1. Dr Alok Tripathi
Department of Biotechnology
aquaimmuno@yahoo.co.in
8/31/2015 6:56:30 AM

2. Our own bodies produce
some 1,00,000 different
proteins and BUT:---how the
immune system produces a
virtually universal range
while at the same time
avoiding reacting to self?
8/31/2015 6:56:30 AM

3. Definitions
We can consider two different
definitions of immunological tolerance.
The strict definition might
be that
immunological tolerance
occurs when an immuno-
competent host fails to
respond to an
immunogenic challenge
with a specific antigen.
A more operational
definition might be that
immunological tolerance
occurs when an
immunocompetent host
fails to respond to the
presence of a specific
antigen.
8/31/2015 6:56:30 AM

4. We have already seen that the adaptive immune system consists of two
distinct clonally variable repertoire expressed by T and B lymphocytes
respectively.
Thus: it is Clear that the essentially random process which
generates these repertoires will produce auto-reactive cells in both
lineages.
How does the immune system prevent the activation of these
cells?
Central Tolerance - this occurs
during lymphocyte development.
Peripheral Tolerance - occurs
after lymphocytes leave the
primary organs.
8/31/2015 6:56:30 AM
We can divide the mechanisms of immune system uses
into two main types:

5. I. Overview
• Billingham neonatal graft
experiments demonstrate
learning of tolerance
during development
• Shift of self-nonself
paradigm to “danger
theory”: immune response
generated in presence of
danger (e.g. PRRs
activated by PAMPs) and
tolerance in absence
Signal 1 vs. 2
• Signal 1
• TCR recognition of Ag
• Alone is tolerogenic,
leads to anergy or
deletion
• Signal 2
• Costimulatory molecule
engagement
• With signal 1, leads to T
cell activation
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T Cell Tolerance

6. 8/31/2015 6:56:30 AM
A) Central tolerance: Mediated by negative
selection in thymus
In T cell development, the
process of –ve selection
leads to the deletion of those
thymocytes whose T cell
receptors have 'high affinity'
for self.
One aspect we did not
consider was what other
parameters influenced this
selection event?
•The only one we need to discuss is
antigen concentration.
For T cells, antigen means
the complex of specific
peptide and MHC.
so the density of any given
peptide-MHC complex is
determined by the
abundance of the peptide
and it's affinity for the
particular MHC allele.
The number of molecules of
any given peptide-MHC
complex on the surface of a
thymic APC probably varies
from 1-50K.
Now if there is only a mean
of 1 complex/cell then even
the highest affinity T cells
might fail to be deleted
whereas for the most
abundant peptide-MHC
complexes quite low affinity
T cells would be deleted.
Thus we would predict that
threshold affinity of TCR for
peptide-MHC required to
produce deletion =1/ the
abundance of that peptide-
MHC complex.
Using transgenic mice and
organ culture techniques it
has been possible to provide
evidence for this hypothesis.

7. • - Autoantigen level is so
low that anti-self T cells
are not activated
• - Poor mechanism for
tolerance: deleterious T
cell is still present in
periphery
1)
Ignorance
• - Negative selection can
occur in the periphery
as well in absence of
signal 2
2) Clonal
Deletion
• - In the absence signal
2, T cell can become
anergic
• - Anergic T is
unresponsive to signals
1 + 2, but recovered by
IL-2
3) T cell
anergy
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B) PERIPHERAL TOLERANCE

8. • Absence of CD4+25+ T cells
causes autoimmune disease
• These cells act by inhibiting
IL-2 production
• Other cells are also
regulatory (e.g. TH3
secretes TGF-, TR1 secretes
IL-10)
4) T regulatory
cells
• - True role of TH3 and TR1
is to negatively regulate
immune response
5)
Immunoregulation
8/31/2015 6:56:30 AM
B) PERIPHERAL TOLERANCE-cont.

9. -can be extended to inhibit
autoimmunity and therefore
promote tolerance
- Providing Ag through gut
(or lungs) can activate these
cells and mediate tolerance
challenge
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Function

10. ……And also…..
Almost any other parameter which affects
the overall strength of interaction between
a clone of thymocytes and the thymic
APC also affects the positive/ negative
selection decision.
Thus the conc. of TCR and of CD4 or CD8
also influences this reaction. One
mechanism of escape for 'autoreactive'
cells into the periphery that has been seen
in TCR transgenic mice is down-regulation
of the T cell receptor, co-receptor or both.
8/31/2015 6:56:30 AM

11. During B cell development, when the complete antigen
receptor (IgM) is first expressed on 'immature' B cells & if those
cells encounter their target antigen in a form which can cross-
link their sIgM, then such cells are programmed to die (deleted
from the repertoire).
It has been discovered that injection of PCA anti-IgM from
birth prevented the development of B cells, resulting in a 'B-
less' mouse. The requirement for crosslinking means that the
antigen has to be polyvalent, the most obvious example of this
being cell-surface mol
This has been directly demonstrated by using transgenic mice
expressing rearranged Ig genes specific for natural or artificial
membrane bound molecules. Presumably other multivalent self
antigens to which immature B cells are exposed also induce
deletion of self reactive cells.
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B cells

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13. Peripheral Tolerance
Because many of the proteins which the body uses are not expressed in thymus or
serum, and in some cases not expressed until after the immune system has matured
there need to be some mechanisms to prevent autoreactivity of lymphocytes after they
have emigrated from the thymus/bone marrow. In fact there are several such
mechanisms:
Ignorance Suppression Anergy
Split
Tolerance
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14. Ignorance
in fact both T cells and B
cells specific for
autoantigens present in
circulation.
In some cases these cells are
quite capable of making a
response but are unaware of
the presence of their
autoantigen.
This arises for 2 reasons.
the antigen may simply be
present in too low conc.
Since all lymphocytes have a
threshold for receptor
occupancy which is required
to trigger a response, then
very low conc. of antigen (in
the case of T cells these are
very low) will not be sensed.
Some antigens are
sequestered from the
immune system in locations
which are not freely exposed
to surveillance, called
immunologically
privileged sites. e.g. eye,
CNS and testis.
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15. Split Tolerance=that some part of the immune
system is tolerant and some other is not.
The most frequent ex. is that where T cell tolerance has been established but
autoreactive B cells are still present.
This arises because T and B cells have different thresholds for activation and
therefore tolerance.
In this situation the B cells are 'helpless'. i.e. for most of antigens, B cells
require help from an antigen-specific T cell in order to make a response.
Thus autoreactive B cells can be present without being able to become
activated provided that there is no T cell help available.
If T cell help is provided, for example by injecting the autoantigen chemically
coupled to an immunogenic foreign carrier, then these B cells will mount a
response.
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16. Experiments of this kind indicate that it takes 100 -1000 × more antigen
to tolerate B cells than T cells. As a result this type of split tolerance
situation is reasonably common for self serum proteins.
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17. Anergy - T cells
 As mentioned in a previous lecture, naive T cells need co-
stimulatory signals to become activated. The expression of
these co-stimulatory molecules is restricted so that most
tissue cells lack either B7.1/B7.2 or CD40 or both. Such cells
also normally lack class II MHC molecules. Thus tissue
cells normally present a spectrum of peptides from their
endogenously synthesised proteins on self MHC class I in
the absence of co-stimulation. Interaction of such cells
with autoreactive T cells leads to the T cell becoming
refractive to later encounter with the same antigen even
when co-stimulation is present. This refractory state is
termed anergy.
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19.  Anergy - B cells
 As implied from the section on split tolerance, there is a
mechanism for tolerising B cells to soluble antigens if they are
present at sufficiently high concentration. The general rules for
this tolerance mechanism were worked out using mice
transgenic for rearranged immunoglobulin molecules and a
transgenic soluble protein whose concentration in serum could
be regulated. It is apparent that the critical parameter is receptor
(surface Ig) occupancy. When more than 5%* of the sIgM
molecules are normally occupied by monomeric soluble antigen
the B cell becomes anergic. This anergic state can be recognised
in the case of B cells by the downregulation of surface IgM.
Note the level of surface IgD remains unaffected and the precise
explanation for why such B cells are refractory to stimulation
even when T cell help is available is not known.
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21. Suppression
 In some cases there are autoreactive T cells present
which are capable of reacting to their cognate antigen
as presented within the host
 [ ie. they are not anergic or ignorant], yet do not express
this reactivity in the normal intact animal.
 These cells appear to be prevented from reacting by
the presence of other T cells, a phenomenon which has
been termed Dominant Regulation or Suppression.
The mechanism is again obscure, but the following
experiments make it likely that this form of tolerance
is at least as important as anergy.
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23. Timing
Some 50 years ago, Owen observed two types of non-identical twin cattle,
those that had shared a haemopoietic system in utero were tolerant of blood
cells from each other and those who had not, were not cross-tolerant.
Burnet postulated that there was a temporal window of tolerance such
that antigens encountered while the immune system was immature
tolerised the relevant lymphocytes.
Medewar subsequently investigated the effects of transferring haemopoietic
cells from histoincompatible 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.
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24. 8/31/2015 6:56:31 AM

25. Timing is an important parameter on the
tolerisability of the immune system
In essence bone marrow stem
cells establish chimaerism of
the host.
Some of these cells
differentiate into APC and
migrate to the thymus where
they tolerate developing
thymocytes by deletion
(central tolerance).
Lifelong chimaerism is
needed to maintain tolerance
and even a low level of
chimaerism is sufficient.
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26. Timing is an important parameter on the
tolerisability of the immune system
For the first few days of the mouse's life there are
too few post-thymic T cells to sustain a response
and these are tolerised either by peripheral
deletion or some other mechanism.
If the transfer is done later the number and
maturity of the peripheral T cell pool is
sufficient to destroy the donor stem cells before
they can engraft.
Even antigens which cannot establish
chimaerism can establish a state of tolerance in
neonatal mice, this is less permanent however.
8/31/2015 6:56:31 AM

27. The Danger Hypothesis - Matzinger versus
Medewar?
Matzinger has proposed that
“there is not a special window
for tolerance during neonatal
life
but the fact that whether
encounter with an antigen
results in tolerance or an
immune response is
determined by whether the
prevailing host environment
promotes a response via
nonspecific cues 'sensing'
danger.
She has further suggested that the
controlled death process of apoptosis is
critical in preventing autoimmunity when
old or surplus cells are disposed off.
The notion that the normal, default
pathway of the immune system is
tolerance rather than response is not a
new idea to immunologists - antigens
usually fail to elicit a response unless
given with adjuvants, whose purpose is
probably to generate stimulatory cues
(cytokines).
8/31/2015 6:56:31 AM

28. The Danger Hypothesis - Matzinger versus
Medewar?....contd
 Recent experiments have shown that not only can adults be tolerised
under certain circumstances, but that neonates can make effective
immune responses if the antigen is presented in sufficiently
immunogenic form.
 I believe that the supposed conflict between Matzinger and Medewar
is rather 'hyped up' and essentially a matter of detail. Neonatal T cells
are not intrinsically tolerisable but the systemic neonatal environment
does predispose to tolerance. Nevertheless, I think that her hypothesis
has drawn the attention of a wider audience to current ideas about
tolerance induction and the factors determining immune
responsiveness.
 Which of the 4 mechanisms of peripheral tolerance referred to above
do you think might be inducible by inhalation of peptide?
 blockade of B7 (B7.1 and B7.2)?
 What factors can you list which would influence the effectiveness
(immunogenicity) of a new subunit (purified protein) vaccine?
8/31/2015 6:56:31 AM