The maturation of B and T lymphocytes involves a series of events that occur in the primary (also called generative or central) lymphoid organs (Fig. 1). These events include the following:
• Commitment of progenitor cells to the B-lymphoid or T-lymphoid lineage.
• Proliferation of progenitors and immature committed cells at specific early stages of development, providing a large pool of cells that can generate useful lymphocytes.
• The sequential and ordered rearrangement of antigen receptor genes and the expression of antigen receptor proteins. (The terms rearrangement and recombination are used interchangeably.)
• Selection events that preserve cells that have produced functional antigen receptor proteins and eliminate potentially dangerous cells that strongly recognize self antigens. These selection processes during development ensure that the lymphocytes that mature and enter the peripheral immune system will express functional receptors with useful specificities.
• Differentiation of B and T cells into functionally and phenotypically distinct subpopulations. B cells develop into follicular, marginal zone, and B-1 cells; and T cells develop into CD4+ and CD8+ αβ T lymphocytes, γδ T cells, natural killer T (NKT) cells, and MAIT cells.
Fig1. Stages of lymphocyte maturation. Development of both B and T lymphocytes involves the sequence of maturational stages shown. B-cell maturation is illustrated, but the basic stages of T-cell maturation are similar.
Commitment to the B- and T-Cell Lineages and Proliferation of Progenitors
Multipotent stem cells in the fetal liver and bone marrow, known as hematopoietic stem cells (HSCs), give rise to all lineages of blood cells, including lymphocytes. Some HSCs mature into common lymphoid progenitors that can give rise to B cells, T cells, and innate lymphoid cells (Fig. 2). The maturation of B cells from progenitors committed to this lineage occurs before birth in the fetal liver and after birth in the bone marrow, with the final steps being completed in the spleen. Fetal liver–derived stem cells give rise mainly to a type of B cell called a B-1 cell, whereas bone marrow–derived HSCs give rise to the majority of circulating B cells (follicular B cells) as well as a subset of B cells called marginal zone B cells. Precursors of T lymphocytes emerge from the fetal liver before birth and from the bone marrow later in life and circulate to the thymus, where they complete their maturation. T cells that express γδ TCRs arise only from fetal liver HSCs, and the majority of T cells, which express αβ T-cell receptors (TCRs), develop from bone marrow–derived HSCs. In general, the B and T cells that are generated early in fetal life have less diverse antigen receptors. Despite their different anatomic locations, the early maturation events of both B and T lymphocytes are fundamentally similar.
Fig2. Multipotent stem cells give rise to distinct B and T lineages. Hematopoietic stem cells (HSCs) give rise to distinct progenitors for various types of blood cells. One of these progenitor populations (shown here) is called a common lymphoid progenitor (CLP). CLPs give rise to B and T cells and also contribute to natural killer (NK) cells and innate lymphoid cells (ILCs). Pro–B cells can eventually differentiate into follicular (FO) B cells, marginal zone (MZ) B cells, and B-1 cells. Pro–T cells may commit to either the αβ or γδ T-cell lineages. Commitment to different lineages is driven by various transcription factors, indicated in italics.
Commitment of common lymphoid progenitors to the B- or T-cell lineage depends on transcriptional regulators that drive development toward either B cells or T cells. Key events in the commitment of precursor cells to the B-cell or T-cell lineage are expression of the proteins involved in antigen receptor gene rearrangements, described later in the chapter, and making particular antigen receptor gene loci accessible to these proteins. In the case of developing B cells, the immunoglobulin (Ig) heavy-chain locus, initially in a closed chromatin configuration, is opened up so that it becomes accessible to the proteins that will mediate Ig gene rearrangement and expression. In developing αβ T cells, the TCR β gene locus is made accessible first by specific lineage-determining transcription factors.
Numerous transcription factors are involved in the maturation of T and B cells (see Fig. 2). NOTCH1 and GATA3 commit developing lymphocytes to the T-cell lineage. The NOTCH family of proteins are cell surface molecules that are proteolytically cleaved when they interact with specific ligands on neighboring cells. The cleaved intracellular portions of NOTCH proteins migrate to the nucleus and modulate the expression of specific target genes. NOTCH1 is activated in lymphoid progenitor cells and, together with GATA3, it induces the expression of a number of genes that are required for the further development of αβ T cells. Some of these genes encode components of the pre–T-cell receptor (pre-TCR) and the RAG1 and RAG2 proteins, which are required for V(D)J recombination, described later. The EBF, E2A, and PAX5 transcription factors induce the expression of genes required for B-cell development. These include genes encoding the RAG1 and RAG2 proteins, components of the pre–B-cell receptor, and proteins that contribute to signaling through the pre–B-cell receptor and the B-cell receptor.
During B- and T-cell development, committed progenitor cells proliferate first in response to cytokines and later in response to signals generated by a preantigen receptor that select cells that have successfully rearranged the first set of antigen receptor genes. Proliferation ensures that a large enough pool of progenitor cells will be generated to eventually produce a highly diverse repertoire of mature, antigen-specific lymphocytes. In mice, widely used for basic research of lymphocyte development, the cytokine interleukin-7 (IL-7) drives proliferation of both early T- and B-cell progenitors; in humans, IL-7 is required for the proliferation of T-cell progenitors but not of progenitors in the B lineage. IL-7 is produced by stromal cells in the bone marrow and by epithelial and other cells in the thymus. Mice with targeted mutations in the gene encoding either IL-7 or the IL-7 receptor show defective maturation of lymphocyte precursors beyond the earliest stages and, as a result, profound deficiencies in mature T and B cells. Mutations in the human gene for the common γ chain, a protein that is shared by the receptors for several cytokines, including IL-2, IL-7, and IL-15 among others, give rise to an immunodeficiency disorder called X-linked severe combined immunodeficiency disease (X-SCID). This disease is characterized by a block in T-cell and NK cell development, but normal B-cell development, because in humans, IL-7 is required for T-cell development and IL-15 for NK cells.
The greatest proliferative expansion of lymphocyte pre cursors occurs after successful rearrangement of the genes encoding one of the two chains of the T- or B-cell antigen receptor, producing a preantigen receptor. Signals generated by preantigen receptors are responsible for far greater proliferation of developing lymphocytes that have successfully rearranged the Ig heavy-chain gene or the TCR β chain gene than the earlier proliferation driven by cytokines such as IL-7.
Role of Epigenetic Changes and MicroRNAs in Lymphocyte Development
Many steps in lymphocyte development are regulated by epi genetic mechanisms. Epigenetics refers to the control of gene expression and phenotypes by mechanisms other than changes in the coding sequences themselves. Epigenetic mechanisms regulate the accessibility and activity of genes by inducing changes in promoter and enhancer regions of genes. These changes include the methylation of DNA on cytosine residues in specific sites, a modification that generally silences genes; posttranslational modifications of the histone tails of nucleosomes (e.g., acetylation, methylation, and ubiquitination) that may render genes either active or inactive depending on the histone modified and the nature of the modification; active remodeling of chromatin by protein machines called remodeling complexes that can also either enhance or suppress gene expression; and the silencing of gene expression by noncoding RNAs.
Some critical components of lymphocyte development are regulated by epigenetic mechanisms.
• Histone modifications in antigen receptor gene loci are required for recruitment of proteins that mediate gene recombination to form functional antigen receptor genes. This process is discussed later in the chapter.
• Commitment of developing T cells to the CD4 or CD8 lineage depends on epigenetic mechanisms that silence the expression of, for example, the CD4 gene in CD8+ T cells. Silencing involves chromatin modifications that place the CD4 gene into a heterochromatin state that is inaccessible to transcription factors.
• we discussed microRNAs (miRNAs) in the context of T-cell activation. They contribute in significant ways to modulating gene and protein expression during development as well. Dicer is a key enzyme in miRNA generation. Deletion of Dicer in the T lineage results in a preferential loss of regulatory T cells and the consequent development of an autoimmune phenotype similar to that seen in the absence of FOXP3. The loss of Dicer in the B lineage results in a block at an early stage of development, primarily due to enhanced apoptosis of developing cells. Gene ablation studies have revealed that many specific miRNAs are involved in lymphocyte development.
Antigen Receptor Gene Rearrangement and Expression
The rearrangement of antigen receptor genes is an essential event in lymphocyte development, and this process is responsible for the generation of a diverse adaptive immune repertoire. As we discussed in previous chapters, each clone of B or T lymphocytes produces an antigen receptor with a unique antigen-binding structure. In any individual, there may be 107 to 109 different B- and T-lymphocyte clones, each with a unique receptor. The ability of each individual to generate these large and diverse lymphocyte repertoires has evolved in such a way that a relatively small number of inherited gene segments can give rise to a vast number of functional genes encoding distinct Ig and TCR molecules, each capable of binding to a different antigen. Functional antigen receptor genes are produced in immature B cells in the bone marrow and in immature T cells in the thymus by a process of gene rearrangement. In this process, segments of antigen receptor genes are randomly recombined and nucleotide sequence variations are introduced at the joints, resulting in the pro duction of a large number of variable region–encoding exons. The DNA rearrangement events that lead to the production of antigen receptors are not dependent on or influenced by the presence of antigens. In other words, as the clonal selection hypothesis had proposed, diverse antigen receptors are generated and expressed before encounter with antigens.
Selection Processes That Shape the B- and T-Lymphocyte Repertoires
The process of lymphocyte development has numerous steps, sometimes called checkpoints, at which the developing cells are tested and continue to mature only if a preceding step in the process has been successfully completed. One of these developmental checkpoints is based on the successful production of one of the polypeptide chains of the two-chain antigen receptor protein, and a second checkpoint requires the second chain and thus assembly of a complete receptor. The requirement for traversing these developmental checkpoints ensures that only lymphocytes that produce complete antigen receptors, and are therefore likely to be functional, are selected to mature. Additional selection processes operate after antigen receptors are expressed and serve to eliminate potentially harmful, self reactive lymphocytes and to commit developing cells to particular lineages. (Note that the term “checkpoints” is also used to describe very different phenomena in the context of peripheral immune activation and cancer immunotherapy. We will next summarize the general principles of these events.
Preantigen receptors and antigen receptors deliver signals to developing lymphocytes that are required for the survival of these cells and for their proliferation and continued maturation (Fig. 8.3). Preantigen receptors, called pre–B-cell receptors (pre-BCRs) in B cells and pre-TCRs in T cells, are signaling molecules expressed during B- and T-cell development that contain only one of the two polypeptide chains present in a mature antigen receptor. Cells of the B-lymphocyte lineage that successfully rearrange their Ig heavy-chain genes express the µ heavy-chain protein and assemble a pre-BCR. In an analogous fashion, developing T cells that make a sucessful TCR β chain gene rearrangement synthesize the TCR β chain protein and assemble a pre-TCR. The assembled pre BCR and pre-TCR form complexes with proteins that generate signals for survival, proliferation, and the phenomenon of allelic exclusion (discussed later), and for the further development of B and T cells. Because of the random addition of nucleotides at junctions between segments of antigen receptor genes that are joined together during lymphocyte development and the triplet base pair code for determining amino acids, only about one in three antigen receptor gene rearrangements is in frame and, therefore, capable of generating a proper full-length protein. Such a successful rearrangement is sometimes called a “productive” rearrangement. If cells make out-of-frame or “nonproductive” gene rearrangements at the Ig µ or TCR β chain loci, the preantigen receptors are not expressed, the cells do not receive necessary survival signals, and they undergo programmed cell death. Thus, expression of the preantigen receptor is the first checkpoint during lymphocyte development.
Fig3. Checkpoints in lymphocyte maturation. During development, the lymphocytes that express receptors required for continued proliferation and maturation are selected to survive, and cells that do not express functional receptors die by apoptosis. Positive selection and negative selection further preserve cells with useful specificities. The presence of multiple checkpoints ensures that only cells with useful receptors complete their maturation.
In the next step of maturation, developing B and T cells express complete antigen receptors and the cells are selected for survival. Lymphocytes that have successfully navigated the preantigen receptor checkpoint go on to rearrange and express genes encoding the light chain of the BCR or the α chain of the TCR and express the complete antigen receptor while they are still immature. At this immature stage, cells that express useful antigen receptors may be preserved and potentially harmful cells that strongly recognize self antigens may be eliminated or induced to alter their antigen receptors (see Fig.3).
A process called positive selection facilitates the survival of potentially useful lymphocytes. In the T-cell lineage, positive selection ensures the maturation of T cells whose receptors recognize self major histocompatibility complex (MHC) molecules. Positive selection also ensures that the expression of the coreceptor on a T cell (CD8 or CD4) is matched to the recognition of the appropriate type of MHC molecule (MHC class I or class II, respectively). Mature T cells whose precursors were positively selected by self MHC molecules in the thymus are able to recognize foreign peptide antigens displayed by the same self MHC molecules on antigen-presenting cells (APCs) in peripheral tissues. In the B-cell lineage, positive selection pre serves receptor-expressing cells and is coupled to the generation of different B-cell subsets.
Negative selection is the process that eliminates or alters developing lymphocytes whose antigen receptors bind strongly to self antigens present in the generative lymphoid organs. Both developing B and T cells are susceptible to negative selection during a short period after antigen receptors are first expressed. Developing T cells with a high affinity for self antigens are eliminated by apoptosis, a phenomenon known as clonal deletion, but some self-reactive CD4+ T cells are not deleted and instead differentiate in the thymic medulla into CD4+ regulatory T cells. In the B lineage, strongly self-reactive immature B cells may be induced to make further Ig gene rear rangements and thus generate new antigen receptors that are not self-reactive. This phenomenon is called receptor editing. If editing fails, the self-reactive B cells may die, which is also called clonal deletion. Negative selection of immature lymphocytes is an important mechanism for maintaining tolerance to many self antigens; this is also called central tolerance because it develops in the central (generative) lymphoid organs.
