo several rounds of proliferation, which
cell transition. If successful, pro-B cells mature into pre-B cells that expands the size of the clone that expresses a particular µ-heavy
express the pre-BCR. The second transition occurs as pre-B cells chain. Rag gene expression is also suppressed in these proliferating
mature into B cells that express the BCR. cells, which contributes to allelic exclusion. However, at some point
these pre-BCR-expressing cells exit the cell cycle and reactivate the
recombinatorial machinery so that light chain gene recombination
The Pro-B- to Pre-B-Cell Transition can commence.
Recent studies indicate that a highly regulated balance between
The key event during the pro-B- to pre-B-cell transition is the signaling through the IL-7 receptor (discussed later) and the pre-BCR
rearrangement and expression of the Ig heavy chain genes. It is ensures that proliferation and Ig recombination are mutually exclu-
important to recognize that not all pro-B cells successfully navigate sive events. IL-7 receptor signaling stimulates cell proliferation and
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this transition. The principal reason for this is that not all Ig gene inhibits light chain gene rearrangements. In contrast, pre-BCR
recombination events are successful. For example, Ig heavy chain gene signaling represses proliferation, likely through activation of the
rearrangements are productive in only around one-third of pro-B RAS–extracellular-signal-regulated kinase (ERK) pathway as well as
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cells. Those cells with nonproductive Ig gene rearrangements undergo via limiting PI3K activity through BLNK-mediated signals. This in
apoptosis and are eliminated from the BM by resident macrophages turn leads to the expression of transcription factors, including E2A,
and stromal cells. IRF4, and PAX5, that induce cell cycle exit, RAG expression, and
However, if Ig heavy chain recombination is productive and µ light chain gene recombination. These events are strongly influenced
heavy chain protein is expressed, it appears on the surface of the by the movement of developing B-cell progenitors through various
pre-B cells in association with two additional molecules referred to as niches wherein they are exposed to different environmental signals
the surrogate light chains. The surrogate light chain proteins, Vpre-B including, most notably, transiting from a setting of high to low IL-7
and λ5, are encoded by genes located on chromosome 16 in mice availability (Fig. 20.4).
and on chromosome 22 in humans, and are noncovalently linked to Once the recombinatorial machinery is reactivated, light chain
one another. λ5 in turn is covalently linked to the CH1 domain of gene rearrangement and expression occurs. As with heavy chain