BOX 7-1 Genetic Blocks in Lymphocyte Maturation
Studies of natural and targeted gene mutations in mice, as well as identification
of the affected genes in several inherited human immunodeficiency diseases,
have contributed to our understanding of the role of individual molecules in the
development of mature B and T lymphocytes. The genes identified by these
approaches encode transcription factors, components of the pre-T and pre-B
antigen receptor complexes, enzymes and adapter proteins involved in signal
transduction in lymphocyte precursors, and proteins required for the formation
of ligands involved in positive selection of lymphocytes. Some of these genetic
blocks, especially mutations in transcription factors, have helped establish the
existence of stem cells and early precursors committed to differentiate into
either B or T lineages. Other mutations have contributed to our understanding
of different stages of B or T lineage maturations, often referred to as
checkpoints because these mutations block development of cells that are not
able to express useful antigen receptors. Not surprisingly, many of these
checkpoint mutations are found in genes encoding the structural or signaling
components of the pre-B or pre-T receptors. Analyses of mutations in
transcription factors and signaling molecules are also beginning to reveal the
essential signaling pathways involved in lymphocyte maturation. In several
cases, the mutations in homologous genes in mice and humans result in
different degrees of developmental blockade. For example, mutations in the IL2 receptor common γ chain, the cause of X-linked severe combined
immunodeficiency disease in humans, result in a block only in T cell
development in humans, but knockout of the same gene in mice results in
failure of both T cell and B cell maturation. Mutations in the Btk kinase, the
cause of X-linked agammaglobulinemia, result in a complete absence of
mature B cells in humans but only a partial failure of B cell development in
mice. Such observations suggest that maturation of lymphocytes is dependent
on distinct sets of stimuli to different extents in different species. Thus, for
maturation of B cells, signals from the pre-B cell receptor, which involve the Btk
kinase, may be more important in humans, and signals delivered by cytokines,
such as IL-7, which uses the IL-2R γ chain, may be critical in mice.
The following table includes a summary of many of the identified genetic blocks
in lymphocyte maturation.
Maturation block
Defective
gene/product
Stem cell committed B or Ikaros
T lineage precursor
PU-1
IL-7
Experimental model or
human disease
Transcription factor
Mouse gene knockout
Transcription factor
Mouse gene knockout
Cytokine: growth factor
Mouse gene knockout
for immature lymphocytes
IL-7 receptor α chain IL-7-induced signaling
Mouse gene knockout
Common cytokine
receptor α chain
IL-7-and other cytokineinduced signaling
Mouse gene knockout, human Xlinked SCID (no B cell maturation
defect in humans)
Jak 3
IL-7 receptor-associated
tyrosine kinase
Mouse gene knockout, human
autosomal SCID
Transcription factor
Mouse gene knockout
Transcription factor
Mouse gene knockout
EBF
Transcription factor
Mouse gene knockout
Sox-4
Transcription factor
Mouse gene knockout
Igα
Pre-B receptor complex
signaling
Rare human agammaglobulinemia
RAG-1,RAG-2
Lymphocyte-specific
Mouse gene knockout, human
Stem cell ⇒ committed B Pax-5
precursor, pro-B cell
E2A
Pro-B cell ⇒ pre-B cell
Function of
encoded protein
components of V(D)J
recombinase
autosomal SCID
Scid (DNA-protein
kinase)
V(D)J recombinase
component
Natural mouse mutation
Ku80
V(D)J recombinase
component
Mouse gene knockout
μ heavy chain
Ig heavy chain
component of pre-B
receptor
Mouse gene knockout, rare human
agammaglobulinemia
λ5
Surrogate light chain
component of pre-B
receptor
Mouse gene knockout, rare human
agammaglobulinemia
Syk
Protein tyrosine kinase
involved in pre-B cell
receptor signaling
Mouse gene knockout
B cell tyrosine kinase Tyrosine kinase
(Btk)
Human X-linked agammaglobulinemia, mouse gene knockout
B cell linker protein
(BLNK)/SLP-65
Adapter protein
Mouse gene knockout, rare human
agammaglobulinemia
Phospholipase Cγ2
Enzyme involved in B cell Mouse gene knockout
receptor signaling
Pre-B cell ⇒ immature B κ light chain gene
cell
Component of Ig B cell
antigen receptor
Mouse gene knockout
B cell receptor complex
signaling
Mouse gene knockout
Stem cell ⇒ committed T Winged helix nude
lineage precursor
Transcription factor
(expressed in thymic
epithelium)
Natural mouse mutation
Committed T lineage
GATA-3
precursor ⇒ early doublenegative thymocyte
Transcription factor
Mouse gene knockout
Early pre-T ⇒ doublepositive thymocyte
RAG-1 or RAG-2
Lymphocyte-specific
component of V(D)J
recombinase
Mouse gene knockout, rare human
immunodeficiency
Artemis
DNA repair enzyme
Human autosomal SCID
Scid (mouse)
DNA-dependent kinase
Natural mouse mutant
Pre-Tα
Component of pre-T cell
receptor
Mouse gene knockout
TCRβ
Component of pre-T
receptor and mature T
cell antigen receptor
Mouse gene knockout
CD3ε
Signaling chain of the pre- Mouse gene knockout
T and mature T cell
antigen receptor complex
CD3γ
Signaling chain of the pre- Mouse gene knockout
T and mature T cell
antigen receptor complex
CD3δ
Signaling chain of the pre- Human autosomal SCID
T and mature T cell
antigen receptor complex
Lck
CD4-and CD8-associated Mouse gene knockout
protein tyrosine kinase
Itk
Protein tyrosine kinase
involved in pre-T and
mature TCR signaling
TCRα enhancer
Regulates expression of α Mouse gene knockout
chain of TCR
CD3γ
Signaling component of
TCR
Mouse gene knockout
ZAP-70
Protein tyrosine kinase
involved in pre-T and
Mouse gene knockout, rare human
immunodeficiency
Immature B cell ⇒
mature B cell
Double-positive
thymocyte ⇒ singlepositive thymocyte
Igβ
Mouse gene knockout
mature TCR signaling
MHC class I or II
Component of TCR ligand Mouse gene knockout
required for positive
selection
CIITA and RFX
genes
Required for class II MHC Mouse gene knockouts, human
gene transcription
bare lymphocyte syndrome
TAP-1 or TAP-2
Peptide transporter
required for class I MHC
assembly
CD4 or CD8
Component of TCR ligand Mouse gene knockout, rare human
required for positive
immunodeficiency (CD8)
selection
Mouse gene knockout, rare human
immunodeficiency
Abbreviation: SCID, severe combined immunodeficiency.
page 142
BOX 7-2 Chromosomal Translocations in Tumors of Lymphocytes
Reciprocal chromosomal translocations in many tumors, including lymphomas
and leukemias, were first noted by cytogeneticists in the 1960s, but their
significance remained unknown until almost 20 years later. At this time,
sequencing of switch regions of IgH genes in B cell tumors revealed the
presence of DNA segments that were not derived from Ig genes. This was first
observed in two tumors derived from B lymphocytes, human Burkitt's
lymphoma and murine myelomas. The "foreign" DNA was identified as a
portion of the c-myc protooncogene, which is normally present on chromosome
8 in humans. Protooncogenes are normal cellular genes that often code for
proteins involved in the regulation of cellular proliferation, differentiation, and
survival, such as growth factors, receptors for growth factors, or transcriptionactivating factors. In normal cells, their function is tightly regulated. When these
genes are altered by mutations, inappropriately expressed, or incorporated into
and reintroduced in cells by RNA retroviruses, they can exhibit either enhanced
or aberrant activities and function as oncogenes. Dysfunction of oncogenes is
one important mechanism leading to increased cellular growth and, ultimately,
neoplastic transformation. The most common translocation in Burkitt's
lymphoma is t(8;14), involving the Ig heavy chain locus on chromosome 14;
less commonly, t(2;8) or t(8;22) translocations are found, involving the κ or λ
light chain loci, respectively. In all cases of Burkitt's lymphoma, c-myc is
translocated from chromosome 8 to one of the Ig loci, which explains the
reciprocal 8;14, 2;8, or 8;22 translocations detectable in these tumors. The myc
gene product is a transcription factor, and translocation of the gene leads to its
dysregulated expression. Increased Myc activity is believed to lead to
uncontrolled cell proliferation at the expense of differentiation, but the precise
mechanism of this effect is not known. Thus, Myc is an example of a
transcription factor that serves as an oncoprotein.
Chromosomal translocation
Genes involved in
translocation
Burkitt's lymphoma
t(8;14) (q24.1;q32.3) most common
c-myc, IgH
Acute lymphoblastic leukemia
(pre-B ALL)
t(12;21) (p13;q22) (25% of childhood
cases)
TEL1, AML1
Pre-B ALL; also chronic
myelogenous leukemia
t(9;22) (q34;q11.2) (25% of adult ALL, 3% bcr, abl (Philadelphia
of childhood ALL)
chromosome)
Type of tumor
B cell derived
Follicular lymphoma
t(14;18) (q32.3;q21.3)
bcl-2, IgH
Diffuse B cell lymphoma, large
cell type
t(3;14) (q27;q32.3)
bcl-6 (DNA-binding protein), IgH
Mantle cell lymphoma
t(11;14) (q13;q32.3)
cyclin D1, IgH
Pre-T cell acute lymphoblastic
leukemia
t(1;14) (p32;q11.2) (5% of cases);
del(1p32) (20% of cases)
TAL1, TCRα; TAL1, SCL
T cell lymphoma, anaplastic
large cell type
t(2;5) (p23;q35)
ALK (tyrosine kinase), NPM
(unknown function)
T cell derived
This box was written with the assistance of Dr. Jon Aster, Department of Pathology, Brigham & Women's Hospital and Harvard
Medical School, Boston.
These are some examples of chromosomal translocations that have been molecularly cloned in human lymphoid tumors. Each
translocation is indicated by the letter t. The first pair of numbers refers to the chromosomes involved, for example, (8;14), and
the second pair to the bands of each chromosome, for example, (q24.1; q32.3). The normal chromosomal locations of antigen
receptor genes (indicated in bold) are IgH, 14q32.3; Igκ, 2p12; Igλ, 22q11.2; TCRαδ, 14q11.2; TCRβ, 7q34; and TCRγ, 7p15.
page 142
page 143
Because antigen receptor genes normally undergo several genetic
rearrangements during lymphocyte maturation, they are likely sites for
accidental translocations of distant genes. DNA sequencing of antigen receptor
genes involved in chromosomal translocations has suggested that these
mistakes may occur at the time of attempted V(D)J rearrangement in pre-B and
pre-T cells, during switch recombination in germinal center B cells, and
possibly during the process of somatic hypermutation (also in germinal center
B cells). In some translocations associated with lymphoid tumors, DNA
breakage near protooncogenes occurs at sites that resemble heptamer and
nonamer sequences, suggesting that they may be caused by an aberrant
V(D)J recombinase activity. However, in most instances (and, more generally,
in all translocations found in nonlymphoid tumors), such sequences are lacking
and the cause of DNA breakage within or adjacent to proto-oncogenes is
uncertain.
Since the discovery of myc translocations in B cell lymphomas, the genes
involved in translocations in many other lymphoid (and nonlymphoid) tumors
have been identified (see Table). In most cases, these translocations result in
dysregulation of transcription factors, cell survival proteins, or signaltransducing molecules such as kinases. One gene that has proved to be
particularly important in acute lymphoid and myeloid leukemias is AML1, a
gene on chromosome 21 that encodes a member of the runt family of
transcription factors. About 25% of childhood acute lymphoblastic leukemias
have a balanced (12;21) translocation that produces a fusion gene encoding
the DNA-binding portion of AML-1 and the dimerization domain of TEL-1, a
member of the Ets family of transcription factors. Similarly, about 20% of acute
myelogenous leukemias have a balanced (8;21) translocation involving a
different Ets-like transcription factor, ETO, and AML-1. In both instances, the
oncogenic AML-1 fusion proteins appear to have "dominant negative" activity
that inhibits normal AML-1 function and results in a block in differentiation. This
implies that the normal role of AML-1 is to promote the terminal differentiation
of blood cell progenitors, which is supported by the observation that AML-1
knockout mice die during embryogenesis because of a failure to produce blood
cells. The "Philadelphia chromosome," found in chronic myelogenous leukemia
and some acute lymphoblastic leukemias, is created by a balanced t(9;22)
translocation. This yields a fusion gene consisting of the c-abl and bcr genes
that encodes a novel tyrosine kinase. The t(14;18) translocation found in the
most common B cell lymphoma, follicular lymphoma, leads to overexpression
of a survival gene, bcl-2, and prevention of programmed cell death. In another
type of B cell lymphoma, mantle cell lymphoma, the bcl-1 gene is translocated
to the IgH locus; bcl-1 codes for cyclin D, a protein that regulates cell cycle
progression. The most frequent chromosomal rearrangements in T cell acute
leukemias lead to inappropriate expression of TAL1, a gene encoding a basic
helix-loop-helix transcription factor that appears to specifically interfere with T
cell differentiation.
Table 7-1. Contributions of Different Mechanisms to the Generation of
Diversity in Ig and TCR Genes
Immunoglobulin TCR αβ
TCR γδ
Heavy chain κ
α
γ
δ
Variable (V) segments
45
35
45 50
5
2
Diversity (D) segments
23
0
0
2
0
3
D segments read in all three reading frames
Rare
--
--
Often
--
Often
N region diversification
V-D, D-J
None
V-J V-D, D-J V-J V-D1, D1-D2, D1-J
Joining (J) segments
6
5
55 12
Mechanism
Total potential repertoire with junctional diversity
∼1011
β
5
4
∼1016
∼1018
The potential number of antigen receptors with junctional diversity is much greater than the number that can be generated only
by combinations of V, D, and J gene segments. Note that although the upper limit on the numbers of Ig and TCR proteins that
may be expressed is very large, it is estimated that each individual contains on the order of 107 clones of B and T cells with
distinct specificities and receptors; in other words, only a fraction of the potential repertoire may actually be expressed.
Table 7-2. Development of MHC Restriction in Bone Marrow plus Thymus
Chimeras
Chimera
Specific killing of virus-infected
targets from
Host strain and treatment
Bone marrow
donor
Thymus
donor
Strain A
Strain B
(A ×B)F1 irradiated
(A × B)F1
None
+
+
A irradiated
(A × B)F1
None
+
-
A irradiated and
thymectomized
(A × B)F1
A
+
-
A irradiated and
thymectomized
(A × B)F1
B
-
+
Bone marrow chimeras are created by reconstituting an irradiated mouse of one strain with bone marrow progenitors from
another strain. In this example, the strain A and B mice have different class I MHC alleles. The MHC restriction specificity of
mature T cells in these mice is tested by assaying the ability of cytolytic T lymphocytes generated in response to viral infection to
kill virus-infected target cells from different mouse strains in vitro. These experiments demonstrate that the host MHC type, and
not the bone marrow donor type, determines the restriction specificity of the mature T cells and that the thymus is the site where
self MHC restriction is learned.