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Immunological Tolerance


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When specific lymphocytes encounter:

antigens immunogens antigens -tolerogens

the lymphocytes activated the lymphocytes maybe

inactivated or eliminated

immune response tolerance

Note: A single antigen maybe an immunogen or a tolerogen,depending on the conditions in which it is displayed tospecific lymphocytes e.g. in the presence or absence,respectively, of inflammation and innate immune responses.


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Immunological Tolerance:Central & Peripheral Tolerance


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Self-tolerance maybe induced in immatureself-reactive lymphocytesin the generative lymphoid organs (central tolerance) or in maturelymphocytes in pheripheral sites (peripheral tolerance).

Central tolerance occurs during the maturation of lymphocytes in thecentral generative lymphoid organs where all developing lymphocytespass through a stage at which encounter with antigen may lead to celldeath or replacement of a self-reactive antigen receptor with a newone.

Peripheral tolerance occurs when, as a consequence of recognisingself antigens, mature lymphocytes become incapable of responding tothat antigen, or are induced to die by apoptosis or mature T cells areactively suppressed by regulatory T cells.

Whether lymphocytes that recognise antigens become activated ortolerant is determined by: the properties of the antigens, the stateof maturation of the antigen-specific lymphocytes, and the types of

stimuli received when these lymphocytes encounter self antigens.


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T lymphocyte tolerance

Tolerance in CD4+ helper T lymphocytes is an effective way of

preventing immune responses to protein antigens because helperT cells are necessary inducers of both cell-mediated andhumoral immune responses to proteins.

Central T cell tolerance

During their maturation in the thymus, many immature T cellsthat recognize antigens with high avidity are deleted (negativeselection) and some of the surviving cells in the CD4+lineagedevelop into regulatory T cells specific for these antigens thatenter peripheral tissues.

Note: Affinitymeasures the strength of interaction between an epitope and an antibodysantigen binding site.

Aviditygives a measure of the overall strength of an antibody-antigen complex. It isdependent on three major parameters:

Affinity of the antibody for the epitope (see above)

Valency of both the antibody and antigen Structural arrangement of the parts that interact


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Negative selection (clonal deletion)

occurs in thymus. eliminates thymocytes bearing high affinity receptors for self-

MHC molecules alone or self-antigen presented by self-MHC. irreversible loss of activity since antigen-reactive cells are

eliminated.

various experimental demonstration:

1. by Kappler and Marrack negative selection ofthymocytes that binds strongly to self.

2. by von Boehmer and Kisielow negative selection ofthymocytes requires both self-antigen and self-MHC.(see figure 10.7)


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Question: How does clonal deletion occur? What causes death?- below results of experiment done by Kappler and Marrack- negative selection was studied using the V17a T cell receptor model.

V17a is expressed on cortical thymocytes. Thymocytes expressing

TCRs bearing V17a bind too strongly to a conserved region of IEor IE(i.e. IE+ mouse strains). This causes inappropriate signaling,and cells are deleted. Therefore are lost on mature thymocytes dueto negative selection.Table1: Utilisation of V17a in IE+ and IE- mouse strains

% cells with V17a:Cell Population: SJL (IE-) B10.BR (IE+)Immature thymocytes (CD4+8+) 2.9 2.6Mature thymocytes

(CD4+) 2.4 0.06(CD8+) 0.4 0.01Periph T cells 13.9 0.09Conclusion: Cells with V17a is never expressed on peripheral T cellsin mice that have MHC II IE because of negative selection in the

thymus (clonal deletion) to prevent auto-reactivity.


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von Boehmer and Kisielow negative selection of thymocytes requires bothself-antigen and self-MHC.


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Question: Where does negative selection occur? Bone marrow chimeras used as tested cells responsible for negative

selection.To test : transferred bone marrow from various donors into irradiated micerecipients, where all cells in irradiated recipients that were derived fromendogenous bone marrow were killed by irradiation.

Table 2 : Bone marrow derived cells control clonal deletion in the thymusMarrow donor Recipient % periph T cells

(thym epith) V17a+IE- IE- 13.6IE+ IE- 0.1

IE- IE+ 16.2IE+ IE+ 0.08

Bone marrow derived-cells reseeding the irradiated thymus determine clonaldeletion of self-reactive thymocytes.

What happens?

Host DC die when mice are irradiated. Replaced by DC from donor marrow. DC display both class I and II MHC. If thymocyte binds IE on these DC, and is activated, it dies and not seen in

the periphery.

Conclusion: In bone marrow chimaeras - Positive selection is on host thymicepithelial cells and Negative selection occurs on donor-derived DC

P i i l i h i i h li l ll


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Positive selection occurs on thymic epithelial cells.

P tid i l i l d i ti l ti


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Peptide in groove also involved in negative selection


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Peripheral T cell Tolerance

Peripheral tolerance is the mechanism by which mature T cells

that recognize self antigens in the peripheral tissues arerendered incapable of subsequently responding to theseantigens.

The antigens could be:

1. tissue-specific self-antigens that are not abundant in the

thymus

2. tolerogenic forms of foreign antigens

The mechanisms of peripheral tolerance are:anergy (functional unresponsiveness)

suppression

deletion (cell death)

DBA/2 mouse (H-2d): T cells with V 6 TCR autoreact with IEd


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DBA/2 mouse (H-2 ): T cells with V6 TCR autoreact with IE During the first week of life, autoreactive T cells are released from thymus. In normal adult, as mouse ages, negative selection initiates, and this process stops. Neonatal thymectomy freezes the early situation, V6 remains in periphery lack of

negative selection.

Table3: Peripheral tolerance in DBA/2 mice

Table4: % of V6 in adult, neonatal (newborn) and adult neo-thymectomised mice (NTx)

In neonatal mouse, many T cells with potential reactivity for self MHC (IEd) leave thymusand go to periphery.

In adult neonataly thymectomised animal, the autoreactive cells are still there, as aresult of lack of negative selection.

Question: What is their immune status when they get there? Do they react with self?


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Table 5:Reactivity of these autorective cells

Day 3 thymocytes can respond to IEdhowever peripheral cells in the thymectomizedmouse neither proliferate or produce IL-2 in response to IEd.They have becomeanergized.

How do these cells become anergised? We know that proper T cell signaling requires twosignals; aTCR-mediated signal and a co-stimulatory signal.


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deletion- the induced death of members ofa clone of T and B lymphocytes withinappropriate receptors i.e. those thatreact strongly with self during an immuneencounter


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anergy- a physiological state in which cellsare unable to be activated by antigen e.g.in a T cell marked by the inability of the

cells to proliferate in response to apeptide-MHC complex.


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Anergy (Functional Unresponsiveness)

Exposure of mature CD4+ T cells to an antigen in the absence ofco-stimulation or innate immunity may make the cells incapable

of responding to that antigen. In a normal response, T cell are induced when the cells recognise

an antigen presented by a professional APC and activatingreceptors on the T cells (such as CD28) recognise co-stimulatorson the APCs/dendritic cells (DC) (such as B7).

If the T cell recognise a self antigen without co-stimulation, theT cell becomes unresponsive to the antigen because of a block insignaling from the TCR complex or engagement of inhibitoryreceptors (such as CTLA-4).

The signaling block may be the result of recruitment of

phosphatases to the TCR complex or the activation of ubiquitinligases that degrade signaling proteins.

Therefore the T cell remains viable but is unable to respond tothe self antigen.


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When T cells recognise self-antigens, theymay engage inhibitory receptors of the

CD28 family, whose function is toterminate T cell responses


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Note: T cell help to B cells is a fundamental aspect of adaptive immunity and the generation ofimmunological memory. Follicular helper CD4 T (T(FH)) cells are the specialized providers of Bcell help. T(FH) cells are important for the formation of germinal centers. Once germinalcenters are formed, T(FH) cells are needed to maintain them and to regulate germinal centerB cell differentiation into plasma cells and memory B cells.


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suppression - cells are there and thesuppression is a rapidly reversible statei.e. if remove suppressing agent, responseof T suppressor cells or regulatory T

cells reappears.

Question: What are T suppressor cells or

regulatory T cells?


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Regulatory T cells

Regulatory T lymphocytes are a subset of CD4+T cells whosefunction is to suppress immune responses and maintain self-tolerance.

Regulatory T cells are generated by self antigen recognition in thethymus (sometimes called natural regulatory cells) and (probably toa lesser extent) by antigen reconition in peripheral lymphoid organs(called inducible or adaptive regulatory cells).

The development and survival of these regulatory T cells requireIL-2 and the transcription factor FoxP3 and STAT5.


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Regulatory T cells In peripheral tissues, regulatory T cells produce IL-10 and TGF-

both of which inhibit immune responses.

They also inhibit the ability of APCs to stimulate T cells.

Activation of the regulatory T cell is Ag specific. Suppression bythe regulatory T cell is non-specific, if they are removed, Ag-reactivity ensues.


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B lymphocyte tolerance

Tolerance in B lymphocytes is necessary for maintainingunresponsiveness to thymus-independent self-antigens, such aspolysaccharides and lipids.

They are also important in preventing antibody responses toprotein antigens.

Experimental studies revealed multiple mechanisms of Blymphocyte tolerance induced when encountered with self

antigens.


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Central Tolerance in B cells

Clonal deletion also occurs for B cells. When a self reactive BCR

is expressed in the bone marrow, negative selection of the self-reactive immature B cells occurs. The selected cells are deletedby apoptosis or undergo receptor editing to produce non-selfreactive mIg.

Receptor editing: if immature B cells recognise self antigensthat are present at high concentration in the bone marrow andespecially if the antigen is displayed in multivalent form (eg oncell surfaces) many antigen receptors on each B cell are cross-linked, thus delivering strong signals to the cells. One

consequence of such signaling is that the B cells reactivate theirRAG1 and RAG2 genes and initiate a new round of VJrecombination in the Ig light chain gene locus. The selfreactive immature B cell is deleted and a new Ig light chain isexpressed, thus creating a B cell receptor with a new

specificity.


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Central Tolerance in B cells


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Copyright 2011 by Saunders, an imprint of Elsevier Inc.Abbas, Lichtman, and Pillai. Cellular and Molecular Immunology, 7thedition. Copyright 2012 by Saunders, an imprint o


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If the rearrangement at the locus is non-productive,rearrangements at the light chain loci may follow.

Deletion: if editing fails, the immature B cells may be deletedvia apoptosis.

Anergic: if developing B cells recognise self-antigens weakly/lowaffinity, the cells become functionally unresponsive (anergic)and exit the bone marrow in this unresponsive state.

Anergy is due to downregulation of antigen receptor expression

as well as a block in antigen receptor signaling.


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Peripheral Tolerance in B cells

Mature B lymphocytes that recognise self antigens in peripheral

tissues in the absence of specific helper T cells may berendered functionally unresponsive or die by apoptosis.

Anergy: Some self-reactive B cells that are repeatedlystimulated by self antigens become unresponsive to furtheractivation.

Deletion: As a result, they have a shortened life span and areeliminated more rapidly than cells that have not recognised selfantigens via mitochondrial apoptosis.

Fas mediated elimination: These B cells may be activelyeliminated by the interaction of FasL on helper T cells with Fas

on the activated B cells. Signaling by inhibitory receptors: B cells that recognise self

antigens with low affinity may be prevented from responding bythe engagement of various inhibitory receptors (clonalignorance).

Peripheral Tolerance in B cells


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Copyright 2011 by Saunders, an imprint of Elsevier Inc.Abbas, Lichtman, and Pillai. Cellular and Molecular Immunology, 7thedition. Copyright 2012 by Saunders, an imprint o

p

Clonal deletion occurs in B cells


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Clonal deletion occurs in B cells

B cells eliminated if specific for cell surface self Ag seen in bonemarrow of transgenic mouse ( transgenic using rearranged H and Lchains for an Ab recognising Kb). The B cells are clonally deletedthrough central tolerance.

Peripheral deletion of B cells


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Transgenic mouse #1 H-2k mouse with H and L chain transgenes for KbAb.

Transgenic mouse #3 - Kbtransgene into H-2kmouse under liver

promoter, that is, limits expression to liver.Mate these two mice F1 will be H-2k. In bone marrow, 95% of B cellshave transgenic receptor. In periphery, there are almost no B cells atall. Since B cells are not in the immature state, therefore negativeselection must have occurred at mature B cell stage in the periphery.

B cell tolerance to soluble self Ag


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g

Many B cells will leave bone marrow with self reactivity to solublesecreted self Ag in blood and lymph. How are these cells controlled?

In an experiment by Basten et al:

1.Transgenic mouse #1 used rearranged H & L genes for Ab specificfor hen egg white lysozyme (HEL), endogenous gene rearrangementsuppressed; >95% of B cells are HEL-specific.

2. HEL-transgenic mouse transgenic # 2 - as adults, make soluble HEL(hen egg lysozyme) and secrete into serum. T cells completely tolerantto this foreign protein, no T cell responses thus no Ab.3. Mate the two transgenic F1 inherit HEL transgenes & anti-HEL Ab transgenes. Makes and secrete soluble HEL Ag, normal numbers

of B cells in all compartments No clonal deletion.But B cells are not functional, they are anergic.4. Transfer B cells from transgenic #1 into normal adult mice. Highlevels of soluble self Ag cause alterations of B cell phenotype. Bcells are not deleted, become low, high..anergized

B cell tolerance to soluble self Ag


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Conclusion:B cells specific for self are not deleted but leads to a state

of anergy.


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Traced this rescue effect to molecule on B cell surface CD40 interact withmolecule on T cell surface called CD40L. B cells are eliminated if interact withself cell surface marker in periphery. It is therefore not deleted in bone marrowbut deleted in periphery. This is linked to failure to activate CD4 help. CD40 on

B cell must be ligated by CD40L on activated T cell, or B cell dies.


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Tolerance induced by foreign protein antigens

Foreign antigens may be administered in ways the preferentiallyinduce tolerance rather than immune responses.

In general, protein antigens administered subcutaneously orintradermally with adjuvants favour immunity, whereas high

doses of antigens administered systemically without adjuvantstend to induce tolerance.

The likely reason for this is that adjuvants stimulate innateimmune responses and the expression of co-stimulators on APCs

and in the absence of these second signals, T cells thatrecognise the antigen may become anergic or die or maydifferentiate into regulatory cells.


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FcRIIBis the only inhibitory Fc receptor. It controls many aspects of immune

and inflammatory responses


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marginal zoneprecursor B cells

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