B-Cell Development

Murine B-Cell Development

As the progeny of murine HSCs differentiate, they generate multipotent progenitors (MPPs). MPPs include four phenotypically separable subsets; MPP1s are short-term HSCs, MPP2s and MPP3s are myeloerythroid biased, and MPP4s primarily generate lymphoid progeny. MPP4s are presumably the precursors of most lineage-negative (Lin ⁻ ) CD117(c-kit) ^low Sca-1 ^low CD127 ⁺ (interleukin-7 receptor α) common lymphoid progenitors (CLPs). Lin ⁻ indicates that the cells lack expression of determinants present on mature myeloid, erythroid, and lymphoid lineage cells. CLPs then mature through pre-pro-B, pro-B, pre-B-, and B-cell stages of development that can be phenotypically resolved based on their expression of various cell surface and cytoplasmic determinants ( Fig. 22.1 ). The stages of development depicted in Fig. 22.1 are based on the scheme originally defined by Hardy.

Human B-Cell Development

The human MPP compartment is not as well defined as in the mouse, but various downstream stages of development which parallel those in the mouse can be phenotypically identified. For example, CLPs sequentially generate pro-B, pre-B, and B cells each of which expresses distinct combinations of cell surface determinants. Fig. 22.1 shows that human pre-B and newly produced B cells express CD20. It is relevant to note this determinant because antibodies that recognize it (rituximab) are in widespread clinical use for the treatment of lymphoma and, increasingly, autoimmune diseases. Developing and mature B-lineage cells express additional cell surface determinants that include CD21, CD22, CD24, CD38, and CD40, several of which are linked to critical intracellular signaling pathways.

Transcriptional Regulation of B-Cell Development

The generation of lymphoid cells from HSCs is dependent on expression of the Ets family member PU.1. Mice in which Spi1 , the gene encoding PU.1, is not expressed produce erythroid and megakaryocytic but not monocytic, granulocytic, and lymphoid cells and die during the fetal or neonatal period. PU.1 regulates several events that promote B lineage specification, including expression of CD135 (Flt3), which is expressed on immature progenitors such as the CLP, CD45R(B220), and CD127 (IL-7Rα). However, deletion of Spi1 at the CLP stage of development has no negative effect on B-cell development, indicating that PU.1 expression is not required once B-cell specification has initiated.

Further B lineage specification is dependent on expression of additional transcription factors including early B-cell factor (EBF) and the E2A-encoded splice variants E12 and E47. Each of these DNA-binding proteins regulates the expression of a variety of B-lineage target genes. For example, Ebf1 regulates the expression of Igα, VpreB, λ5, and Pax5 and represses genes associated with alternative lineage fates. Mice in which EBF and E2A are not expressed exhibit an almost complete block in B-cell development at the pro-B-cell stage.

Ebf1 and E2A-expressing progenitors can still exhibit some non-B lineage potential, indicating that the expression of these DNA binding proteins does not result in absolute B lineage commitment. Instead, this is dependent on expression of the Pax5 transcription factor. Phenotypically identifiable B-cell precursors are present in Pax5 knock-out mice, and these cells can differentiate into myeloid, T, and natural killer (NK) cells. However, if the gene encoding Pax5 is introduced into Pax5 -deficient precursors, this developmental promiscuity ceases. Thus, a critical function of Pax5 is to suppress non-B lineage potential. For example, Pax5 may repress expression of myeloid growth factor receptors and inhibit T-cell potential by antagonizing expression of Notch1, a cell-surface receptor required for commitment to the T-cell lineage. Continued Pax5 expression is necessary to maintain lineage fidelity even in relatively mature B cells.

Additional transcription factors, such as Ikaros, Satb1, Foxo1, IRF4, IRF8, c-Myb, Gfi1, Miz-1, Bcl6, and Bach2, function at various times during B-cell development. For example, IRF4 is involved with Ig recombination and the attenuation of IL-7 signaling, thus promoting the transition from the pre-B to B cell stages of maturation. IRF8, along with PU.1, regulates EBF expression. Ikaros plays a role in regulating expression of key B lineage genes such as IL-7Rα and EBF and promoting B lineage commitment. c-Myb has been shown to synergize with PU.1 to activate IL-7 receptor (IL-7R) gene transcription. Focused reviews should be consulted for a full discussion of these and additional transcriptional regulators of B lymphopoiesis.

MicroRNAs

MicroRNAs (miRNAs) are 19 to 23 nucleotide-long RNA molecules that are processed from longer RNA precursors. miRNAs act post-transcriptionally, by promoting degradation of mRNA targets or by blocking their translation. However, like transcriptional regulators, a single miRNA can potentially regulate many targets to provide coordinated and simultaneous regulation of a network of genes.

Mice with conditional deletion of Dicer, an enzyme necessary for miRNA synthesis do not develop B lymphocytes, indicating the importance of miRNA regulatory mechanisms during B-cell differentiation. Work is now ongoing to identify the role of specific miRNAs at distinct stages of B-cell development. For example, deletion of miR-17~92 cluster blocks pro-B-cell maturation.

Heavy Chain Gene Rearrangement

The initial Ig rearrangement event occurs at the heavy chain locus located on human chromosome 14 ( Fig. 22.2 ). The Ig heavy chain locus includes multiple variable (V), diversity (D), joining (J), and constant (C) region gene segments that are separated from one another by introns. The V region genes are located at the 5′ end of the Ig heavy chain locus, and each consists of approximately 300 base pairs. These genes, which are separated by short intron sequences, are organized into families based on sequence homology. There are ~25 human D region genes located 3′ to the V region that are also grouped into families based on sequence homology. Downstream of the D region are six human J region genes. Finally, 10 C region genes representing alternative Ig isotypes are arranged in tandem.

The transcription of the unrearranged heavy chain locus occurs prior to actual Ig gene recombination. This results in production 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 proteins. A mechanistic link between transcription and Ig gene rearrangement has been hypothesized. For example, transcription could make unrearranged Ig genes accessible to both RNA polymerase and V(D)J recombinase, the germline transcripts could function in the rearrangement reaction, or transcription could alter structural characteristics of DNA, making the recombination signal sequences described below better targets for recombination.

The initial event during heavy chain gene rearrangement occurs by the CLP stage and juxtaposes a D region segment to a J region segment. Although in theory any D region gene can join with equal frequency to any J region gene, there may be preferential utilization of selected D and J region genes at various times during fetal and adult B-cell development. The next recombination event involves the rearrangement of a V region gene to the D–J complex, and this occurs at the pro-B-cell stage of development. The biased usage of J proximal V genes may occur in the newly generated repertoire of neonatal mice and humans. The heavy chain C region remains separated from the rearranged VDJ complex by an intron, and this entire sequence is transcribed. RNA processing subsequently leads to deletion of the intron between the VDJ complex and the most proximal C region genes. After translation, μ heavy chain protein is expressed in the cytoplasm of pre-B cells (see Fig. 22.2 ).

The E2A encoded transcription factors are particularly important for Ig gene recombination and mediate their effects via binding to specific promoter sequences located 5′ of each heavy chain V region and enhancer regions located 3′ of the J region genes and downstream from the CH region genes (see Fig. 22.2 ). Before Ig gene rearrangement, E12 and E47 proteins may be in an inactive state owing to their heterodimeric association with another protein known as Id. Successful transition from the pro-B to pre-B-cell stage is dependent on cessation of Id expression, a conclusion consistent with the fact that mice expressing an Id transgene have a complete block in B-cell differentiation.

Each pro-B cell has two Ig heavy chain genes, but only one of these encodes heavy chain protein in any given cell. This phenomenon is known as allelic exclusion. One theory for how this occurs is that functional Ig rearrangements are rare, so the chance that two functional rearrangements will occur in an individual cell is extremely low. Alternatively, the expression of heavy chain protein from a successfully rearranged allele may inhibit rearrangements at the other heavy chain allele.