BOX 10-1 Transgenic Mouse Models for the Analysis of Tolerance and
Autoimmunity
The experimental analysis of self-tolerance is confounded by two important
technical problems. First, it is not possible to identify self-reactive lymphocytes
by functional assays because these cells are normally deleted or functionally
inactive (anergic). Second, in normal animals or humans, it has been difficult or
impossible to define the nature, tissue distribution, and levels of expression of
self antigens, particularly MHC-associated peptide antigens for T cells. For
these reasons, much of our early understanding of tolerance was based on
administering tolerogenic forms of foreign antigens to animals and studying
subsequent immune responses to immunogenic forms of the same antigens.
Conclusions about self-tolerance were largely extrapolations from these studies
with foreign antigens. Transgenic technology has provided a valuable tool for
studying self-tolerance in mice. Rearranged antigen receptor genes can be
expressed as transgenes in T or B lymphocytes (see Appendix III). Because
these antigen receptor genes inhibit recombination at other, endogenous,
antigen receptor gene loci (the phenomenon of allelic exclusion), a large
fraction of the T or B lymphocytes in these mice express the introduced,
transgene-encoded antigen receptor. Therefore, lymphocytes with a known
specificity may be detected and followed quantitatively for the life of a mouse.
The second application of transgenic technology is to express known proteins
in different tissues. These transgene-encoded antigens are present throughout
the development of the animal, and therefore they are effectively self antigens
for the mouse.
Transgenic approaches may be used to study self-tolerance in many ways.



The maturation and functional responsiveness of self antigen-specific
lymphocytes may be followed in mice by expressing an antigen
receptor specific for a normally expressed self antigen. This was first
done by expressing a class I MHC-restricted TCR specific for the male
antigen H-Y in CD8+ T cells. The T cells fail to mature in male mice
because they are negatively selected in the thymus when the immature
cells encounter H-Y peptides (see Chapter 7, Fig. 7-21). The same
principle has been exploited to study B cell tolerance to a self class I
MHC molecule by expressing a membrane Ig specific for one class I
allele in B cells. In mice containing that class I allele, the B cells are
eliminated in the bone marrow or they change their specificity.
A variation of this approach is to express transgenic antigen receptors
specific for self antigens that are targets of autoimmune diseases.
Examples include mice expressing a TCR specific for a protein in
pancreatic islet β cells (a target for autoreactive T cells in type I
diabetes), a TCR specific for myelin basic protein (which is a central
nervous system autoantigen), and Ig specific for self DNA (involved in
the autoimmune disease lupus). These transgenic mice are useful for
defining not only the mechanisms of self-tolerance but also the
pathogenesis of reactions that serve as models of human autoimmune
diseases.
Transgenic models may be made even more amenable to analysis by
coexpressing both the antigen receptors of T or B lymphocytes and the
antigen that is recognized by these receptors. Two examples we
mention in the text are T cells specific for a viral glycoprotein
expressed in islet β cells and B cells specific for hen egg lysozyme
expressed in different tissues. By changing the promoters used to drive
transgene expression, it is possible to vary the site of expression of the

antigen. The use of inducible promoters allows investigators to turn the
expression of the antigen on and off during the life of the mouse. It is
also possible to express the same antigen in different forms (secreted,
membrane bound, and cytoplasmic) and thus to analyze tolerance to
different types of self antigens.
Genes encoding particular immunoregulatory molecules, such as
costimulators and cytokines, may be coexpressed with antigens, thus
modeling the consequences of local alterations in the tissues where
particular self antigens are present. In addition, by breeding antigen
receptor transgenics with appropriate knockout mice, investigators
have generated mice in which lymphocytes of known specificities lack
genes encoding lymphocyte regulatory molecules, such as CTLA-4
and Fas ligand. This results in selective defects in lymphocyte
regulation and provides models for studying the effects of such
changes on defined lymphocyte populations as well as the
pathogenesis of disorders associated with mutations in these
regulatory genes.
Experimental systems using transgenic and knockout mice have allowed
investigators to analyze the types of antigens that induce central and peripheral
tolerance in T and B cells, the mechanisms of these pathways of tolerance, and
the genetic control of self-tolerance. Experimental protocols have been
developed to compare the consequences of self antigen recognition by
immature or mature lymphocytes. For instance, as described in the text, by
mating one mouse expressing a transgenic antigen receptor with another
mouse expressing the antigen, in the offspring, the immature lymphocytes are
exposed to the antigen throughout development. Alternatively, mature
lymphocytes expressing the antigen receptor may be transferred into mice
expressing the antigen as a self protein, and the consequences of this
encounter may be analyzed.
Despite the value of transgenic technology, several important caveats should
be mentioned. Expression of a single antigen receptor markedly limits the
normal lymphocyte repertoire. Transgene-encoded protein antigens are often
expressed at higher concentrations than are normal self proteins. Transgeneencoded immunoregulatory molecules not only are expressed at high levels but
also are expressed constitutively and constantly, which is rarely the case with
normal immunoregulatory molecules. Therefore, many of the normal controls
on lymphocyte activation and regulation may be lost in these transgenic mice.
BOX 10-2 Apoptosis in Lymphocytes
There are many situations in biology when cells normally die and are
eliminated by phagocytosis without eliciting harmful inflammatory reactions. For
instance, the process of embryogenesis involves modeling of tissues and
organs by balanced cell division and cell death, and physiologic reductions in
circulating hormone levels lead to death of hormone-dependent cells. In these
situations, cell death occurs by a process called apoptosis, which is
characterized by DNA cleavage, nuclear condensation and fragmentation,
plasma membrane blebbing, changes in membrane lipid distribution, and
detachment of cells from the extracellular matrix. Apoptotic cells are rapidly
phagocytosed because they express membrane molecules that are recognized
by a variety of receptors on phagocytes. This type of physiologic cell death
contrasts with necrosis, in which plasma membrane integrity breaks down and
cellular contents are enzymatically degraded and released, resulting in
pathologic inflammation. In the immune system, apoptosis is important for
eliminating unwanted and potentially dangerous lymphocytes at many stages of
maturation and after activation of mature cells. In the following sections, we
describe the mechanisms and regulation of apoptosis, focusing on
lymphocytes. The physiologic roles of apoptosis in the immune system are
discussed in this and other chapters.
INDUCTION OF APOPTOSIS IN LYMPHOCYTES The induction of apoptosis
involves the activation of cytosolic enzymes called caspases. Caspases are
cysteine proteases (i.e., proteases with cysteines in their active sites), so
named because they cleave substrates at aspartic acid residues. Caspases are
present in the cytoplasm of most cells in an inactive form (also called a
zymogen, referring to an inactive enzyme). In its inactive state, a caspase
exists as a single polypeptide chain with a prodomain and a catalytic domain.
Caspases are themselves activated by cleavage after aspartic residues, and
the active caspase that is produced is a dimer with two catalytic subunits.
Fourteen caspases have been identified, and the number is likely to increase.
Some caspases function as initiators, to start the process of apoptosis often by
cleaving and thereby activating more caspases. These activated caspases
function as effectors or executioners, cleaving many substrates and leading to
nuclear fragmentation and the other changes of apoptosis. In lymphocytes and
most other cells, caspase activation and subsequent apoptosis may be induced
by two distinct pathways, one of which is associated with mitochondrial
permeability changes and the other with signals from death receptors in the
plasma membrane. The mechanisms of induction of these two pathways are
illustrated in Figure A, and their biochemical mechanisms are in Figure B.
Cell death as a result of loss of survival stimuli: the intrinsic, or
mitochondrial, pathway of apoptosis. If lymphocytes are deprived of
necessary survival stimuli, such as growth factors or costimulators (for T cells),
the result is rapid increase in the permeability of mitochondrial membranes and
release of several proteins, including cytochrome c, into the cytoplasm.
Cytochrome c functions as a cofactor with a protein called apoptosis activating
factor-1 (Apaf-1) to activate an enzyme, called caspase-9, that initiates the
apoptotic pathway. Other proteins released from mitochondria may directly
block the normal anti-apoptotic activities of Bcl family members (described
later), again resulting in cell death. This pathway of apoptosis has been called
passive cell death, implying that it does not require active signals resulting from
the engagement of death receptors. (Note, however, that all pathways of
apoptosis are actively induced by enzymes and protein degradation.) This form
of apoptosis is also called programmed cell death, or death by neglect,
implying that many cells are programmed to die unless protected by survival
stimuli, and they will die if neglected (not provided survival stimuli). DNA
damage caused by irradiation, certain chemotherapeutic drugs, and
glucocorticoids may induce apoptosis of target cells by the mitochondrial
pathway. In addition, self antigen recognition may trigger mitochondrial
translocation of proapoptotic members of the Bcl family (such as Bim; see text),
which block the protective actions of the anti-apoptotic members, again
resulting in cell death by the mitochondrial pathway.
Activation-induced cell death mediated by death receptors: the extrinsic, or receptorinitiated, pathway of apoptosis. The second pathway of apoptosis in lymphocytes is triggered by
the binding of ligands to death-inducing membrane receptors. The best defined death receptors
belong to a family of proteins with homologous cysteine-rich extracellular domains. The first
members of this family to be identified were receptors for the cytokine tumor necrosis factor (TNF),
and the family includes a large number of proteins, such as Fas and CD40. The cytoplasmic regions
of different members of this family contain either a conserved "death domain" or a domain that
binds signaling molecules and activates transcription factors. (The pathway of transcriptional
activation by TNF receptors is described in Chapter 11, Box 11-1.) The two best described death
domain-containing receptors are Fas (CD95) and the type I TNF receptor; we focus on Fas as the
prototype because its role in lymphocyte regulation is better established. Fas was identified as a 36kD surface protein that, on cross-linking by specific antibodies, triggered apoptosis of cells that
expressed it. Lymphoid cells and many other cell types express Fas. Fas ligand (FasL) is a
homotrimeric membrane protein that is expressed mainly on T lymphocytes after activation by
antigen and IL-2. When mature T lymphocytes are repeatedly stimulated by antigens, they
coexpress Fas and FasL. FasL binds to Fas on the same or adjacent cells, clustering three or more
Fas molecules. The intracellular death domains of the clustered Fas receptors bind a cytosolic
death domain-containing adapter protein called FADD (for Fas-associated death domain). FADD, in
turn, binds the inactive form of a caspase, caspase-8 (see Figure B). Caspase-8 undergoes
autocatalytic activation and is then able to activate effector caspases and trigger apoptosis. This
pathway of apoptosis is called activation-induced cell death because it is induced by lymphocyte
activation (and not by the absence of survival stimuli). The type I TNF receptor probably triggers a
similar pathway of cell death. Although the type I TNF receptor does not bind FADD directly, its
cytoplasmic domain does bind a homologous protein, called TRADD (for TNF receptor-associated
death domain), that can recruit FADD to the complex. The physiologic role of TNF receptors in
regulating lymphocyte survival and in self-tolerance is not established. Note that apoptosis induced
by antigen recognition, and involving the mitochondrial pathway, has also been called activationinduced cell death, because it follows activation by the antigen. This pathway of apoptosis does not
involve death receptors.
Passive cell death by the mitochondrial pathway and activation-induced cell
death by the death receptor pathway differ in how they are induced and, as we
shall see, in their regulation and principal physiologic roles (see Table). In
some nonlymphoid cells, such as hepatocytes, Fas-induced signals result in
increased mitochondrial permeability, and the two death pathways may act
cooperatively to trigger apoptosis. Many of the proteins known to induce and
regulate apoptosis were identified as the products of genes that are
homologous to genes first shown to regulate apoptosis in the worm
Caenorhabditis elegans. During the development of this worm, particular cells
die in a precise sequence, so that the consequences of genetic manipulations
on death or survival of cells can be accurately defined. Several different ced
genes (for "cell death abnormal" genes) are known, and their mammalian
homologues have been identified. Caspase-9 is homologous to Ced-3, Apaf-1
to Ced-4, and the anti-apoptotic protein Bcl-2 (see below) to Ced-9.
Effector mechanisms of apoptosis. Once caspase-9 or caspase-8 becomes
proteolytically active, it in turn cleaves and activates other downstream effector
caspases, including caspase-3 and caspase-6. These enzymes act on a variety
of substrates, including nucleases and proteins of the nuclear envelope, to
initiate DNA fragmentation and nuclear breakdown, the hallmarks of apoptosis.
(Note that not all mammalian caspases are involved in cell death. The first of
these enzymes to be identified, now called caspase-1, functions to convert the
precursor form of the cytokine IL-1β to its active form and was therefore
originally called the IL-1-converting enzyme [ICE]. On the basis of this name,
caspase-8 was originally called FADD-like ICE or FLICE.)
REGULATION OF APOPTOSIS Programmed death of lymphocytes (passive
cell death) is prevented by various activating stimuli, including specific antigen
recognition, growth factors (such as the cytokine IL-2), and costimulation (e.g.,
engagement of CD28 on T cells by B7 molecules on APCs). All these stimuli
function by inducing the expression of anti-apoptotic proteins of the Bcl family.
The first member of this family to be identified was called Bcl-2 because it was
the second oncogene found in a human B cell lymphoma. Bcl-2, and its
homologue Bcl-x, inhibit apoptosis by blocking the release of proapoptotic
proteins like cytochrome c from mitochondria and by inhibiting the activation of
caspase-9 (see Figure B). Several other Bcl family members have been
identified that form homodimers and heterodimers and can be phosphorylated
in response to growth factors, thereby regulating their activities. The details of
these interactions are being investigated in many laboratories. Some proteins
related to Bcl proteins are proapoptotic. For instance, a protein called Bid
(which contains a domain that is homologous to domains found in Bcl proteins)
binds to and blocks the activity of Bcl-2. Therefore, Bid promotes apoptosis.
Another related protein called Bim, which may be induced by antigen
recognition, antagonizes Bcl-2 and thus promotes apoptosis. Many other
proapoptotic and anti-apoptotic members of this complex family are known. Bcl
proteins do not appear to block Fas/TNF receptor-induced apoptosis in most
cell types.
Activation-induced cell death by the Fas pathway is prevented by a protein
called FLIP (for FLICE-inhibitory protein) that has a death domain but lacks a
caspase domain. FLIP may bind to the adapter protein FADD or to inactive
caspase-8 in the cytoplasm, but it cannot activate the caspase. Thus, it
competitively inhibits the binding of caspase-8 to the Fas-associated protein
complex and blocks the apoptotic signal. Naive T cells contain high levels of
FLIP, and activation of the cells in the presence of IL-2 reduces the expression
of FLIP. This is why the Fas pathway is inactive in naive T cells, allowing
antigen-stimulated responses to develop, but becomes active after T cell
stimulation, functioning to prevent responses to repeated antigen encounter.
Table I01-1. Pathways of Apoptosis in Lymphocytes
Features
Induced by
"Passive" cell death (death by
neglect)
Deficiency of survival stimuli (antigen,
growth factors, costimulators)
Activation-induced cell death
Repeated lymphocyte activation
Membrane
None
receptors involved
Death receptors (e.g., Fas)
Early caspases
involved
Caspase-8 (and caspase-10 in humans)
Caspase-9
Effect of IL-2 on T Prevents apoptosis
cells
Enhances apoptosis
Role of Bcl
proteins
Prevents apoptosis
No effect in most cell types
Principal
physiologic roles
Death of immature lymphocytes that fail
positive selection
Death of mature cells that do not
encounterantigen, and after antigen is
eliminated
Elimination of some self-reactive mature
lymphocytes that repeatedly encounter self
antigens
Negative selection of immature lymphocytes
(largely not Fas or TNF-R mediated)
Abbreviation: TNF-R, tumor necrosis factor receptor.
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PHYSIOLOGIC ROLES OF APOPTOSIS IN LYMPHOCYTES Programmed, or
passive, cell death plays an essential role in controlling the size of the
lymphocyte pool at many stages of lymphocyte maturation and activation.
Immature lymphocytes that do not express functional antigen receptors or are
not positively selected die by neglect (see Chapter 7). After their maturation, if
naive lymphocytes do not encounter the antigen for which they are specific, the
naive cells die by apoptosis. After activation by antigen, many of the progeny of
the activated cells also die as the antigen is eliminated. In all these situations,
the lymphocytes do not receive the survival stimuli that would protect them
from programmed cell death. As expected, overexpression of Bcl-2 or Bcl-x as
a transgene in T or B lymphocytes results in enhanced survival of immature
lymphocytes and prolonged immune responses. Thus, this pathway of
apoptosis is critical for maintaining homeostasis in the immune system.
Fas-mediated activation-induced cell death appears to be most important for
preventing uncontrolled activation of lymphocytes (e.g., by abundant and
persistent self antigens). Great interest in the function of Fas was spurred by
the demonstration that in two inbred mouse strains that develop autoimmune
disease as a result of recessive single-gene mutations, the defects lie in either
Fas or FasL. These were the first systemic immune diseases shown to be a
result of a failure of apoptosis. A small number of humans with similar disorders
have been described. We will return to a discussion of these autoimmune
disorders in Chapter 18. The negative selection of immature thymocytes that
encounter high concentrations of self antigens is also due to activation-induced
cell death, but it does not appear to rely on either Fas or TNF receptors. The
Fas pathway may prevent uncontrolled lymphocyte activation in response to
persistent infections (e.g., some viral infections), but the importance of this
pathway in normal homeostasis is not established. In addition to its role in the
maintenance of peripheral tolerance to self antigens, Fas:FasL-mediated cell
death may serve other functions. Cytolysis by CD8+ cytolytic T lymphocytes
(CTLs) is in part mediated by FasL on the CTLs binding to Fas on target cells
(see Chapter 13). Two tissues known to be sites of immune privilege, namely,
the testes and the eyes, may constitutively express FasL. It is postulated that
FasL kills leukocytes that enter the tissues, thus preventing local immune
responses, which is the hallmark of immune privilege. However, these findings
are controversial and may not apply to all species. The physiologic value of
immune privilege in these tissues, and in the central nervous system, is not
understood.
Table 10-1. Factors That Determine the Immunogenicity and Tolerogenicity of
Protein Antigens
Factor
Factors that favor stimulation of
immune responses
Factors that favor tolerance
Amount
Optimal doses that vary for different
antigens
High doses
Persistence
Short-lived (eliminated by immune
response)
Prolonged (repeated T cell stimulation
induces apoptosis)
Portal of entry; location
Subcutaneous, intradermal; absence from
generative organs
Intravenous, oral; presence in
generative organs
Presence of adjuvants
Antigens with adjuvants: stimulate helper T Antigens without adjuvants:
cells
nonimmunogenic or tolerogenic
Properties of
High levels of costimulators
Low levels of costimulators and
antigenpresenting cells
cytokines
Table 10-2. Self-Tolerance in T and B Lymphocytes
Feature
T lymphocytes
B lymphocytes
Principal sites of
tolerance induction
Thymus (cortex); periphery
Bone marrow; periphery
Tolerance-sensitive
stage of maturation
CD4+CD8+ (double-positive) thymocyte
Immature (IgM+IgD-) B lymphocyte
Stimuli for tolerance
induction
Central: high-avidity recognition of antigen in Central: high-avidity recognition of
thymus
multivalent antigen in bone marrow
Peripheral: antigen presentation by APCs
Peripheral: antigen recognition without T
lacking costimulators; repeated stimulation by cell help
self antigen
Principal mechanisms Central tolerance: clonal deletion (apoptosis) Central tolerance: clonal deletion
of tolerance
(apoptosis), receptor editing
Peripheral tolerance: anergy, apoptotic cell
death, suppression
Peripheral tolerance: block in signal
transduction (anergy); failure to enter
lymphoid follicles