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BOX 8-1 Methods for Studying T Cell Activation
Our current knowledge of the cellular events in T cell activation is based on a
variety of experimental techniques in which different populations of T cells are
activated by defined stimuli and functional responses are measured. In vitro
experiments have provided a great deal of information on the changes that
occur in a T cell when it is stimulated by antigen. More recently, several
techniques have been developed to study T cell proliferation, cytokine
expression, and anatomic redistribution in response to antigen activation in
vivo. The new experimental approaches have been particularly useful for the
study of naive T cell activation and the localization of antigen-specific memory
T cells after an immune response has waned.
POLYCLONAL ACTIVATION OF T CELLS Polyclonal activators of T cells
bind to many or all TCR complexes regardless of specificity and activate the T
cells in ways similar to peptide-MHC complexes on APCs. Polyclonal activators
are mostly used in vitro to activate T cells isolated from human blood or the
lymphoid tissues of experimental animals. Polyclonal activators can also be
used to activate T cells with unknown antigen specificities, and they can evoke
a detectable response from mixed populations of naive T cells, even though the
frequency of cells specific for any one antigen would be too low to elicit a
detectable response. The polymeric plant proteins called lectins, such as
concanavalin-A (Con-A) and phytohemagglutinin (PHA), are one commonly
used group of polyclonal T cell activator. Lectins bind specifically to certain
sugar residues on T cell surface glycoproteins, including the TCR and CD3
proteins, and thereby stimulate the T cells. Antibodies specific for invariant
framework epitopes on TCR or CD3 proteins also function as polyclonal
activators of T cells. Often, these antibodies need to be immobilized on solid
surfaces or beads or cross-linked with secondary anti-antibodies to induce
optimal activation responses. Because soluble polyclonal activators do not
provide costimulatory signals that are normally provided by APCs, they are
often used together with stimulatory antibodies to receptors for costimulators,
such as anti-CD28 or anti-CD2. Superantigens, another kind of polyclonal
stimulus, bind to and activate all T cells that express particular types of TCR β
chain (see Chapter 15, Box 15-1). T cells of any antigen specificity can also be
stimulated with pharmacologic reagents, such as the combination of the
phorbol ester PMA and the calcium ionophore ionomycin, that mimic signals
generated by the TCR complex.
ANTIGEN-INDUCED ACTIVATION OF T CELLS Polyclonal populations of
normal T cells that are enriched for T cells specific for a particular antigen can
be derived from the blood and peripheral lymphoid organs of individuals after
immunization with the antigen. The immunization serves to expand the number
of antigen-specific T cells, which can then be restimulated in vitro by adding
antigen and MHC-matched APCs to the T cells. This approach can be used to
study antigen-induced activation of a mixed population of previously activated
("primed") T cells expressing many different TCRs, but the method does not
permit analysis of responses of naive T cells.
Monoclonal populations of T cells, which express identical TCRs, have been
useful for functional, biochemical, and molecular analyses. The limitation of
these monoclonal populations is that they are maintained as long-term tissue
culture lines and therefore may have phenotypically diverged from normal T
cells in vivo. One type of monoclonal T cell population that is frequently used in
experimental immunology is an antigen-specific T cell clone. Such clones are
derived by isolating T cells from immunized individuals, as described for
polyclonal T cells, followed by repetitive in vitro stimulation with the immunizing
antigen plus MHC-matched APCs and cloning of single antigen-responsive
cells in semisolid media or in liquid media by limiting dilution. Antigen-specific
responses can easily be measured in these populations because all the cells in
a cloned cell line have the same receptors and have been selected for growth
in response to a known antigen-MHC complex. Both helper and CTL clones
have been established from mice and humans. Other monoclonal T cell
populations used in the study of T cell activation include antigen-specific T cell
hybridomas, which are produced like B cell hybridomas (see Box 3-1, Chapter
3), and tumor lines derived from T cells have been established in vitro after
removal of malignant T cells from animals or humans with T cell leukemias or
lymphomas. Although some tumor-derived lines express functional TCR
complexes, their antigen specificities are not known, and the cells are usually
stimulated with polyclonal activators for experimental purposes. The Jurkat line,
derived from a human T cell leukemia cell, is an example of a tumor line that is
widely used as a model to study T cell signal transduction.
TCR transgenic mice are a source of homogeneous, phenotypically normal T
cells with identical antigen specificities that are widely used for in vitro and in
vivo experimental analyses. If the rearranged α and β chain genes of a single
TCR of known specificity are expressed as a transgene in mice, a majority of
the mature T cells in the mice will express that TCR. If the TCR transgene is
crossed onto a RAG-1-or RAG-2-deficient background, no endogenous TCR
gene expression occurs and 100% of the T cells will express only the
transgenic TCR. TCR transgenic T cells can be activated in vitro or in vivo with
a single peptide antigen, and they can be identified by antibodies specific for
the transgenic TCR. One of the unique advantages of TCR transgenic mice is
that they permit the isolation of sufficient numbers of naive T cells of defined
specificity to allow one to study functional responses to the first exposure to
antigen. This advantage has allowed investigators to study the in vitro
conditions under which antigen activation of naive T cells leads to
differentiation into functional subsets such as T H1 and TH2 cells (see Chapter
12). Naive T cells from TCR transgenic mice can also be injected into normal
syngeneic recipient mice, where they home to lymphoid tissues. The recipient
mouse is then exposed to the antigen for which the transgenic TCR is specific.
By use of antibodies that label the TCR transgenic T cells, it is possible to
follow their expansion and differentiation in vivo and to isolate them for
analyzing recall (secondary) responses to antigen ex vivo.
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METHODS TO ENUMERATE AND STUDY FUNCTIONAL RESPONSES OF
ANTIGEN-SPECIFIC T CELLS IN VIVO Fluorescent dyes can be used to
study proliferation of T cells in vivo. T cells are first labeled with chemically
reactive lipophilic fluorescent esters and then adoptively transferred into
experimental animals. The dyes enter cells, form covalent bonds with
cytoplasmic proteins, and then cannot leave the cells. One commonly used dye
of this type is 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE),
which can be detected in cells by standard flow cytometric techniques. Every
time a T cell divides, its dye content is halved, and therefore it is possible to
determine whether the adoptively transferred T cells present in lymphoid
tissues of the recipient mouse have divided in vivo and to estimate the number
of doublings each T cell has gone through.
Peptide-MHC tetramers are used to enumerate T cells with a single antigen
specificity isolated from blood or lymphoid tissues of experimental animals or
humans. These tetramers contain four of the peptide-MHC complexes that the
T cell would normally recognize on the surface of APCs. The tetramer is made
by producing a class I MHC molecule to which is attached a small molecule
called biotin by use of recombinant DNA technology. Biotin binds with high
affinity to a protein called avidin, and each avidin molecule binds four biotin
molecules. Thus, avidin forms a substrate for assembling four biotin-conjugated
MHC proteins. The MHC molecules can be loaded with a peptide of interest
and thus stabilized, and the avidin molecule is labeled with a fluorochrome,
such as FITC. This tetramer binds to T cells specific for the peptide-MHC
complex with high enough avidity to label the T cells even in suspension. This
method is the only feasible approach for identifying antigen-specific T cells in
humans. For instance, it is possible to identify and enumerate circulating HLAA2-restricted T cells specific for an HIV peptide by staining blood cells with a
tetramer of HLA-A2 molecules loaded with the peptide. The same technique is
being used to enumerate and isolate T cells specific for self antigens in normal
individuals and in patients with autoimmune diseases. Peptide-MHC tetramers
that bind to a particular transgenic TCR can also be used to quantify the
transgenic T cells in different tissues after adoptive transfer and antigen
stimulation. The technique is now widely used with class I MHC molecules; in
class I molecules, only one polypeptide is polymorphic, and stable molecules
can be produced in vitro. This is more difficult for class II molecules, because
both chains are polymorphic and required for proper assembly, but class IIpeptide tetramers are also being produced. The use of peptide-MHC tetramers
to analyze the expansion of T cells is mentioned in the text.
Cytokine secretion assays can be used to quantify cytokine-secreting effector
T cells within lymphoid tissues. The most commonly used methods are
cytoplasmic staining of cytokines and single-cell enzyme-linked immunosorbent
assays (ELISPOT). In these types of studies, antigen-induced activation and
differentiation of T cells take place in vivo, and then T cells are isolated and
tested for cytokine expression in vitro. Cytoplasmic staining of cytokines
requires permeabilizing the cells so that fluorochrome-labeled antibodies
specific for a particular cytokine can gain entry into the cell, and the stained
cells are analyzed by flow cytometry. Cytokine expression by T cells specific for
a particular antigen can be determined by additionally staining T cells with
peptide-MHC tetramers or, in the case of TCR-transgenic T cells, antibodies
specific for the transgenic TCR. By use of a combination of CFSE and
anticytokine antibodies, it is possible to examine the relationship between cell
division and cytokine expression. In the ELISPOT assay, T cells freshly
isolated from blood or lymphoid tissues are cultured in plastic wells coated with
antibody specific for a particular cytokine. As cytokines are secreted from
individual T cells, they bind to the antibodies in discrete spots corresponding to
the location of individual T cells. The spots are visualized by adding secondary
enzyme-linked anti-immunoglobulin, as in a standard ELISA (see Appendix III),
and the number of spots is counted to determine the number of cytokinesecreting T cells.
BOX 8-2 The B7 and CD28 Families of Costimulators and Receptors
The best defined costimulators for T lymphocytes are the B7 family of
molecules on APCs that bind to members of the CD28 family of receptors on T
cells. The first proteins to be discovered in these families were CD28 and B7-1.
The existence and functions of B7-1 and CD28 were originally surmised from
experiments with monoclonal antibodies specific for these molecules. The
cloning of the genes encoding B7-1 and CD28 opened the way for a variety of
experiments in mice that have clarified the role of these molecules and led to
the identification of additional homologous proteins involved in T cell
costimulation. For example, residual costimulatory activity of APCs from B7-1
knockout mice suggested the existence of additional costimulatory molecules,
and homology-based cloning strategies led to the identification of the B7-2
molecule. B7-1 and B7-2 both bind to CD28 and together account for the
majority of costimulatory activity provided by APCs for the activation of naive T
cells. This is evident from the phenotype of B7-1/B7-2 double knockout mice,
which have profound defects in adaptive immune responses to protein
antigens.
Although costimulatory pathways were discovered as mediators of T cell
activation, it is now clear that homologous molecules are involved in inhibiting T
cell responses. The first and best defined example of an inhibitory receptor on
T cells is CTLA-4, a member of the CD28 family, which was discovered and
characterized by the use of monoclonal antibodies. The name CTLA-4 is based
on the fact that this molecule was the fourth receptor identified in a search for
molecules expressed in CTLs, but its expression and function are not restricted
to CTLs. CTLA-4 is structurally homologous to CD28 and is a receptor for both
B7-1 and B7-2. Unlike CD28, CTLA-4 serves as a negative regulator of T cell
activation. CTLA-4 knockout mice show excessive T cell activation,
proliferation, and systemic autoimmunity.
Several other proteins structurally related to B7-1 and B7-2 or to CD28 and
CTLA-4 have recently been identified by homology-based cloning strategies,
and their functions are now being studied. These proteins can be considered
members of the B7 and CD28 families, respectively. Three recently discovered
members of the CD28 family are called ICOS (inducible costimulator), PD-1
(programmed death-1, because this molecule was thought to regulate
programmed cell death of T cells), and BTLA (as it is similar to CTLA-4). The
available data indicate that CD28 is important for activating naive T cells and
ICOS plays a greater role in effector responses, particularly IL-10 and IL-4
production. CTLA-4, PD-1, and BTLA are all inhibitory receptors. How diverse
signals from this receptor family are integrated during immune responses is a
major question in the field.
Members of the B7 family include B7-1, B7-2, ICOS-ligand (L), B7-H1 (PD-L1),
B7-DC (PD-L2), B7-H3, and B7-H4 (see Figure). All these proteins are
transmembrane proteins consisting of two extracellular Ig-like domains,
including an N-terminal V-like domain and a membrane proximal C-like domain.
B7-H3 may be expressed in alternative forms, with either one pair of V and C
domains like the other B7 family members or two tandem pairs of V and C
domains. The main function of these molecules is to bind to CD28 family
receptors on T cells, thereby stimulating signal transduction pathways in the T
cells. There is no compelling evidence that the cytoplasmic tails of the B7
family members transduce activating signals in the cells on which they are
expressed. B7-1 and B7-2 are mainly expressed on APCs, including dendritic
cells, macrophages, and B cells. Although there is some constitutive
expression on dendritic cells, expression of both molecules is strongly induced
by various signals including endotoxin, inflammatory cytokines (e.g., IL-12, IFNγ), and CD40-CD40 ligand interactions. The temporal patterns of expression of
B7-1 and B7-2 differ; B7-2 is expressed constitutively at low levels and induced
early after activation of APCs, whereas B7-1 is not expressed constitutively and
is induced hours or days later. The other members of the B7 family, ICOS
ligand and PD-L1 and PD-L2, are also expressed on APCs, but in addition,
there is significant expression on a variety of nonlymphoid tissues. PD-L1 and
PD-L2, for example, are expressed on endothelial cells and cardiac myocytes.
The nonlymphoid expression of these molecules suggests they play a role in
regulating T cell activation in peripheral tissues and perhaps in the
maintenance of self-tolerance.
The CD28 family of receptors are all expressed on T cells, but PD-1 is also
expressed on B cells and myeloid cells. These receptors are transmembrane
proteins, all of which include a single Ig V-like domain and a cytoplasmic tail
with tyrosine residues. CD28, CTLA-4, and ICOS exist as disulfide-linked
homodimers; PD-1 is expressed as a monomer. Both CD28 and CTLA-4 bind
both B7-1 and B7-2, with different affinities, and share a sequence motif,
MYPPPY, in the V domain, which is essential for B7 binding. ICOS has an
FDPPPF motif in the corresponding position, which mediates binding to ICOSligand but not to B7-1 or B7-2. PD-1 has neither motif and binds to PD-L1 or
PD-L2 but not to B7-1, B7-2, or ICOS-ligand. The cytoplasmic tails of the CD28
family molecules contain structural motifs that mediate interaction with
signaling molecules, although the signal pathways that are engaged are
incompetently understood. Tyrosine residues in the tails of CD28 become
phosphorylated on B7-1 or B7-2 binding, and they serve as docking sites for
PI-3 kinase and Grb-2. PI-3 kinase activation is linked to the activation of the
serine/threonine kinase Akt. PI-3 kinase binding and activation also occur on
the cytoplasmic tail of CTLA-4, and perhaps ICOS. Consistent with their roles
in negative regulation of cellular activation, both CTLA-4 and PD-1 have
variations of the immunoreceptor tyrosine inhibitory motifs (ITIMs) in their
cytoplasmic tails (see Box 8-3) and recruit the tyrosine phosphatase SHP-2.
CD28 is constitutively expressed on CD4+ and CD8+ T cells. ICOS is
expressed on CD4+ and CD8+ T cells only after TCR binding to antigen, and its
induction is enhanced by CD28 signals.
X-ray crystallographic analyses of B7-1 and B7-2 interactions with CTLA-4 suggest that these molecules form a
repeating lattice structure in the immunological synapse. B7-1 or B7-2 may dimerize in the APC membrane, and each
monomeric subunit would bind to one chain of a different CTLA-4 dimer. This arrangement would enhance the local
concentration of the ligands and receptors and
presumably enhance signaling. It is not known if such an arrangement also applies to other B7:CD28 family ligandreceptor interactions.
The functional consequences of signaling by CD28 family members differ for
each molecule. CD28 signaling promotes T cell IL-2 production and clonal
expansion of naive T cells and increased BCL-XL expression resulting in
enhanced T cell survival. As a built-in positive amplification mechanism, CD28
signals also up-regulate CD40 ligand expression, which leads to CD40
signaling in APCs, which in turn up-regulates the expression of the CD28
ligands B7-1 and B7-2. In contrast, ICOS signaling promotes expression of
effector cytokines such as IL-10 and IL-4, but not of the T cell growth factor IL2. It appears that ICOS is more important for costimulation of effector T cell
responses, particularly TH2 responses, than for clonal expansion of antigenstimulated T cells. It is not known whether CTLA-4 and PD-1 play distinct or
overlapping roles in inhibiting immune responses. The CTLA-4 knockout
mouse develops severe enlargement of lymphoid organs and infiltration of
many tissues by activated lymphocytes, whereas the PD-1 knockout develops
an unusual dilated cardiomyopathy thought to be antibody mediated. These
findings suggest that CTLA-4 and PD-1 do indeed inhibit different types of
immune responses.
BOX 8-3 Protein Tyrosine Kinases and Phosphatases
Protein-protein interactions and the activities of cellular enzymes are often
regulated by phosphorylation of tyrosine residues. Inducible phosphorylation of
tyrosines is much less common than phosphorylation of serine or threonine
residues and is usually reserved for critical regulatory steps. It is not surprising,
therefore, that the kinases that catalyze tyrosine phosphorylation are essential
components of many intracellular signaling cascades in lymphocytes and other
cell types. These protein tyrosine kinases (PTKs) mediate the transfer of the
terminal phosphate of adenosine triphosphate (ATP) to the hydroxyl group of a
tyrosine residue in a substrate protein. A particular PTK will phosphorylate only
a limited set of substrates, and this specificity is determined by the amino acid
sequences flanking the tyrosine as well as by the tertiary structural
characteristics of the substrate protein.
Some tyrosine kinases are intrinsic components of the cytoplasmic tails of cell
surface receptors, such as the receptors for platelet-derived growth factor
(PDGF) and epidermal growth factor (EGF). Examples of receptor tyrosine
kinases that are involved in hematopoiesis include c-Kit, a receptor for stem
cell factor, and the receptor for monocyte colony-stimulating factor (M-CSF).
Many PTKs of importance in the immune system, however, are cytoplasmic
proteins that are noncovalently associated with the cytoplasmic tails of cell
surface receptors or with adapter proteins. Sometimes this association is only
transiently induced in the early stages of signaling cascades. Three families of
cytoplasmic PTKs that are prominent in B and T cell antigen receptor signaling
cascades, as well as Fc receptor signaling, are the Src, Syk/ZAP-70, and Tec
families (see Figure). The Janus kinases are another group of nonreceptor
PTKs involved in many cytokine-induced signaling cascades and are discussed
in Chapter 11.
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Src kinases are homologous to, and named after, the transforming gene of
Rous sarcoma virus, the first animal tumor virus identified. Members of this
family include Src, Yes, Fgr, Fyn, Lck, Lyn, Hck, and Blk. All Src family
members share tertiary structural characteristics that are distributed among
four distinct domains and are critical for their function and regulation of
enzymatic activity. The amino terminal end of most Src family kinases contains
a consensus site for addition of a myristic acid group; this type of lipid
modification serves to anchor cytoplasmic proteins to the inner side of the
plasma membrane. Otherwise, the amino terminal domains of Src family PTKs
are highly
variable among different family members, and this region determines the ability
of each member to interact specifically with different proteins. For example, the
amino terminal region of the Lck protein is the contact site for interaction with
the cytoplasmic tails of CD4 and CD8. The Src family PTKs have two internal
domains called Src homology 2 (SH2) and Src homology 3 (SH3) domains,
each of which has a distinct three-dimensional structure that permits specific
noncovalent interactions with other proteins. SH2 domains are about 100
amino acids long and bind to phosphotyrosines on other proteins. Each SH2
domain has a unique binding specificity, which is determined by the three
residues amino terminal to the phosphotyrosine on the target protein. SH2
domains are also found on many signaling molecules outside the Src family.
SH3 domains are approximately 60 amino acids long and also mediate proteinprotein binding. It is believed that SH3 domains bind to proline residues and
may function cooperatively with the SH2 domains of the same protein binding
to phosphotyrosines. The critical role of SH3 domains in PTK function is
illustrated by mutations in the SH3 domain of Bruton's tyrosine kinase; these
mutations do not alter the protein's stability or kinase activity but result in
defective B cell receptor signaling and agammaglobulinemia. The carboxyl
terminal SH1 domain in Src PTKs is usually the tyrosine kinase enzymatic
domain. It contains binding sites for the substrate and for the ATP phosphate
donor and an autophosphorylation site (a tyrosine residue) whose
phosphorylation is needed for kinase activity. The carboxyl terminal domain
has a second tyrosine that serves as a regulatory switch for the PTK. When it is
phosphorylated, the PTK is inactive, and when the phosphate is removed by
tyrosine phosphatases, the enzyme activity is turned on. The PTKs that
phosphorylate Src family PTKs at this inhibitory site form yet another subfamily
of PTKs, the best described member being c-Src kinase-1 (Csk-1).
The Syk/ZAP-70 family of PTKs includes only two identified members so far.
Syk is abundantly expressed in B lymphocytes but is also found in lower
amounts in other hematopoietic cells including some T cells. ZAP-70 is
expressed in only T lymphocytes and NK (natural killer) cells. Syk and ZAP-70
play similar roles in membrane Ig and TCR signaling cascades, respectively.
There are two SH2 domains in the Syk/ZAP-70 PTKs, but no SH3 domain and
no inhibitory tyrosine. Syk and ZAP-70 are inactive until they bind, through their
two SH2 domains, to paired phosphotyrosines within immunoreceptor tyrosinebased activation motifs (ITAMs) of antigen-receptor complex proteins (see
text). Furthermore, activation of ZAP-70 probably requires Src family PTK (e.g.,
Lck)-mediated phosphorylation of one or more tyrosine residues.
The Tec PTKs are a third group of nonreceptor kinases that are activated by
antigen receptors in B and T cells as well as by many other signals in other cell
types. This family includes Btk, Tec, Itk, DSrc29, Etk, and Rlk. Btk is expressed
in all hematopoietic cells, but mutations of the Btk gene primarily affect B cell
development in humans, causing X-linked agammaglobulinemia. The major
Tec kinases expressed by T cells are Itk, Rlk, and Tec. All the Tec family
members contain a Tec homology (TH) domain, which includes sequences
unique to this family (Btk homology domain) and sequences homologous with
GTPase-activating protein. The TH domain mediates interactions with other
signaling molecules. In addition, Tec kinases share structural similarities with
the Src family kinases, including SH3 and SH2 domains that mediate
interactions with other signaling molecules and a catalytic SH1 domain. Unlike
the Src kinases, the Tec kinases do not have N-terminal myristoylation sites. In
contrast, most Tec family kinases contain a pleckstrin homology (PH) domain,
which promotes membrane localization by binding to PIP3 generated by PI-3
kinase. The Tec kinases also do not contain a C-terminal regulatory tyrosine.
Activation of a Tec kinase requires membrane localization, phosphorylation by
a Src kinase, and autophosphorylation of the SH3 domain. The SH2 domains
of Btk and Itk bind the phosphorylated adapter proteins BLNK/SLP-65 and
SLP-76, in B and T cells, respectively, shortly after antigen recognition. Once
bound to these adapter proteins, Btk and Itk can phosphorylate and activate
phospholipase C.
Protein tyrosine phosphatases (PTPs) are enzymes that remove phosphate
moieties from tyrosine residues, countering the actions of PTKs. PTPs may be
membrane receptors or cytoplasmic proteins. One important receptor PTP,
which has a role in lymphocyte activation, is CD45, a membrane-bound protein
that removes autoinhibitory C-terminal phosphates from Src family kinases,
such as Lck and Fyn, and is essential for T and B cell activation. The
phosphatase activity of CD45 appears to require dimerization for activation,
and mutations that prevent dimerization result in dysregulated activation of
lymphocytes. Cytoplasmic PTPs are loosely associated with receptors or are
recruited to tyrosine phosphorylated receptors through SH2 domains. These
enzymes include two PTPs commonly called SH2-containing phosphatase-1
and SH2-containing phosphatase-2 (SHP-1 and SHP-2). SHP-1 is particularly
abundant in hematopoietic cells. SHP-1 is a negative regulator of lymphocytes
and contributes to the inhibitory signals mediated by inhibitory receptors of the
immune system, such as killer inhibitory receptors of NK cells (see Chapter
12). These inhibitory receptors bind SHP-1 through special motifs called
immunoreceptor tyrosine inhibitory motifs (ITIMs) by analogy to the ITAMs of
the TCR and other activating receptors. The central role of SHP-1 in regulating
lymphocyte function is illustrated by the "moth-eaten" mouse strain, which
carries a mutation in the SHP-1 gene and is characterized by severe
immunodeficiency and autoimmunity. SHP-2 plays a positive role in signaling
by certain receptors, such as the EGF receptor, but may also be involved in
negative regulation in CTLA-4 signaling.
Other phosphatases act on phosphorylated inositol lipids and inhibit cellular
responses; an example is SHIP-1, an inositol phosphatase that is associated
with an Fc receptor of B cells and serves to terminate B cell responses (see
Chapter 9, Fig. 9-19).
BOX 8-4 Transcriptional Regulation and NF-κB
A common theme in immunology is that activating stimuli, such as antigens and
cytokines, result in the initiation of transcription of genes that were previously
silent or transcribed at very low rates in the responding cells. The set of genes
that can be transcribed in any given differentiated cell type is determined by
numerous mechanisms, including the accessibility of the DNA encoding a gene
to soluble protein factors (i.e., the "openness of the chromatin"), chemical
modifications of the DNA in the regulatory regions of a gene (e.g., by
methylation of particular nucleotides), and protein factors that bind to the DNA
and promote or repress transcription. When a gene is available for active
transcription, a complex of proteins, including the RNA polymerase enzyme,
assemble near the site where transcription begins. The DNA sequence that
binds this basal transcription complex of proteins is called the promoter.
However, binding of the basal transcription complex to the promoter is not
sufficient by itself to initiate transcription efficiently, and additional signals are
provided by interactions with DNA-binding proteins called transcription factors.
Transcription factors have dual functions: they must recognize and bind to
specific DNA sequences, and they must interact with proteins of the basal
transcription complex, either directly or through a coactivator protein. The
specific DNA sequences that bind these transcription factors must be located
on the same segment of DNA as the promoter and are therefore called cisacting sequences. In some cases, these sequences must be near and in a
particular orientation to the transcriptional start site and are often considered to
be part of the promoter. In other cases, these cis-acting regulatory sequences
can be as much as several thousand nucleotides away and will work in either
orientation. Such movable regulatory DNA sequences are called enhancers.
The transcription factors that bind to promoter or enhancer sequences are
usually encoded by genes that are distant from the promoter or enhancer
sequences (e.g., on a different chromosome), and therefore transcription
factors are sometimes called trans-acting factors, meaning they need not be
encoded by the same piece of DNA as the regulated gene. In fact, many
transcription factors are multimeric proteins, and their subunits may be
encoded by widely separated genes on separate chromosomes.
Transcription factors may be activated in different ways in response to various
stimuli.
We discuss the transcription factor called nuclear factor κB (NF-κB) as a
prototype because of its central role in many immunologic reactions. The
functional NF-κB transcription factor is a dimer of either identical or structurally
homologous protein subunits of about 50 to 75 kD. The common structural
motif shared by these proteins is called a rel homology domain because this
sequence was first identified in the retroviral transforming gene v-rel. The
mammalian proteins that share this motif are p50 (also called NF-κB1), p52
(NF-κB2), p65 (RelA), c-Rel, and RelB. Drosophila express a member of this
family called dorsal. Members of the NF-κB/Rel family are expressed in almost
every cell type; RelB is expressed in lymphocytes and a few other cells. All of
these proteins can participate in either homodimer or heterodimer formation,
except for RelB, which only forms heterodimers with p50 or p52.
In resting cells, functional NF-κB dimers are present in an inactive state in the
cytoplasm, bound to one or more inhibitory proteins called inhibitors of κB
(IκBs). The activation of NF-κB is initiated by the signal-induced degradation of
IκB proteins. The details of this process are not fully known, but it involves
phosphorylation of specific serine residues in IκB by an enzymatic complex
called IκB kinase (IKK). How IKK activation is linked to upstream signals
generated by the TCR or cytokine receptors is not fully understood but
probably involves additional kinases as well as TRAF family adapter molecules
(see Box 11-1). IKK is composed of at least two kinases called IKK-α and IKKβ and a noncatalytic regulatory subunit called NF-κB essential modulator
(NEMO) or IKK-γ. IKK-mediated phosphorylation targets IκB for ubiquitination
and, finally, degradation of the polyubiquitinated IκB by the cytoplasmic
proteasome. Once NF-κB is freed from IκB, it is able to enter the nucleus,
where it binds to specific DNA sequences in target genes and stimulates their
transcription. NF-κB may interact with other proteins to activate transcription
optimally.
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Molecule Function
Phenotype of gene knockout in mice
p50
Component of NF-κB transcription
factor
Defects in antibody and innate immune responses to
infection
p52
Component of NF-κb transcription
factor
Impaired antibody responses to T-dependent antigens;
absence of B cell follicles, follicular dendritic cell networks,
and germinal centers in secondary lymphoid organs
p65 (RelA) Component of NF-κB transcription
factor
Embryonic lethal due to hepatocyte apoptosis
RelB
Component of NF-κB transcription
factor
Inflammation of multiple organs, myeloid hyperplasia and
extramedullary hematopoiesis, impaired cellular immunity
c-Rel
Component of NF-κB transcription
factor
Defects in lymphocyte proliferation, humoral immunity, and
interleukin-2 expression
IκB-α
Inhibitor of NF-κB; blocks nuclear
localization
Perinatal lethality with increased basal NF-κB activity in
hematopoietic organs, excess granulopoiesis, and
dermatitis
IκB-β
Inhibitor of NF-κB; blocks nuclear
localization
IκB-ε
Inhibitor of NF-κB; blocks nuclear
localization
Mild phenotype with altered Ig production
Bcl-3
Inhibitor of NF-κB; blocks nuclear
localization
Impaired antibody responses to T-dependent antigens;
absence of B cell follicles, follicular dendritic cell networks,
and germinal centers in secondary lymphoid organs
p100
Inhibitor of NF-κB; blocks nuclear
localization
Gastric hyperplasia and an impaired proliferative response
in lymphocytes
IKK-α
Catalytic component of IκB kinase,
Perinatal lethality with defective differentiation of the
which phosphorylates IkB, leading to epidermis and related keratinizing tissues
its degradation and activation of NF-κB
IKK-β
Catalytic component of IκB kinase,
Embryonic lethal due to hepatocyte apoptosis
which phosphorylates IκB, leading to
its degradation and activation of NF-κB
IKKγ(NEMO)
Regulatory component of IκB kinase, Male embryonic lethality (X-linked) due to hepatocyte
which phosphorylates IκB, leading to apoptosis; heterozygous female micedevelop inflammatory
its degradation and activation of NF-κB and proliferative skinlesions similar to human genetic
disorder incontinentia pigmenti
NF-κB was so named because the first identified binding site for this protein is
located within an intronic enhancer in the Ig κ light chain gene. Although NF-κB
is not required for Igκ chain expression, multiple roles for NF-κB in the immune
system have been determined by molecular studies and the phenotype of
several knockout mice (see Table). NF-κB is involved in T cell activation,
contributing to IL-2 transcription (see text), and in the responses of many
diverse cell types to microbial products. Toll-like receptors, which recognize
microbial products (Chapter 12), and proinflammatory cytokines such as TNF
and IL-1 (see Chapter 11) activate NF-κB. In fact, many of the cellular
responses seen in innate immune reactions are dependent on the activity of
this transcription factor. NF-κB also activates the transcription of genes whose
products protect cells from apoptotic death and is thus a component of a basic
survival mechanism of cells. Knockout of the genes encoding the p65 subunit
(RelA) or IKK-β in mice results in embryonic lethality due to massive liver cell
apoptosis.
1. Transcription factors may not be present in a resting cell, and
activation signals induce their de novo synthesis. An example of this
kind of regulation is the synthesis of c-Fos, a component of
transcription factor AP-1, in response to antigenic stimulation of
lymphocytes.
2. Transcription factors may require assembly into multimeric complexes
to bind to DNA, and this step may be regulated. An example of this
kind of regulation is the assembly of cytokine-responsive STAT
proteins into dimers, which is induced by the phosphorylation of these
proteins by Janus kinases (see Chapter 11, Box 11-2).
3. Transcription factors may require covalent modification to interact with
the proteins of the basal transcription complex, and this step may be
regulated. For instance, in the case of c-Jun, regulation involves
phosphorylation of an amino acid side chain in the amino terminal
transactivating region of the protein by the enzyme c-Jun N-terminal
kinase.
4. Transcription factors may preexist in a form that is capable of binding
DNA and of mediating transactivation, but the factor may not have
access to the cis-acting regulatory sequences of the target gene. In the
case of nuclear factor of activated T cells (NFAT), regulation involves
removal of a phosphate moiety from an amino acid side chain, thereby
exposing an amino acid sequence required for the NFAT protein to
enter the nucleus. The protein phosphatase that mediates this reaction,
calcineurin, is activated in the T cell by antigen recognition (see text).