basic glimpse about immune tolerance , University of Dhaka

2. What is Immune Tolerance?
Immune tolerance refers to the state of
a biological system where there should
be an immune response, but there is
none

3.  Tolerance refers to ‘a state of specific unresponsiveness’
to a specific antigen or failure to mount an immune response
to an antigen
 it is an active response to a particular epitope and is just as
specific as an immune response
 It is induced by prior exposure to that antigen
 does not necessarily mean total lack of immune response
 Antigens that induce tolerance are called ‘tolerogens’

4. Highly desirable Tolerance

5. Undesirable tolerance

7.  In 19th Century, Paul Ehrlich coined the term ‘Horror
Autotoxicus’ means ‘horror of self toxicity’
 it was realized that there must be a mechanism to prevent
auto antibody formation
 a normal body does not mount immune response against its
own tissues
 Implied the need for ‘regulating contrivance’ to stop
production of auto antibodies

8. Discovery of Immune tolerance
•In 1938, Traub inoculated mice in Utero with lymphocytic choriomeningitis virus
•Resulted in symptomless, carrier state
•Virus was present in blood and other organs, but no antibody was produced
•If mature virus was inoculated, they produced antibody
•

9. Experiment contd….
 Ray Owen was the first to observe the phenomenon of
immunological tolerance in vivo
 He observed that non-identical dizygotic twin cattle shared
each other’s RBC through their common placenta
 Each twin had its own red blood cells and a second set of
cells derived from the other twin
 Neither twin made antibodies against the blood cells of the
other
 Owen concluded that exposure of the immature immune
system to a foreign antigen resulted in specific tolerance to
that antigen

10. Burnet’s clonal selection
theory Following Owen’s observation, scientists Burnet and Fenner
postulated that age of animal at time of first encounter against
non-self antigen is a critical factor
 Antigens encountered before birth result in deletion of specific
clones at some stage in early embryonic development (Clonal
deletion)
 It means that if immune system encounters antigens, while it was
immature the relevant lymphocytes become tolerized
 would ensure that
 self-reactive antibody-forming cells are physically eliminated
before birth and
 Only antibody-forming cells able to react to ‘not-self’ persist

11. Clonal selection
(1) A hemapoietic stem cell undergoes differentiation
and genetic rearrangement
(2) immature lymphocytes are produced with many
different antigen receptors
(3) Those that bind to antigens from the body's own
tissues are destroyed
(4) the rest mature into inactive lymphocytes
(5) those that encounter a foreign antigen are
activated
(6) Produce many clones of themselves

12. Medawar’s Experiment
 In 1953, Peter Medawar and his colleagues induced
immunological tolerance to skin allografts in mice by
neonatal injection of allogenic cells
 (grafts that are genetically non identical but are from same
species)
 The hypothesis was that “mammals and birds never develop,
or develop to only a limited degree, the power to react
immunologically against foreign homologous tissue cells to
which they have been exposed sufficiently early in foetal life’’
 Consistent with Burnet’s theory

13.  Strain A mice normally rejects
graft from strain B
 neonatal injection of spleen cells
from strain B into strain A
 Newborn strain A mice show
tolerance to skin grafts from
strain B
 but reject grafts from strain C

14. Lederberg’s modfication of
clonal theory
 Burnet’s hypothesis implied that all antibody-forming cells
(lymphocytes) are generated during fetal development
 However, it soon became clear that lymphocytes are
generated throughout life
 In 1959, Lederberg suggested modification of Burnet’s theory

15.  States that, responsiveness is not determined by the
developmental stage of individual
 Rather, It is the state of maturity of the lymphocytes at the
time of encountering antigen
 According to this modification, if lymphocytes contact antigen
in its immature stage they are subjected to ‘clonal abortion’
(removal of immature lymphocytes that interact with
antigens, via cell death)
 If encountered when in mature state, they become activated

16. The Danger Hypothesis
 In 1994 Polly Matzinger suggested a new immunologic
model
 states that the immune system does not distinguish between
self and nonself
 discriminates between dangerous and safe by recognition of
pathogens or alarm signals from injured or stressed cells and
tissues
 pathogen or cell-associated stress compounds, can induce
immune cells

18. Self vs non-self
 Immunological ‘SELF’ implies to all epitopes encoded by the
individual’s DNA
 All others are considered non-self
 Other factors that also determines self or non-self include
 The stage of differentiation when lymphocytes first
encounter the epitopes
 The site of the encounter
 The nature of cells presenting the epitopes
 The number of lymphocytes responding to the epitopes

19. Ways to prevent responding to self
Ag
 Five possible ways-
1. Self-reactive cells may be deleted at certain stages of
development
2. Self reactive cells may be unable to respond
3. Self- reactive T cells in circulation may ignore self Ags
4. Response to self Ag may be suppressed if the Ag is in a
privileged site
5. Tolerance can be maintained by immune regulation

20.  Which of these mechanisms would work depends on
 The stage of maturity of the lymphocyte
 The affinity of the receptor for the self Ag
 The nature of the Ag
 Concentration of the lymphocyte
 Tissue distribution of lymphocyte
 Pattern of expression of lymphocyte

21. The kinds of tolerance
 Tolerance is classified into
1. Central tolerance: tolerance of T or B cells induced in
during development in the primary lymphoid organs (the
bone marrow for B cells and the thymus for T cells)
2. Peripheral tolerance: induced in other tissues and lymph
nodes
 The mechanisms by which these forms of tolerance are
established are distinct, but the resulting effect is similar.

23.  Central tolerance of T cells takes place during their
development within the thymus
 depends on a number of checkpoints through which cells
have to pass in order to develop
 The process of generating new T cell receptors involves
gene rearrangement to generate a highly diverse T cell
receptors
 Such a broad variety is necessary to provide protection
against different infectious agents

24. Stages of T cell
Development
 Double Negative (DN) stage
 Double Positive (DP) Stage
 Single Positive (SP) stage

25. Double Negative (DN)
Stage
 Immature T cells enter the thymus and express neither
CD4 nor CD8 co-receptors, hence called double
negative (DN) cells
 The TCR b chain genes start recombination

26. Double Positive (DP) Stage
 expression of both CD4 and CD8 co-receptors occurs
 T cells mature into CD4 and CD8 Double positive (DP)
cells
 α chain rearrangement initiates
 TCR structure is completed
 These cells come into contact with cortical thymic epithelial
cells that express high levels of class I and class II MHC
molecules on their surface
 These self-MHC molecules present self-peptides, which are
derived from intracellular or extracellular proteins that are
degraded in the normal course of cellular metabolism

27. Thymic Selection of the T cell
Repertoire
 Cells undergo two selection Processes-
1. Positive Selection
2. Negative Selection

28.  Cells whose TCR fail to interact with MHC-self peptide
molecules undergo programmed cell death (Death by
neglect)
 Cells that bind too strongly to MHC/self-peptide complexes,
also die
 Only cells that recognize MHC molecules with moderate/low
affinity survive (positive selection)
 Some are able to rescue failed positive selection by receptor
editing
 positive selection ensures that T cells recognize antigen only
in association with MHC
Positive selection

29. Negative selection
 Selected cells then mature to (SP) single positive (CD4 or
CD8) T lymphocytes and migrate to the medulla
 those that bind with high affinity with self-peptide-MHC
complexes are induced to undergo apoptosis (clonal
deletion)
 Results in self-tolerance
 After negative selection, these SP cells pass from the thymus
into the circulatory system
 Only 2% to 5% of DP thymocytes actually exit the thymus as
mature T cells

32.  investigations in the late 1990s revealed that the thymus had
an extraordinary capacity to express and present proteins
from all over the body
 some medullary epithelial cells of Thymus express a unique
protein, AIRE, that allows cells to express, process, and
present proteins that are ordinarily only found in other
specific organs

33.  Aire promotes the expression of organ-specific genes in
medullary thymic epithelial cells (mTECs)
 These organ-specific proteins are presented on the surface
of mTECs by MHC molecules to T cells developing in the
thymus
 Thymocytes that recognize these organ-specific proteins in
the context of MHC molecules undergo negative selection
 Medullary dendritic cells can acquire these antigens by
engulfing mTECs, and mediate negative selection
 The role of Aire is therefore to limit the generation of self
reactive T cells

34. Aire Protein

36. Checkpoints in T cell
development
 b selection checkpoint- only cells with a rearranged b chain
mature from DN to DP
 a selection checkpoint- cells expressing ab chains must
interact with MHC to survive
 Lineage commitment checkpoint- cells must repress
expression of CD4 or CD8 to develop into SP cells
 Negative selection checkpoint-cells that interact with
MHC-self molecules are deleted

37.  The decision to undergo positive or negative selection is
directly related to the avidity of TCR for a particular MHC-
peptide complex
 This depends on
 The level of expression and stability of the MHC-peptide
on APC
 Affinity of TCR for this complex
 Low avidity interaction promotes positive selection
 High avidity interaction promotes negative selection
 Experiment shows that the same peptide will induce positive
selection at low concentration and negative selection at high
concentration

38. Avidity Model of Thymic
Positive and Negative Selection

39.  Also depends on-
 The architecture of thymus
 The nature of APCs in the cortex vs the medulla
 The type of Ag that these cells can present

40. Other Mechanisms of Central
Tolerance
 Clonal arrest: thymocytes that express autoreactive T-cell
receptors are prevented from maturation
 clonal anergy: autoreactive cells are inactivated, rather than
deleted
 clonal editing: autoreactive cells are given a second or third
chance to rearrange a TCR gene
 clonal deletion is probably the most common mechanism
responsible for thymic negative selection.

41. factors that promote tolerance
 fetal exposure
 High doses of antigen
 Long-term persistence of antigen in the host
 Intravenous or oral introduction
 Absence of adjuvants (compounds that enhance
the immune response to antigen)
 Low levels of costimulation
 Presentation of antigen by immature or
unactivated antigen-presenting cells (APCs)

42. Escape from central tolerance
 Two factors contribute to this
(1) not all self antigens are expressed in the central lymphoid
organs where negative selection occurs, and
(2) there is a threshold requirement for affinity to self antigens
before clonal deletion is triggered

44. Mature self-reactive lymphocytes that recognize
self antigens in peripheral tissues are
inactivated, killed or suppressed

46. Sequestration
 Self Ag may be sequestered in some tissues and will never
be available to T-cells
 allows these antigens to avoid encounter with reactive
lymphocytes under normal circumstances;
 Two ways
1. Physical barrier: location of antigen in privileged sites
2. Immunological barrier: never processed by functional APCs

47. Privileged sites
 Cells ignore self antigens if they are expressed in
Immunologically privileged sites
 The brain
 the anterior chamber and lens of the eye
 testes
 In these sites pro-inflammatory lymphocytes are controlled
by
 Apoptosis
 Cytokine secretion

48. Apoptotic cell death
 Extremely important for maintaining immune homeostasis in
healthy individuals
 by two mechanisms
1. Activation induced cell death (AICD): deletion of cells
with high avidity for Ag
2. Programmed cell death (PCD): deletion of cells when
immune response is no longer required

49. Activation-Induced Cell Death
(AICD)
 External stimulus mediates apoptosis
 T-cells having unusually high avidity for antigen are killed this
way
 Mediated by ligation between Fas-receptor and Fas-ligand
(FasL)
 Interaction between Fas and FasL activates signal that
induce apoptosis in cells
 people with mutated Fas or FasL suffer from autoimmune
lymphoproliferative syndrome (ALPS)
 IL-2 stimulates Fas mediated AICD by enhancing
transcription of FasL

51. AICD can be fratricidal or suicidal

52.  For example, The epithelial cells lining the anterior chamber
of eye express Fas Ligand (FasL)
 allows interaction with T-cells expressing Fas (CD95)
 Induce apoptosis of T-cells
The fluid of the anterior chamber contains cytokines, eg.,
Transforming Growth factor b (TGF b)

53. Homeostasis

54. Balance of signals in T-cell
activation
•Signal 1: TCR-MHC-peptide interaction
•Signal 2: Ligation between co-stimulatory
molecules CD28 and B7 (CD80 & CD86)
•Expression of CTLA-4 on T-cell blocks B7
•and reduce the auto reactivity of T-cells
•T-cell activation is a competition between
stimulating and inhibiting signals

56.  Kidney cells do not express the costimulatory ligands
required for activating a CD4 or a CD8 T cell
 if a T-cell specific for a peptide made by a kidney cell
escaped from the thymus, it will not be activated unless that
peptide were presented on a professional APC
 a high-affinity interaction with MHC/peptide combinations on
the surface of kidney cell, in the absence of costimulatory
ligands, could result in T-cell anergy

57. APC TCR
T cell
CD28
Activated
T cells
APC TCR
Functional
unresponsiveness
Normal T cell
response
Anergy
Apoptosis
(activation-induced
cell death)APC
Deletion
APC
Block in
activation
Suppression
Regulatory
T cell
Peripheral tolerance
Off signals
Activated
T cell

58. APC
TCR
Naïve
T cell
Immunogenic
antigen
(microbe,
vaccine)
Tolerogenic
antigen (e.g.
self)
Effector and
memory cells
Tolerance: functional
inactivation or cell death,
or sensitive to suppression
Antigen (peptide + HLA):
signal 1
Costimulation
(signal 2)
Peripheral tolerance
58

59. Dendritic cells in peripheral
tolerance
 Dendritic cells uptake antigens in their immature state, but
can’t present to T cells
 Present antigen to T-cells only when they are mature
 Activate T-cells
 HOWEVER- if immature dendritic cell process and present
antigen to T-cell, it leads to
 Anergy (unresponsiveness)
 Deletion by apoptosis
 Generation of regulatory T-cells
 This never happens with non-self antigens, however-
because non-self antigens induce maturity of DC

60. Regulatory T cells (TREG cells)

61.  Act in secondary lymphoid tissues and at sites of
inflammation
 TREG cells recognize specific self antigens, and sometimes
foreign antigens
 they down-regulate immune processes when engage with
these antigens in the periphery

62. Regulatory CD4+ T cells
 Can be generated
 naturally in the thymus (nTREG cells), and
 after induction by antigen in the periphery (iTREG cells)
 Some scientists postulate that
 nTREG cells regulate responses against self antigen to
inhibit autoimmune disease
 iTREG cells control reactions against benign foreign
 antigens at mucosal surfaces

63. nTreg cells
 arise from a subset of T cells expressing receptors with
intermediate affinity for self antigens in the thymus
 some of these cells upregulate the transcription factor FoxP3
and migrate out of thymus
 Suppress reaction to self antigens
 Characterized by expression of the a chain of the IL-2
Receptor (CD25)

65.  Whether cells will die by negative selection or
develop into nTreg determined may be by
 the binding of CD28 with CD80/86 or
 Binding of CD40 with CD40L or the
 presence of certain cytokines
 FoxP3, is also imprortant for induction of
immunosuppressive function,

66. Mechanisms of TREG cells
 both contact-dependent and contact independent processes have
been observed
1. kill APCs or effector T cells directly, by using granzyme and
perforin
2. TREG cells express high levels of CTLA-4 which interact with
CD80/86 on an APC and inhibit APC function
3. These APCs begin to express soluble factors (including
indoleamine-2,3-dioxygenase) that inhibit local immune cells
4. TREG cells also secrete immune inhibitory cytokines, such as IL-
10, TGF-a, and IL-35, suppressing the activity of other nearby T
cells and APCs
5. TREG cells express only the low-affinity IL-2R (CD25) but not the
or subunits, which are required for signal transduction
6. they can absorb this growth and survival-promoting cytokine and
discourage expansion of local immunostimulatory effector T cells.

68.  Normlly, TREG cells inhibit
 APCs presenting their cognate antigen or
 effector T cells that share their same antigen specificity
 Do not inhibit T cells with a different specificity
 However, CD4 Treg cells inhibit T cells recognizing other
antigens, when both the TREG cell and the second T cell
recognizing another antigen interact with the same APC

70. Regulatory CD8+ T cells
 use a range of mechanisms to inhibit other cells
from responding to antigen
 three main pathways seem to exist:
 APC lysis,
 Inhibition of APC function, and
 regulation of effector T cells that share cognate
antigen with the CD8 TREG cell.

71. 71

72. Developmental checkpoints for
B cells
72

73. Central Tolerance
 Tolerance begins when IgM appears on B
cell
 eliminate approximately 90% of the self-
reactive B cell pool
 Different mechanisms
 Receptor editing
 Clonal deletion
 Clonal anergy
73

74. •replaces self reactive receptor with new, non-autoreactive
receptor
•When the IgM receptor on an immature B cell reacts with self
antigen further cell differentiation is blocked, but light chain
rearrangement can continue
•permits the B cell to edit its receptor and rescue potentially
auto-reactive cells from death
•if receptor editing fails they are eliminated by apoptosis (Clonal
deletion)
Receptor editing of B cells

75. Clonal anergy of B cells
 autoreactive B cells that recognize soluble self antigens
within the bone marrow may do not die
 their ability to express IgM on surface is lost
 They survive to escape the bone marrow, migrate to
periphery only expressing IgD, which are unable to
respond to antigen
 These B cells are called anergic B cells
75

76. B cell self tolerance: clonal deletion
Immature
B cell recognises
MULTIVALENT
self Ag
B
Clonal deletion by
apoptosis
YYB
Immature
B
B
Small
pre-B
Small pre-B cell
assembles Ig

77. B cell self tolerance: anergy
B
B
Anergic B cell
IgD normal IgM low
Immature
B cell recognises
soluble self Ag
No cross-linking
YY
B
Immature
B
B
Small
pre-B
Small pre-B cell
assembles Ig
IgM
IgD
IgD
IgD

78. Receptor editing
A rearrangement encoding a self specific receptor can be replaced
V CD JVV V
BB
!!Receptor
recognises
self antigen!!
B Apoptosis
or anergy
BB
Edited receptor now recognises
a different antigen and can be
rechecked for specificity
CD JVV VV
Arrest development
And initiate
receptor editing

79. Peripheral tolerance
79

80. Clonal deletion in spleen
 B cells leaving the bone marrow are relatively immature
 These cells migrate from the bone marrow to the outer T cell
zone of the spleen
 immature B cells are classified into two subpopulations of
transitional B cells based on their cell-surface expression
of immunoglobulin receptors and membrane markers
 T1: mIgMhigh, mIgDlow
 T2: mIgMlow, mIgDhigh
 These transitional B cells act sequentially as the precursors
to the fully mature B cell
80

81. 81
•In T-cell zone, T1 cells will
mature into the T2 state
•T2 B cells are then able to
enter the follicles nd develop
into mature, B cells

82.  the most significant amount of negative selection takes
place in these cells
 If T1 B cells encounter multivalent self antigen they are
eliminated by apoptosis
 in healthy adults, fully 55% to 75% of immature B cells
are lost by this process
 once the B cell has matured into a T2 transitional B cell,
it becomes resistant to antigen-induced apoptosis
 These T2 cells also express BAFF-R, the receptor for
the B-cell survival factor
 receive stimulatory survival signal survive (Positive
selection)
82

85. Somatic hypermutation
 Somatic hypermutation is a cellular
mechanism by which immune system
adapts to new foreign elements that
confronts it. (e.g. microorganism)
 It diversifies B cell receptors used to
recognize foreign elements. (e.g.
antigen) and allows to adapt immune
response to new threats.
 It involves mutation affecting V regions
of Ig genes.
85

86.  When B cells recognizes any antigen, they
proliferate and during this proliferation,
BCR (B Cell Receptors) genes undergo
extremely high rate of somatic
hypermutation (105-106 fold greater than
normal mutation rates).
 Somatic hypermutation occurs in
hypervariable region (CDR).
 Via hypermutation, B cells express
receptors possessing enhanced ability to
recognize and bind specific Ag.
86

87. Somatic hypermutation
87

88. Process of somatic hypermutation
 Antigen-activated B cells differentiate into
centroblasts that undergo clonal expansion in
the dark zone of the germinal centre.
 During proliferation, somatic hypermutation
(SHM) induces base-pair changes into the
V(D)G region of the rearranged genes
encoding the immunoglobulin variable region
of the heavy and light chain, some of these
base-pair mutations lead to a change in the
amino-acid sequence.
88

89.  Centroblasts then differentiate into centrocytes
and move to the light zone, where the
modified antigen receptor, with help from
immune helper cells including T cells and
follicular dendritic cells (FDCs), is selected for
improved binding to the immunizing antigen.
 Newly generated centrocytes that produce an
unfavorable antibody undergo apoptosis and
are removed. A subset of centrocytes
undergoes immunoglobulin class-switch
recombination (CSR).
89

90.  Cycling of centroblasts and centrocytes
between dark and light zones seems to
be mediated by a chemokine gradient,
presumbly established by stromal cells
in the respective zones. Antigen-
selected centrocytes eventually
differentiate into memory B-cells or
plasma cells.
90

91. B-cell response to thymus-
dependent (TD) antigen
91

92. 92

93. T-cell derived soluble factors that influence clonal
expansion and maturation
93

94. Class switching
 Class switching mainly occurs to produce
antibody of identical specificity (same Ag binding
region or CDR) but different Ig isotype (different
heavy chain).
 Class switching depends on three factors:
i) Switch region: DNA flanking regions with
multiple copies of short repeat (2-3 kb
upstream).
ii) Switch recombinase: A protein or system of
protein that carries out DNA recombination and
recognizes switch region.
iii) Switch factor: Cytokine signals from helper T
cells that dictates the isotype to which B cell
switches.
94

95. Class switching
95

96. Events of class switching
 Antigenic stimulation
 Cytokine release
 Heavy chain DNA undergoes rearrangement
 V(D)J combines to any CH segment, according
to the cytokine signal, with the help of switch
region and switch recombinase
 Class switching and new heavy chain
transciption
96

97. RNA processing to produce Ig
heavy chain
97

98. Experimental induction of
tolerance
 Protein product encoded by ‘transgene’
is treated by immune system as auto
antigen and its effects can be studied ‘in
vivo’ without trauma and inflammation
associated with grafting foreign cells or
tissues.
 Parent strain and transgenic strain ideal
for control experiments because they
are congenic (differ at only one locus)

99. Experimental induction of
tolerance
 Also, by using targeted mutagenesis ,
immunologists can ‘knock out’ specific
genes to study the role of their gene
products during immunological
tolerance.

100.  Tolerance can be induced with soluble
antigens, when rabbits are injected with
bovine serum albumin (BSA) without
adjuvant at birth and fail to make antibodies
against this protein later in life
 Medawar investigated the effects of
transferring haemopoietic cells from histo
incompatible mice at different times after
birth.
 He found that if the cells were transferred in
the first few days of life (but not later) the
recipient mouse acquired lifelong tolerance
to the antigens of the donor

101. Experimental induction of
tolerance
 The modified theory was later proved
experimentally
 Transgenic methods used to investigate self
tolerance
 Introduction of specific gene into mice of defined
genetic background and to analyse its effects upon
development of immune system
 If introduced gene is linked to tissue-specific
promoter, its expression is confined to specific cell
types

102. Factors
 The stage of differentiation when
lymphocytes first confront the epitopes
 The site of encounter
 The nature of cells presenting epitopes
 The number of cells responding to the
epitopes

103. Importance of induced
tolerance
 to protect us from unpleasant, even dangerous, allergic
reactions to such things as food (e.g. peanuts), insect
stings, grass pollen (hay fever)
 to enable transplanted organs (e.g., kidney, heart, liver) to
survive in their new host (graft rejection)
 to reveal the mechanisms of autoimmunity for designing
treatments for systemic lupus erythematosus (SLE) and
multiple sclerosis (MS)

104. Major factors affectingTolerance
Ag processing Properly
proceesed
Improperly processed

106. Programmed Cell Death
 AKA death by neglect
 LACK of external stimuli induces
apoptosis
 Mediated by cytochrome c release from
mitochondria
 How do we know this? Because mice
lacking components of this pathway
suffer from a serious developmental
disease of the CNS where brain tissues
protrude out of forehead

107. APAF-1

108. Steps in PCD
 A variety of apoptotic stimuli cause
cytochrome c to be expelled from
mitochondria into the cytoplasm
 Cyt. C associates with APAF-1
 APAF-1 undergoes some conformational
changes, allowing dATP/ATP to attach to it
 This leads to the formation of apoptosome
 Apoptosome recruits and activates caspase-
9
 This triggers the caspase apoptotic pathway

109. Summary

110. Regulation of AICD and
PCD
 Independently regulated
 FLIP (FLICE inhibitory protein, FLICE is
something similar to FADD) binds to
FADD or pro-caspase-8 to block AICD
 IL-2 enhances transcription and
expression of FasL and shuts down
FLIP to increase AICD
 BCL-2 antideath proteins bind to
different proteins in PCD pathway to
block PCD

111. Experiment for clonal deletion
 H-2Kb is a foreign MHC class I molecule.
 MET-Kb transgenic: Non-b haplotype mice
that were given the gene for H2-Kb. As the
gene was controlled by the metallothionein
promoter (specific for such sites as the
liver), they were called MET-Kb transgenic.
 Anti-Kb Ig transgenic: Non-b mice, which
had been given the genes for anti- H2-Kb
antibodies (anti-Kb in short).
111

112. 112

113.  Double transgenic: contains genes for both H-
2Kb antigen and Anti-Kb antibody. The result was
to be the production of self reactive B cells for
anti-Kb Ig.
 Double transgenic offspring expressed H-2Kb in
the liver and exported B cells specific for H-2Kb
from the bone marrow.
 However, these self-reactive B cells were
partially deleted in the spleen and entirely
deleted in the lymph nodes and thus no
autoantibody was produced – no idiotype
corresponding to the anti-Kb Ig was detectable.
 Conclusion: In peripheral B cells, tolerance
was induced by clonal deletion.
113

114. Experiment for clonal anergy
 HEL (Hen Egg Lysozyme) transgenic: A
mouse was given the HEL gene linked to a
tissue specific promoter. The HEL (largely
soluble) induced B cell and T cell tolerance.
 Anti-HEL Ig transgenic: A second transgenic
line (anti-HEL Ig) carried rearranged heavy
and light chain genes encoding a high-affinity
HEL antibody.
 An allotypic marker (IgHa) distinguished this
from endogenous immunoglobulin (IgHb).
114

115. 115

116.  The majority of B cells in these transgenics
carried IgM and IgD of the ‘a’ allotype.Double
transgenic offspring were highly HEL
tolerant, producing neither anti-HEL antibody
nor antibody-secreting B cells.
 Conclusion: HEL-binding (self reactive) B
cells were not, however, deleted, but had
downregulated surface IgM, but not IgD,
receptors. They behaved as anergic cells.
116

117. TH1 and TH2 suppress each
other
 Cytokines secreted by TH1: IFNgamma, TNFalpha
etc.
 Cytokines secreted by TH2: IL-4, IL-5, IL-6, IL-10
etc.
 IFNgamma prevents production of TH2 cells
 IL-10 downergulates macrophage effector
functions e.g. Ag presentation to TH1

118. Case in point: DTH
 Delayed-type hypersensitivity involves local
accumulation of a LOT of non-specific immune
cells like macrophages
 “Delayed” because it takes a while (2-3 days) for
the reaction to develop
 “Hypersensitivity” because it causes tissue
damage
 AKA type IV hypersensitivity

120. Simplified mechanism of
DTH
 Involves a lot of cytokines
 TH1-secreted cytokines cause extravasation,
drawing in macrophages
 Activated macrophages present Ag more
efficiently, activating more TH1
 TH1 in turn secretes more cytokines to activate
and draw in macrophages
 This positive feedback is very powerful, like a
chain reaction

121. Luckily…
 Cytokines secreted by TH2 turns off macrophage
effector functions, one of which being Ag
presentation to T-cells
 This is an excellent example of how immune
regulation is crucial to induce desirable tolerance.