FIGURE 8-15 Ig heavy and light chain gene recombination and expression. The sequence of DNA recombination and gene expression events is shown for the Ig |i heavy chain (A) and the Ig k light chain (B). In the example shown in A, the V region of the |i heavy chain is encoded by the exons V1, D2, and J1. In the example shown in B, the V region of the k chain is encoded by the exons V2 and J1.

FIGURE 8-15 Ig heavy and light chain gene recombination and expression. The sequence of DNA recombination and gene expression events is shown for the Ig |i heavy chain (A) and the Ig k light chain (B). In the example shown in A, the V region of the |i heavy chain is encoded by the exons V1, D2, and J1. In the example shown in B, the V region of the k chain is encoded by the exons V2 and J1.

intron. The rearranged Ig heavy chain gene is transcribed to produce a primary transcript that includes the rearranged VDJ complex and the C| exons. The C| nuclear RNA is cleaved downstream of one of two consensus polyadenylation sites, and multiple adenine nucleotides, called poly-A tails, are added to the 3' end. This nuclear RNA undergoes splicing, an RNA processing event in which the introns are removed and exons joined together. In the case of the | RNA, introns between the leader exon and the VDJ exon, between the VDJ exon and the first exon of the C| locus, and between each of the subsequent constant region exons of C| are removed, thus giving rise to a spliced mRNA for the | heavy chain. If the mRNA is derived from an Ig locus at which rearrangement was productive, translation of the rearranged | heavy chain mRNA leads to synthesis of the | protein. For a rearrangement to be productive (in the correct reading frame), bases must be added or removed at junctions in multiples of three. This ensures that the rearranged Ig gene will be able to correctly encode an Ig protein. Approximately half of all pro-B cells make productive rearrangements at the Ig H locus on at least one chromosome and can thus go on to synthesize the | heavy chain protein. Only cells that make productive rearrangements survive and differentiate further.

Once a productive Ig | rearrangement is made, a cell ceases to be called a pro-B cell and has differentiated into the pre-B stage. Pre-B cells are developing B lineage cells that express the Ig | protein but have yet to rearrange their light chain loci. The pre-B cell expresses the | heavy chain on the cell surface, in association with other proteins, as the pre-B cell receptor, which has several important roles in B cell maturation.

The Pre-B Cell Receptor

Complexes of y, surrogate light chains, and signal-transducing proteins called Iga and Igp form the pre-antigen receptor of the B lineage, known as the pre-B cell receptor (pre-BCR). The | heavy chain associates with the X5 and V pre-B proteins, also called surrogate light chains because they are structurally homologous to k and X light chains but are invariant (i.e., they are identical in all pre-B cells) and are synthesized only in pro-B and pre-B cells (Fig. 8-16A). Iga and IgP also form part of the B cell receptor in mature B cells (see Chapter 7). Signals from the pre-BCR drive the pro-B to pre-B transition and are responsible for the largest proliferative expansion of B lineage cells in the bone marrow. It is not known what the pre-BCR recognizes; the consensus view at present is that this receptor functions in a ligand-independent manner and that it is activated by the process of assembly (i.e., the fully assembled receptor is in the "on" mode). The importance of pre-BCRs is illustrated by studies of knockout mice and rare cases of

•Inhibition of H chain recombination (allelic exclusion)

•Proliferation of pre-B cells •Stimulation of k light chain recombination •Shutoff of surrogate light chain transcription

•Inhibition of p chain gene recombination •Proliferation of pre-T cells •Stimulation of a chain recombination •Expression of CD4 and CD8 •Shutoff of pTa transcription

FIGURE 8-16 Pre-B cell and pre-T cell receptors. The pre-B cell receptor (A) and the pre-T cell receptor (B) are expressed during the pre-B and pre-T cell stages of maturation, respectively, and both receptors share similar structures and functions. The pre-B cell receptor is composed of the | heavy chain and an invariant surrogate light chain. The surrogate light chain is composed of two proteins, the V pre-B protein, which is homologous to a light chain V domain, and a X5 protein that is covalently attached to the | heavy chain by a disulfide bond. The pre-T cell receptor is composed of the TCR p chain and the invariant pre-T a (pTa)chain. The pre-B cell receptor is associated with the Iga and IgP signaling molecules that are part of the BCR complex in mature B cells (see Chapter 9), and the pre-T cell receptor associates with the CD3 and Z proteins that are part of the TCR complex in mature T cells (see Chapter 7).

human deficiencies of these receptors. For instance, in mice, knockout of the gene encoding the | chain or one of the surrogate light chains results in markedly reduced numbers of mature B cells because development is blocked at the pro-B stage.

The expression of the pre-BCR is the first checkpoint in B cell maturation. Numerous signaling molecules linked to both the pre-BCR and the BCR are required for cells to successfully negotiate the pre-BCR-mediated checkpoint at the pro-B to pre-B cell transition. A kinase called Bruton's tyrosine kinase (Btk) is activated downstream of the pre-BCR and is required for delivery of signals from this receptor that mediate survival, proliferation, and maturation at and beyond the pre-B cell stage. In humans, mutations in the BTK gene result in the disease called X-linked agammaglobulinemia (XLA), which is characterized by a failure of B cell maturation (see Chapter 20). In mice, mutations in btk result in a less severe B cell defect in a mouse strain called Xid (for X-linked immunodeficiency). The defect is less severe than in XLA because murine pre-B cells express a second Btk-like kinase called Tec that compensates for the defective Btk.

The pre-BCR regulates further rearrangement of Ig genes in two ways. First, if a | protein is produced from the recombined heavy chain locus on one chromosome and forms a pre-BCR, this receptor signals to irreversibly inhibit rearrangement of the Ig heavy chain locus on the other chromosome. If the first rearrangement is nonproductive, the heavy chain allele on the other chromosome can complete VDJ rearrangement at the Ig H locus. Thus, in any B cell clone, one heavy chain allele is productively rearranged and expressed, and the other is either retained in the germline configuration or nonproductively rearranged. As a result, an individual B cell can express Ig heavy chain proteins encoded by only one of the two inherited alleles. This phenomenon is called allelic exclusion, and it ensures that every B cell will express a single receptor, thus maintaining clonal specificity. If both alleles undergo nonproductive Ig H gene rearrangements, the developing cell cannot produce Ig heavy chains, cannot generate a pre-BCR-dependent survival signal, and thus undergoes programmed cell death. Ig heavy chain allelic exclusion involves changes in chro-matin structure in the heavy chain locus that limit accessibility to the V(D)J recombinase.

The second way in which the pre-BCR regulates the production of the antigen receptor is by stimulating k light chain gene rearrangement. However, | chain expression is not absolutely required for light chain gene recombination, as shown by the finding that knockout mice lacking the | gene do initiate light chain gene rearrangements in some developing B cells (which, of course, cannot express functional antigen receptors and proceed to maturity). The pre-BCR also contributes to the inacti-vation of surrogate light chain gene expression as pre-B cells mature.

Immature B Cells

Following the pre-B cell stage, each developing B cell initially rearranges a k light chain gene, and if the rearrangement is in-frame, it will produce a k light chain protein, which associates with the previously synthesized | chain to produce a complete IgM protein. If the k locus is not productively rearranged, the cell can rearrange the X locus and again produce a complete IgM molecule. (Induction of X light chain gene rearrangement occurs mainly when Ig K-expressing B cell receptors are self-reactive, as will be discussed later). The IgM-expressing B cell is called an immature B cell. DNA recombination in the k light chain locus occurs in a similar manner as in the Ig heavy chain locus (see Fig. 8-15B). There are no D segments in the light chain loci, and therefore recombination involves only the joining of one V segment to one J segment, forming a VJ exon. This VJ exon remains separated from the C region by an intron, and this separation is retained in the primary RNA transcript. Splicing of the primary transcript results in the removal of the intron between the VJ and C exons and generates an mRNA that is translated to produce the k or X protein. In the X locus, alternative RNA splicing may lead to the use of any one of the four functional CX exons, but there is no known functional difference between the resulting types of X light chains. Production of a k protein prevents X rearrangement, and, as stated before, X rearrangement occurs only if the k rearrangement was nonproductive or if a self-reactive rearranged k light chain is deleted. As a result, an individual B cell clone can express only one of the two types of light chains; this phenomenon is called light chain isotype exclusion. As in the heavy chain locus, expression of k or X is allelically excluded and is initiated from only one of the two parental chromosomes at any given time. Also, as for heavy chains, if both alleles of both k and X chains are nonfunctional in a developing B cell, that cell fails to receive survival signals that are normally generated by the BCR and dies.

The assembled IgM molecules are expressed on the cell surface in association with Iga and Igp, where they function as specific receptors for antigens. In cells that are not strongly self-reactive, the BCR provides ligand-independent tonic signals that keep the B cell alive and also mediate the shutoff of Rag gene expression, thus preventing further Ig gene rearrangement. Immature B cells do not proliferate and differentiate in response to antigens. In fact, if they recognize antigens in the bone marrow with high avidity, which may occur if the B cells express receptors for multivalent self antigens that are present in the bone marrow, the B cells may undergo receptor editing or cell death, as described later. These processes are important for the negative selection of strongly self-reactive B cells. Immature B cells that are not strongly self-reactive leave the bone marrow and complete their maturation in the spleen before migrating to other peripheral lymphoid organs.

Subsets of Mature B Cells

Distinct subsets of B cells develop from different progenitors (Fig. 8-17). Fetal liver-derived HSCs are the precursors of B-1 B cells, described later. Bone marrow-derived HSCs give rise to the majority of B cells, which are sometimes called B-2 B cells. These cells rapidly pass through two transitional stages and can commit to development either into marginal zone B cells, also described later, or into follicular B cells. The affinity of the B

B-1 B cell

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