B-Cell Development

Introduction

Mammalian b-cell development occurs via a series of sequential developmental transitions that culminate in the establishment of a protective antibody repertoire that is essential for the host’s immunity to microbial pathogens. Primary antibody deficiencies can result from mutations in genes that are expressed in B-lineage cells and are critical for normal development and function. This chapter focuses on the developmental biology of human B-lineage cells, the mechanisms by which B cells generate their diverse B-cell receptor (BCR) and antibody repertoires, and gene mutations that block the development of antibody-mediated immunity.

Sites of Human B-Cell Development

Similar to all blood cells that reside in the periphery, B lymphocytes are derived from hematopoietic stem cells (HSCs). The liver and omentum are the initial sites of B lymphopoiesis in the human fetus, beginning at approximately 8 weeks’ gestation. Between 12 and 15 weeks’ gestation, HSCs begin to home to the bone marrow, at which time multiple stages of B-lineage cells are present. Human B-cell development continues in the marrow from the midtrimester of fetal development through at least the eighth decade of life, in contrast to T cells, which appear to develop primarily until the fourth decade of life.

Stages of Marrow B-CELL Development

Development of mammalian blood cells from HSCs is mediated by cytokines that regulate survival, proliferation, and differentiation; transcription factors that regulate gene expression; and the microenvironment of primary lymphoid organs such as fetal liver and marrow. The stages of mammalian B-cell development have historically been defined by two methods: (1) detection of the rearrangement and expression of immunoglobulin (Ig) gene segments and (2) measurement of cell-surface molecules, termed cluster of differentiation (CD) markers, using monoclonal antibodies.

B-lineage cells in primary lymphoid tissue are derived from lymphohematopoietic progenitors. Multilymphoid progenitors have the capacity to develop into all T, B, and/or natural killer (NK) cells, with limited fitness to develop into myeloid cells. As shown in Fig. 117.1 , one model suggests that multilymphoid progenitors can differentiate into early thymic progenitors, which develop into all T-lineage cells, or a common B/NK progenitor, which develops into B-lineage or NK cells.

Rearrangement of genomic deoxyribonucleic acid (DNA) at the Ig heavy (IgH) and light (IgL) chain loci ^, distinguishes B-lineage cells from all other cells in the body and is thus a signature event in commitment to the B-lineage. The Ig loci are located on three human chromosomes: heavy chain (IGH) is at 14q32, the kappa (κ) light chain (IGK) at 2p11, and the lambda (λ) light chain (IGL) at 22q11; each of these loci contains arrays of variable (V), diversity (D; only for IGH), and joining (J) gene segments, followed by constant (C) region genes. Fig. 117.2 illustrates the rearrangement of V(D)J genes from germline DNA, which together with heavy and light chain constant region genes encode the BCR and antibody proteins. IgH genes are the first to be assembled: first, an IGHD gene segment is joined to an IGHJ gene segment in the CD34 ⁺ /CD10 ⁺ /CD22 ⁺ /CD19 ⁻ progenitor (pro-B) cell. Pro–B cells with IGHD to IGHJ rearrangements on both copies of chromosome 14 then progress to pro–B-1 cells by selecting an IGHV gene segment to join to the DJ rearrangement and expressing the B-lineage–specific cell surface molecule CD19 with continued expression of CD34 and CD10. The next differentiation step to pre–B-cell phenotype includes stopping expression of CD34. Pre–B-1 cells that fail to produce in-frame VDJ rearrangements (i.e., rearrangements that encode a μ heavy chain protein) undergo apoptosis and are eliminated from further development.

If the VDJ rearrangement on either chromosome is in frame, pre–B-1 cells then differentiate into CD34 ⁻ /CD10 ⁺ /CD19 ⁺ large pre–B-2 cells, defined by transient cell surface and constitutive cytoplasmic expression of the rearranged μ heavy chain protein. The cell surface μ heavy chain pairs with the surrogate light chain (a heterodimer of two proteins originally designated VpreB and λ5 in the mouse) as well as associating with anchoring molecules CD79α (Igα) and CD79B (IgB); the complex is referred to as the pre–B-cell receptor (pre-BCR). ^, Pre-BCR expression is required for normal human B-cell development (see further on). A point of considerable debate since the initial discovery of the pre-BCR is whether it has a natural ligand in the marrow microenvironment. Although the structure of the pre-BCR is compatible with a ligand-independent signaling function, compelling evidence in human and mouse studies has revealed that the soluble lectin produced by marrow stromal cells, Galectin-1, can function as a pre-BCR ligand. Cell-surface pre-BCR signals the large pre–B-2 cells to undergo substantial proliferation (estimated to be four to six cell divisions), thereby expanding the number of cells with in-frame VDJ rearrangements. Large pre–B-2 cells eventually cease expression of the surrogate light chain (and hence the pre-BCR) and exit the cell cycle. At this stage they are designated small pre–B-2 cells and undergo V-to-J rearrangement at the IGK locus. If the small pre–B-2 cell makes an in-frame VJ κ rearrangement, it expresses cell-surface μ/κ Ig. If rearrangement of both κ alleles is out of frame, the small pre–B-2 cell can undergo rearrangement at the λ LC locus. Immature B cells harboring in-frame IgL rearrangements express the κ or λ chain in complex with a μ or δ heavy chain on the cell surface as the BCR. Both IgM and IgD with the same VDJ-encoded sequences are expressed on the surface of these cells via alternative splicing of primary ribonucleic acid (RNA) transcripts to join the VDJ gene rearrangement to either IgM or IgD constant region genes. Several B-lineage–restricted cell-surface markers—including CD20, CD21, CD22, and CD40—are expressed at high levels in immature B cells.

Orderly expression of the genes that mediate recombination and diversification of the Ig loci is essential for normal B-cell development. The protein products of recombination activating genes (RAG)1 and RAG2 are required for initiation of Ig germline rearrangement and generation of DNA strand breaks at recognition signal sequences 5′ or 3′ of Ig gene segments. ^, RAG proteins are also essential for rearrangement of T-cell–receptor genes during T-cell development in the thymus. RAG expression is initiated in CD10 ⁺ /CD19 ⁻ pro-B cells, undergoes downregulation in pre–B-1 cells following in-frame heavy chain rearrangements, and is reinduced at the small pre–B-2 cell stage during IGK/L rearrangement. Terminal deoxynucleotidyl transferase (TdT) is another enzyme expressed in early B- and T-cell development. TdT contributes to repertoire diversification by adding random nucleotides in a template-independent manner to the 3′ overhangs during the formation of the VD and DJ junctions of IgH, and at the VJ junction of the IgL chain. Therefore, the combined actions of RAG and TdT are required to form the most diverse parts of the Ig heavy and light chain repertoires, the VDJ and VJ junctional regions that encode the complementarity-determining region-3 (CDR3) loops that are often involved in antigen-binding contacts.

A complex interplay of transcription factors is essential in mediating the continuing restriction that locks B-lineage cells into their developmental fate; these include PU.1 and Ikaros, which function at the myeloid/lymphoid cell junction and the transcription factor E2A (E2A), early B-cell factor (EBF), and paired box protein 5 (PAX5; also known as BSAP ), which regulate irreversible specification and commitment to the B-lineage. Whereas E2A and EBF are essential for the formation of pro-B cells with EBF appearing only after E2A expression, PAX5 is necessary for final commitment to B-cell differentiation and is expressed throughout B-cell development. Other transcription factors (discussed later) that play key roles in terminal B-cell development and differentiation into plasma cells include B-cell lymphoma 6 (BCL-6), B-lymphocyte-induced maturation protein 1 (BLIMP1), X-box-binding protein 1 (XBP1), and interferon regulatory factor 4 (IRF4). A summary of the patterns of gene expression characterizing the individual stages of B-cell development in the marrow is shown in Fig. 117.1 .

It is clear that there are selective pressures on B cells during their development prior to leaving the bone marrow for the peripheral blood. Comparison of BCR sequence features between bone marrow B-cell precursors and mature naïve B cells in human blood show fewer long CDR3 sequences in the heavy chain of naïve B cells, and lower rates of autoreactivity or polyreactivity of the antibodies expressed, consistent with a selection against autoreactive B cells. Other checkpoints may also act at earlier stages in B-cell development.