Dr Alok Tripathi

Department of Biotechnologyaquaimmuno@yahoo.co.in

8/31/2015 6:56:30 AM

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

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

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:

I. Overview

• Billingham neonatal graft experiments demonstrate learning of tolerance during development

• Shift of self-nonselfparadigm 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

8/31/2015 6:56:30 AM

T Cell Tolerance

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.

• - 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) ClonalDeletion

• - 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

8/31/2015 6:56:30 AM

B) PERIPHERAL TOLERANCE

• 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.

-can be extended to inhibit autoimmunity and therefore

promote tolerance

- Providing Ag through gut (or lungs) can activate these cells and mediate tolerance

challenge

8/31/2015 6:56:30 AM

Function

……And also…..

Almost any other parameter which affects the overall strength of interaction between

a clone of thymocytes and the thymicAPC 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

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.

8/31/2015 6:56:31 AM

B cells

8/31/2015 6:56:31 AM

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 AnergySplit

Tolerance

8/31/2015 6:56:31 AM

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, calledimmunologically

privileged sites. e.g. eye, CNS and testis.

8/31/2015 6:56:31 AM

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.

8/31/2015 6:56:31 AM

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.

8/31/2015 6:56:31 AM

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.

8/31/2015 6:56:31 AM

8/31/2015 6:56:31 AM

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 sIgMmolecules 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.

8/31/2015 6:56:31 AM

8/31/2015 6:56:31 AM

Suppression

In some cases there are autoreactive T cells present which are capable of reacting to their cognate antigenas 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.

8/31/2015 6:56:31 AM

8/31/2015 6:56:31 AM

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 haemopoieticcells 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.

8/31/2015 6:56:31 AM

8/31/2015 6:56:31 AM

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.

8/31/2015 6:56:31 AM

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

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

The Danger Hypothesis - Matzinger versus Medewar?....contd

Recent experiments have shown that not only can adults be tolerisedunder 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 Medewaris 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