Overview of Lymphocyte Development

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. 8.1). These events include the following:

• Commitment of progenitor cells to the B lymphoid or T
lymphoid lineage.

• Proliferation of progenitors and immature commi ed 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 lymphocytes that express functional receptors
with useful specificities will mature and enter the
peripheral immune system.

• 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 mucosa-associated invariant T

(MAIT) cells. The properties and functions of these
different lymphocyte populations are discussed in later
chapters.

FIGURE 8.1 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 (see Chapter 2) . HSCs mature into
common lymphoid progenitors that can give rise to B cells, T cells,
NK cells, and innate lymphoid cells (Fig. 8.2). The maturation of B
cells from progenitors commi ed 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) and 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 T cell receptors (TCRs) arise from fetal liver HSCs, and

the majority of T cells, which express αβ 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.

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 the generation of accessibility, at
the level of chromatin, of particular antigen receptor gene loci to
these proteins. In the case of developing B cells, the
immunoglobulin (Ig) heavy chain locus, initially in a closed
chromatin configuration, is opened 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. These changes in the chromatin accessibility of
antigen receptor loci during development are initiated by sets of
lineage-specific transcription factors.

Numerous transcription factors are involved in the maturation of
T and B cells (see Fig. 8.2). 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 (see Fig. 7.2). 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 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

p p g q

cell development. These include genes encoding the RAG1 and

RAG2 proteins, components of the pre-B cell receptor (pre-BCR),

and proteins that contribute to signaling through the pre-BCR and

the BCR. The role of these proteins in T and B cell development will

be considered later in this chapter.

FIGURE 8.2 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, innate lymphoid cells (ILCs), and some

dendritic cells (not depicted here). 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.

During B and T cell development, commi ed progenitor cells
proliferate first in response to cytokines and later in response to
signals generated by a pre-antigen 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 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. The factors
that drive the proliferation of human progenitor B cells remain to
be identified. 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 encoding the common γ
chain, a protein that is shared by the receptors for several
cytokines, including IL-2, IL-7, and IL-15, give rise to an
immunodeficiency disorder called X-linked severe combined
immunodeficiency disease (X-SCID) (see Chapter 21). This disease
is characterized by a block in T cell and NK cell development, but
normal B cell development, because IL-7 is required for T cell

development in humans and IL-15 for NK cells.

The greatest proliferative expansion of lymphocyte precursors
occurs after successful rearrangement of the genes encoding one of
the two chains of the T or B cell antigen receptor, producing a pre-

antigen receptor (described later). Signals generated by pre-antigen
receptors are responsible for far greater proliferation of developing
lymphocytes (which have successfully rearranged the Ig heavy
chain gene or the TCR β chain gene, as the case may be) than the
earlier proliferation driven by cytokines such as IL-7.

Role of Epigenetic Changes and MicroRNAs
in Lymphocyte Development

Many nuclear events in lymphocyte development are regulated by
epigenetic mechanisms. Epigenetics refers to the control of gene
expression and phenotypes by mechanisms other than changes in
the coding sequences themselves. In developing lymphocytes,
epigenetic mechanisms also control antigen receptor gene
rearrangement events. DNA exists in chromosomes tightly bound
to histones and nonhistone proteins, forming what is known as
chromatin. DNA in chromatin is wound around a protein core of
histone octamers, forming structures called nucleosomes, which
may be either well separated from other nucleosomes or densely
packed. Chromatin may therefore exist as relatively loosely packed
structures, called euchromatin, wherein genes can be accessed by
transcription factors and are transcribed, or as tightly packed
heterochromatin in which genes are maintained in a silenced state.
The structural organization of portions of chromosomes varies in

different cells, making certain genes available for transcription
factors to bind to, while these very same genes may be unavailable
to transcription factors in other cells. 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 certain cytosine residues that generally
silences genes; post-translational 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 the CD4 gene in CD8+ T cells. Silencing
involves chromatin modifications that place the CD4 gene
into an inaccessible heterochromatin state.


• In Chapter 7, 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. As mentioned in Chapter 7, 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
(discussed in Chapters 15 and 21). The loss of Dicer in the B
lineage results in a block at the pro-B to pre-B cell transition
(discussed in more detail later), primarily due to enhanced
apoptosis of pre-B 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 fairly small number of genes can give
rise to a vast number of 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 production 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 (see Fig. 1.7). We will discuss the
molecular details of antigen receptor gene rearrangement later in
this chapter.

Selection Processes That Shape the B and T
Lymphocyte Repertoires

The process of lymphocyte development contains numerous steps,
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 is a quality control mechanism that

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 [see Chapter 18]).
We will next summarize the general principles of these events.

Pre-antigen 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) .
Pre-antigen receptors, called pre-BCRs in B cells and pre-TCRs in T
cells, are signaling structures 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 successful 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 pre-antigen receptors are not
expressed, the cells do not receive necessary survival signals, and
they undergo programmed cell death. Thus, expression of the pre-

antigen receptor is the first checkpoint during lymphocyte
development.

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 pre–antigen
receptor checkpoint go on to rearrange and express genes encoding
the second chain of the BCR or 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. 8.3).

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.
Also, the expression of the coreceptor on a T cell (CD8 or CD4) is
matched to the recognition of the appropriate type of MHC
molecule (class I MHC or class II MHC, 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 preserves receptor-expressing cells and is coupled to the

generation of different B cell subsets.

FIGURE 8.3 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 and negative
selection further preserve cells with useful
specificities. The presence of multiple checkpoints
ensures that only cells with useful receptors
complete their maturation.

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.
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.
Strongly self-reactive immature B cells may be induced to make
further Ig gene rearrangements and thus avoid self reactivity. This

phenomenon is called receptor editing. If editing fails, the self-
reactive B cells 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 (see Chapter 15).


With this introduction, we will proceed to a more detailed
discussion of lymphocyte maturation, starting with the key event in
the process, the rearrangement and expression of antigen receptor
genes.

Rearrangement of Antigen Receptor Genes in
B and T Lymphocytes

The genes that encode diverse antigen receptors of individual B and T
lymphocytes are generated by the recombination of different variable
(V) region gene segments with diversity (D) and joining (J) gene
segments. This specialized process of site-specific gene
rearrangement is called V(D)J recombination. Elucidation of the
mechanisms of antigen receptor gene rearrangement, and therefore
of the underlying basis for the generation of lymphocyte diversity,
represents one of the landmark achievements of modern
immunology.

The first insights into how millions of different antigen receptors
could be generated from a limited amount of coding DNA in the
genome came from analyses of the amino acid sequences of Ig
molecules. These analyses showed that the polypeptide chains of
many different antibodies of the same isotype shared identical
sequences at their C-terminal ends (corresponding to the constant
domains of antibody heavy and light chains) but differed
considerably in the sequences at their N-terminal ends that
correspond to the variable domains of antibodies (see Chapter 5).
Contrary to one of the central tenets of molecular genetics,
enunciated as the one gene–one polypeptide hypothesis,
immunologists postulated in 1965 that each antibody chain is


actually encoded by at least two genes, one variable and the other
constant, and that the two are physically combined at the level of
DNA or of messenger RNA (mRNA) to eventually give rise to
functional Ig proteins. Formal proof of this hypothesis came more
than a decade later when Susumu Tonegawa demonstrated that the
structure of Ig genes in the cells of an antibody-producing tumor,
called a myeloma or plasmacytoma, is different from that in
embryonic tissues or in nonlymphoid tissues not commi ed to Ig
production. These differences arise because DNA segments
encoding Ig heavy and light chains are separated within the
inherited (or germline) loci and are brought together and joined
only in developing B cells but not in other tissues or cell types.
Similar rearrangements were found to occur during T cell
development in the loci encoding the polypeptide chains of TCRs.
Antigen receptor gene rearrangement is best understood by first
describing the inherited unrearranged (germline) organization of
Ig and TCR genes and then describing their rearrangement during
lymphocyte maturation.

Germline Organization of Immunoglobulin
and T Cell Receptor Genes

Germline Ig and TCR genes are composed of multiple DNA segments
that are spatially separate in all cells and are combined in
developing lymphocytes. We will first describe the Ig loci and then
the TCR loci.

Organization of Immunoglobulin Gene Loci
Three separate loci encode, respectively, all of the Ig heavy chains,

the Ig κ light chain, and the Ig λ light chain. Each locus is on a
different chromosome. The organization of human Ig genes is
illustrated in Fig. 8.4. Ig genes are organized in essentially the same
way in all mammals, although their chromosomal locations and the
number and order of different gene segments in each locus may
vary.

At the 5′ end of each Ig locus, there is a cluster of variable (V)
gene segments, each about 300 base pairs long. The numbers of
functional V gene segments vary considerably among the different
Ig loci and among different species. For example, in humans there
are about 35 functional V gene segments in the κ light chain locus,
about 30 in the λ locus, and about 45 in the heavy chain locus;
whereas in mice, there are about 30 functional V gene segments in
the κ locus, only two in the λ light chain locus, and about 250 in the
heavy chain locus. In both species V gene segments for each locus
are spaced over large stretches of DNA, up to 2000 kilobases long.
Located 5′ of each V gene segment is a leader exon that encodes the
20 to 30 N-terminal residues of the translated protein. These
residues are moderately hydrophobic and make up the leader (or
signal) peptide. Signal sequences are found in all newly synthesized
secreted and transmembrane proteins and are involved in guiding
nascent polypeptides being translated on ribosomes to bind to a
cytosolic complex that docks these specific ribosomes onto the
endoplasmic reticulum membrane to allow protein translocation
into the lumen of the endoplasmic reticulum. Here the signal
sequences are rapidly cleaved, and they are not present in the
mature proteins. Upstream of each leader exon is a V gene segment
promoter at which transcription can be initiated, but as discussed
later, this occurs most efficiently after rearrangement.


FIGURE 8.4 Germline organization of human
immunoglobulin gene loci.The human heavy chain,
κ light chain, and λ light chain genes are shown.
Only functional gene segments are shown;
pseudogenes have been omitted for simplicity.
Exons and introns are not drawn to scale. Each
CH gene is shown as a single box but is
composed of several exons, as illustrated for Cμ.
Gene segments are indicated as follows: C,
constant; D, diversity; enh, enhancer; J, joining; L,
leader (often called signal sequence); V, variable.
In this and in subsequent figures, the tubular
structures depict double-stranded segments of
chromosomes, with the 5′ and 3′ ends referring to
the coding strands.

At varying distances 3′ of the V gene segments are several joining
(J) segments that are typically 30 to 50 base pairs long and are
separated by noncoding sequences. Between the V and J segments
in the Ig heavy chain (IgH) locus, there are additional segments

known as diversity (D) segments. D segments are not found in Ig
light chain loci. Like V gene segments, the numbers of D and J
segments vary in different Ig loci and different species.

The constant (C) region genes are located 3′ of the J segments.
Each Ig locus has a distinct arrangement and number of C region
genes. In humans, the Ig κ light chain locus has a single C gene
(Cκ), and the λ light chain locus has four functional C genes (Cλ).

The Ig heavy chain locus has nine C genes (CH), arranged in a
tandem array, that encode the C regions of the nine different Ig
isotypes and subtypes (see Chapter 5). The Cκ and Cλ genes are
each composed of a single exon that encodes the entire C domain of
the light chains. In contrast, each CH gene is composed of five or six
exons. Three or four exons (each similar in size to a V gene
segment) each encode a CH domain of the Ig heavy chain, and two
smaller exons code for the carboxy-terminal ends of the membrane
form of each Ig heavy chain, including the transmembrane and
cytoplasmic domains of the heavy chains (Fig. 8.5A).

The V, J, and D (if present) gene segments are brought together
to create the coding sequence for the variable domains of antibody
chains (see Fig. 8.5A). In an Ig light chain protein (κ or λ), the V
domain is encoded by the rearranged V and J gene segments; in the
Ig heavy chain protein, the V domain is encoded by the recombined
V, D, and J gene segments. In the case of Ig heavy chain V domains,
the non-germline junctional residues between the rearranged V and
D segments and the D and J segments, as well as the germline
sequences of the D and J segments themselves, make up the third
hypervariable region, also known as complementarity-determining
region 3 (CDR3) (see Chapter 5). The junctional sequences between
the rearranged V and J segments as well as the J segment itself
make up the third hypervariable region of Ig light chains. CDR1
and CDR2 are encoded in the V gene segment only.

A complete Ig light chain or heavy chain protein contains a V
domain encoded by a rearranged VJ or VDJ exon, fused to a C
domain or domains. The apposition of Ig V and C domains does not


occur at the level of DNA rearrangement but by RNA-splicing of
the rearranged Ig gene transcript.

Noncoding sequences in the Ig loci play important roles in
recombination and gene expression. As we will see later, sequences
that dictate recombination of different gene segments are found
adjacent to each coding segment in Ig genes. Also present are V
gene promoters and other cis-acting regulatory elements, such as
locus control regions, enhancers, and silencers, which regulate gene
expression at the level of transcription.

FIGURE 8.5 Domains of immunoglobulin and T cell
receptor proteins.The domains of immunoglobulin
(Ig) heavy and light chains are shown in A, and
the domains of T cell receptor (TCR) α and β
chains are shown in B. The relationships between
the Ig and TCR gene segments and the domain
structure of the antigen receptor polypeptide
chains are indicated. The V and C regions of each
polypeptide are encoded by different gene
segments. The locations of intrachain and
interchain disulfide bonds (S-S) are approximate.
Areas in the dashed boxes are the hypervariable
(complementarity-determining) regions. In the Ig μ
chain and the TCR α and β chains,
transmembrane (TM) and cytoplasmic (CYT)
domains are encoded by separate exons. C,
Carboxy termini; N, amino termini.

FIGURE 8.6 Germline organization of human T cell

receptor gene loci.The human T cell receptor
(TCR) β, α, γ, and δ chain genes are shown, as
indicated. Exons and introns are not drawn to
scale, and nonfunctional pseudogenes are not
shown. Each C gene is shown as a single box but
is composed of several exons, as illustrated for
Cβ1. Gene segments are indicated as follows: C,
constant; D, diversity; enh, enhancer; J, joining; L,
leader (usually called signal sequence); sil,
silencer (sequences that regulate TCR gene
transcription); V, variable.

Organization of T Cell Receptor Gene Loci
Each germline TCR locus is arranged in a very similar way to the Ig
loci described earlier, with a 5′ cluster of several V gene segments,
followed by D segments (in the β and δ loci only), followed by a
cluster of J segments, all upstream of C region genes (Fig. 8.6). In