2Why is a mechanism for repertoire selection and self tolerance needed? Generation of the TcR repertoire involves many random mechanismsThe specificity of TcR in the immature repertoire is also random & will include cells with receptors that are:T1. HarmfulSelfantigenrecognition2. UselessTTAPC3. UsefulForeignantigenrecognition
3Self proteins enter the endogenous and exogenous antigen processing pathwaysSelf cellularproteinsSelf serum& cellularproteinsProcessing pathways do not distinguish self from non-self
4Self peptides load onto MHC class I & II molecules Purify stable MHC-peptide complexesFractionate and microsequencepeptidesAcid elute peptides>90% of eluted peptides are derived from self proteinsYet self antigens do not usually activate T cells
5The immune system allows a limited degree of self recognitionTcRs recognise the non-self peptide antigen and the self MHC moleculeMHC molecules RESTRICT T cell activationBut how do T cells learn how much self recognition is acceptable?
6T cells are only allowed to develop if their TcR recognise parts of self MHC Explains why T cells of MHC haplotype A do not recognise antigen specific presented by MHC haplotype BMHC AhaplotypeT CELLMHC BhaplotypeAPCMHC AhaplotypeAPC
7Wholly self-reactive and useless T cells are removed MHC-restricted are retained Random TcRrepertoireensures diversityTHYMUSYTYT?YTAPCHarmfulUselessUsefulNegativelyselectPositivelyselectNeglect
8Stroma provides a microenvironment for T cell development & selection The thymusLobulated structure with a STROMA of epithelial cells & connective tissueStroma provides a microenvironment for T cell development & selectionLobules differentiated into an outer CORTEX & inner MEDULLA, both filled with bone-marrow-derived THYMOCYTESThymocyteCortexMedullaCorticalepithelial cellDendritic cellMacrophageMedullaryepithelial cell
9The thymus is required for T cell maturation Athymic mice (nude) and humans (DiGeorge syndrome)are immunodeficient due to a lack of T cellsNo mature T cellsIn adultNeonatal thymectomyMature T cellsIn adultThymus intact
10Roles of the bone marrow and thymus in T cell maturation Defective lymphocyteproductionNormal thymusscid/scidNo mature T cellsIn adultsThymus defectNormal bonemarrownu/nuNo mature T cellsIn adults
11Bone marrow supplies T cells, and they mature in the thymus Marrow defectThymus defectBone marrow transplantThymus colonised by thymocytes from thymus defective, i.e. orange, mouseThymus graftThymus colonised by thymocytes from the thymus defective, i.e. orange, mouse
12The thymus matures T cells after birth, but early in lifeRemove ThymusAdultNeonateT cells not yet left thymusMature T & B cellsNo T cellsMature B cells presentThe thymus is needed to generate mature T cells
13The thymus is most active in the foetal and neonatal periodOVAAdultNeonateKLHT cells vs. OVANo T cells vs.OVAT cells vs. KLHThe thymus is needed for NEONATAL TOLERANCE
14T cells mature in the thymus but most die there. Mousethymus5 x 107 per dayConstant1-2 x 108cells2 x 106 per day98% of cells die in the thymus without inducing any inflammation or any change in the size of the thymus.Thymic macrophages phagocytose apoptotic thymocytes.
15T cell development is marked by cell surface molecule changesAs T cells mature in the thymus they change their expression of TcR-associated molecules and co-receptors.These changes can be used as markers of their stage of maturation98%CD3/TcR-CD4-, 8-DoublenegativeCD3+TcR-chain +pre-TcR+ (pTCD4+, 8+LargedoublepositiveCD3+TcR +CD4+CD8+SmalldoublepositiveCD3+TcR +CD4+SinglepositiveTcR+CD3+CD4-, 8-CD3+TcR +CD8+Singlepositive
16Different developmental stages of thymocytes are present in different parts of the thymusCortexImmaturedoublenegative &positivethymocytesMedullaMaturesingleDNCD25-CD44+DNCD25+CD44+DNCD25+, CD44lowDNCD3+ pT:CD25-, CD44-DPCD3+ pT:DPCD3+ TcR+SPCD3+ TcR+CD8+CD4+
17Germline configuration TcR rearrangementDNCD25+CD44+CD25-VDJCGermline configurationVDJCD-J fusionVDJCV-DJ fusionDNCD25+,CD44lowC region spliced to VDJ fusion and -chain protein produced in cytoplasmNo TcR at cell surface
18Similarities in the development of T and B cells: A B cell reminderLargePre-BSurrogate light chain is transiently expressed when VHDHJH CHm is productively rearrangedTriggers entry into cell cycleExpands pre-B cells with in frame VDJ joins2. Suppresses further H chain rearrangementAllelic exclusion
19Similarities in the development of T and B cells: Pre T cell a receptorpreTcRa-chainTcRb-chainCD8CD4TcRb-chainpreTcRa-chainDNCD3+ very lowpT:CD25- CD44-DPCD3+ lowpT:CD25- CD44-CD4+ CD8+1. Cell proliferates rapidly to yield daughter cells with the same chainExpands only cells with in-frame TcR b chains2. Successful rearrangement shuts off rearrangement on 2nd chromosomeEnsures only one specificity of TcR expressed per cell
20TcR rearrangement DP DP Selection can now begin CD3+ lowpT:CD25- CD44-CD4+ CD8+When proliferation stops,the chain starts to rearrangeGermline TcR JCVV-J rearrangedTcR 1° transcriptSpliced TcR mRNACD3+ TcR+DPT cells can now recognise antigensand interact with MHC class I & IIthrough CD4 & CD8Selection can now begin
21Mousethymus5 x 107 per day2 x 106 per dayHow does the thymus choose which of the cells entering the thymus are useful, harmful and useless
22Sorting the useful from the harmful and the useless Positive selectionNegative selectionRetention of thymocytes expressing TcR that are RESTRICTED in their recognition of antigen by self MHCi.e. selection of the USEFULRemoval of thymocytes expressing TcR that either recognise self antigens presented by self MHC or that have no affinity for self MHCi.e. selection of the HARMFUL and the USELESS
23Antigen can be seen by the TcR only in the context of an MHC molecule MHC restrictionAntigen can be seen by the TcR only in the context of an MHC moleculeTcR will not bind to an MHC molecule unless there is an antigen in the grooveIn the presence of antigen, the TcR must have some affinity for the MHC molecule
24Experimental evidence for MHC restriction as a marker of positive selection CHIMERAOrange strain cells in a blue strain mouseWhich MHC haplotype will restrict the T cells, Orange or blue?Thymus defectBone marrow transplantTransplant reconstitutesmarrow defective mouseMarrow defect
25Studies in bone marrow chimeras show that MHC restriction is learnt in the thymusMHC (AxB)F1Bone marrow donorIrradatedbone marrowrecipientsMHC AMHC (AxB)F1MHC BMHC haplotype of APCABT cellresponseof recipient Tcells to antigenThe MHC haplotype of the environment in which T cellsmature determines their MHC restriction element
26Mice of a particular MHC haplotype only make T cells Explanation of bone marrow chimera experiment:Mice of a particular MHC haplotype only make T cellsrestricted by that haplotypeMHC AMHC BMHC (AxB)F1Able to makeT cells restrictedby MHC AAble to makeT cells restrictedby MHC BAble to makeT cells restrictedby MHC A or BBone marrowmust containpotential to make Tcells restricted byA and B MHC molecules
27Irradiation prevents the bone marrow from generating lymphocytes Explanation of bone marrow chimera experiment:Irradiation prevents the bone marrow fromgenerating lymphocytesIrradiation destroys the immune system but has no effect onthe epithelial or dendritic cells of the thymusMHC AMHC BMHC AMHC BNormal miceMHC AMHC BMice now have an intact, functional thymic stromabut have no thymocytes, T cells or bone marrowThese mice are severely immunodeficient and can only bereconstituted by a bone marrow transplant
28Reconstitution of irradiated mice with (AxB)F1 bone marrow Explanation of bone marrow chimera experiment:Reconstitution of irradiated mice with (AxB)F1 bone marrowMHC (AxB)F1Bone marrow contains thepotential to make Tcells restricted byA and B MHC moleculesTransplant bone marrowto reconstitute immune systemof immunodeficient miceMHC AMHC (AxB)F1MHC BIrradiatedbone marrowrecipients
29MHC restriction is learnt in the thymus by positive selection Explanation of bone marrow chimera experiment:MHC restriction is learnt in the thymus by positive selectionA x B T cellprecursorsMHC A ThymusMature T cellsrestricted onlyby MHC AMouse with an MHC Athymus, but A x Bbone marrowA x B T cellprecursorsMHC B ThymusMature T cellsrestricted onlyby MHC BMouse with an MHC Bthymus, but A x Bbone marrow
30Peripheral T cells are restricted by the MHC type of the Explanation of bone marrow chimera experiment:Peripheral T cells are restricted by the MHC type of thethymus that they mature inMHC (AxB)F1Bone marrow donorMHC AMHC BBone marrowrecipientsT cellresponseof recipient Tcells to antigenABMHC haplotype of antigen presenting cells
31Summary Bone marrow chimeras show that MHC restriction is learnt in the thymusT cells are ‘educated’ in the thymusto recognise antigens only in the contextof self MHCMHC restriction is learnt in thethymus by positive selectionThe MHC haplotype of the environment in which T cellsmature determines their MHC restriction element
32i.e. selection of the HARMFUL and the USELESS Negative SelectionRemoval of thymocytes expressing TcR that either recognise self antigens presented by self MHC or that have no affinity for self MHCi.e. selection of the HARMFUL and the USELESSSuperantigens can be used to probe the mechanisms of negative selection
33Nominal antigens & superantigens Require processing to peptidesTcR and chains are involved in recognition>1 in 105 T cells recognise each peptideRecognition restricted by an MHC class I or II moleculeAlmost all proteins can be nominal antigensSuperantigensNot processedOnly TcR chain involved in recognition2-20% of T cells recognise each superantigenPresented by almost any MHC class II moleculeVery few antigens are superantigensSuggests a strikingly different mechanismof antigen presentation & recognition.
34Superantigens T cell APC V V e.g. Staphylococcal enterotoxins Class II fromMHC A to ZhaplotypesTcR fromMHC AhaplotypeT cellAPCVVe.g. Staphylococcal enterotoxinsToxic shock syndrome toxin I (TSST-1)Staphylococcal enterotoxins SEA, SEB, SEC, SED & SEEDo not induce adaptive responses, but trigger a massive burst of cytokines that may cause fever, systemic toxicity & immune suppressionSevere food poisoning Toxic shock syndrome
35Interaction of SEB with MHC Class II molecules and the TcR MHC class II TcR beta chainTcR beta chain SEBMHC class II SEB
36Exogenous superantigen-V relationship Superantigen Human V regionSEA 1.1, 5.3, 6.3, 6.46.9, 7.3, 7.4, 9.1SEB 3, 12, 14, 15, 17, 20SEC1 12SEC2 12, 13.1, 13.2SED 5, 12SEE 5.1, 6.3, 6.4, 6.9, 8.1TSST-1 2Explains why superantigens stimulate so many T cells
37Effect of TSST-1 on T cells expressing V2 Fresh PBMC unstainedFluorescence intensity(i.e. amount of staining with anti-V antibody)Cell numberFresh PBMC stainedwith anti-V2PBMC cultured with TSST-1Stained with anti-V2Cell numberPBMC cultured with TSST-1Stained with anti-V3
39Superantigens T cell APC Vb V V Mouse mammary tumour viruses (Mtv) TcR fromMHC AhaplotypeT cellAPCClass II fromMHC A to ZhaplotypesVbVVSuperantigensMouse mammary tumour viruses (Mtv)Cell-tethered superantigen encoded by the viral genome
40Endogenous superantigens Mouse mammary tumour viruses (MMTV)Retroviruses that contain an open reading framein a 3’ long terminal repeat that encodes a superantigenassociated with the cell surface of APCMost mice carry 2-8 integrated MMTV proviruses in their genomeIntegrated MMTVMtv-1, 2, 3, 6, 7 (Mls-1a), 8, 9, 11, 13 & 43Infectious and transmitted by milkMMTV (C3H)MMTV (SW)MMTV (GR)
41Endogenous superantigen V-relationship Mtv Murine V regionMtvMtvMtv , 5.2, 11Mtv 6 3, 5.1, 5.2Mtv 1 3Mtv 3 3MtvMtv 7 6, 7, 8.1, 9MMTV SW 6, 7, 8.1, 9MMTV C3H 14MMTV GR 14Stimulate T cells in a similar manner to exogenous supernatigensValuable tools in analysis of self tolerance
42Mtv act in a similar manner to exogenous superantigens in vitro STIMULATOR CELLSMtv-7 +veRESPONDING T CELLSMtv-7 -veIrradiatedTTTAPCTTTMtv-7 superantigenTTOnly T cells with TcR containing V6, V8.1 and V9 proliferateMtv-7 interacts with V 6, V8.1 and V9 and activatesonly cells bearing those TcRSelective expansion of cells bearing certain V chains
43How do pathogens use superantigens? Unfocussed adaptive immune response activates cells of all specificities as well as those specific for the superantigensReduces the possibility that effective T cell clonal selection can eliminate the pathogenUpon resolution, cells activated by the superantigen die, leaving the host immunosuppressedTransmission of infection
44Transmission of infection 2. Massive T cellresponse to MMTV superantigen3. Vigorous T cell help leads to B cell proliferation and differentiation to long-lived B cellsB1. MMTV infected,MHC class IIpositive B cells4. Infected cells traffic to mammary gland and infect young via milk
45Analysis of negative selection in vivo. MtvMtv-7 superantigen negativeTHYMUSMtv-7 superantigen positiveMtv-7 superantigen binds to V6, V8.1 and V9+ve thymocytesImmature CD4+8+ thymocytesexpressing VV8.1 and V9in the thymusImmature CD4+8+ thymocytesexpressing VV8.1 and V9in the thymusNegative selectionNegative selectionMature CD4+ or CD8+VV8.1 and V9T cells in peripheryNo mature CD4+ or CD8+VV8.1 and V9T cells in peripheryPERIPHERY
46Analysis of negative selection in vivo. Milk transmissible superantigens - MMTV (C3H)V14 present?Male or female B10.BRYesMale or female C3HNo
47X Yes X No V14 present? V14 present? Male C3H Female B10.BR F1 offspringYesMale B10.BRFemale C3HXV14 present?F1 offspringNo
48Deletion of V14 T cells in mice infected with MMTV by milk +FosterfemaleB10.BRYoung male orfemale C3HYesV14 present infostered pups?+FosterfemaleC3HYoung male orfemale C3HOr B10.BRNoMMTV transmitted to fostered pups by infected B cells found in milk
49Are the signals that induce positive & negative selection the same, or different? specificityDIFFERENTspecificityTHYMUSImmature thymocytesPositive selectionNegative selectionPeripheral T cellsX
50Hypotheses of self-tolerance Differential signalling hypothesis Avidity hypothesisAffinity of the interaction between TcR & MHCDensity of the MHC:peptide complex on the cell surfaceQuantitative difference in signal to thymocyte.Differential signalling hypothesisType of signal that the TcR delivers to the cellQualitative difference in signal to thymocyte.
51Removal of useless cells Peptide is not recognised or irrelevantThymocyte receives no signal, fails to be positively selectedand dies by apoptosis.WEAK OR NO SIGNALTcRT cellCD8Thymic epithelial cellMHCClass I
52Positive selection PARTIAL SIGNAL Peptide is a partial agonist Thymocyte receives a partial signal and is rescued from apoptosisi.e. the cell is positively selected to survive and mature.PARTIAL SIGNALTcRT cellCD8Thymic epithelial cellMHCClass I
53Negative selection FULL SIGNAL Peptide is an agonist Thymocyte receives a powerful signal and undergoes apoptosisi.e. the cell is negatively selected and dies.FULL SIGNALTcRT cellCD8Thymic epithelial cellMHCClass I
54The thymus accepts T cells that fall into a narrow window of affinity for MHC molecules UselessNeglectUsefulPositively selectHarmfulNegatively selectNumberof cellsLowHighAffinity of TcR/MHC interaction
56How accurate are these models of positive and negative selection? Positive selection:Relied on very complex chimera experimentsRelied on proof of MHC restriction as an outcome which is tested in an ‘unnatural’ response using MHC mismatched presenting cellsNegative selection:Relied on exceptionally powerful superantigens operating outside the normal mechanisms of antigen recognition
57} T Illustration of selection using TcR transgenic mice Generation of transgenic miceTT cell clone with knownTcR specificity and MHC restrictionRe-implantAnalyse offspringfor transgeneexpression.Rearranged chaincDNA constructRearranged chain}Inject into fertilisedmouse ovumIn TcR transgene-expressing mice almost all thymocytes express thetransgenic TcR due to ALLELIC EXCLUSION.
58Cells that fail positive selection die in the thymus (neglect) In TcR transgenic mice expressing an MHC A restricted TcR, all thymocytes express the MHC A restricted TcRMHC BDNCD3-DPCD3+ TcR+Transgenically express MHC A restricted TcR in an MHC B mouseNo single +ve cells arepresent in the peripherySPCD3+ TcR+CD8+ or CD4+Thymocytes die at the double positive stage after failing +ve selection due to a lack of MHC A
59Restriction element and co-receptor expression are co-ordinated Positive selection determines the restriction element of the TcR AND the expression of CD4 or CD8TcR transgenic mouseTcR from MHC class I-restricted T cellTcR transgenic mouseTcR from MHC class II-restricted T cellOnly CD8cells matureOnly CD4cells matureRestriction element and co-receptor expression are co-ordinatedInstructive model: Signal from CD4 silences the CD8 expression & vice versa?Stochastic/selection model: Cells randomly inactivate CD4 or CD8 gene, then test for matching of TcR restriction with co-receptor expression?
60Double positive to single positive transition Double positive thymocyteSingle CD4+ thymocyte-veTcRMHC Class I3TcRMHC Class II2TcRCD4CD8CD8CD4√XMHC Class IMHC Class II32Thymic epithelial cellInstructive model: Signal from CD4 silences the CD8 expression & vice versa
61Double positive to single positive transition Double positive thymocyteSingle CD4+ thymocyteTcRMHC Class II2TcRMHC Class I3CD8CD4CD8CD8CD4CD4CD4√XMHC Class IMHC Class II32Thymic epithelial cellStochastic/selection model: Cells randomly inactivate CD4 or CD8 gene, whilst testing a match of TcR restriction
62Deletion of cells in the thymus: differential effect on the mature and immature repertoireTcR transgenic mouseTcR from T cell specific for hen egg lysosyme (HEL)~100% of T cells/thymocytes express anti-HEL TcRImmunisewith HELAnalyse peripheralT cells:All transgenic T cellsproliferateAnalyse thymus:All transgenic T cells dieby apoptosisThymocytes activated by antigen in the thymic environment dieT cells activated by antigen in the periphery proliferate
63How do we become self tolerant to these antigens? How can the thymus express all self antigens – including self antigens only made by specialised tissues?How do we become self tolerant to these antigens?
65Promiscuous expression of tissue-specific genes by medullary thymic epithelial cells
66How is self tolerance established to antigens that can not be expressed in the thymus?T cells bearing TcR reactive with proteins expressed in the thymus are deleted.Some self proteins are not expressed in the thymus e.g. antigens first expressed at pubertySelf tolerance can be induced outside the thymusPERIPHERAL TOLERANCE or ANERGYA state of immunological inactivity caused by a failure to deliver appropriate signals to T or B cells when stimulated with antigeni.e. a failure of antigen presenting cells to deliver COSTIMULATION
67T helper cells costimulate B cells Two - signal models of activation Signal 1 antigen & antigen receptorACTIVATIONThYBSignal 2 - T cell helpCD40MHC class IIand peptideT cell antigen receptorCo-receptor (CD4)CD40 Ligand (CD154)
68Antigen presentation - T cells are co-stimulated APCThSignal 1 antigen & antigen receptorACTIVATIONSignal 2B7 family members (CD80 & CD86)CD28Costimulatory molecules are expressed by most APC including dendritic cells, monocytes, macrophages, B cells etc., but not by cells that have no immunoregulatory functions such as muscle, nerves, hepatocytes, epithelial cells etc.
69Mechanism of co-stimulation in T cells 1AntigenLow affinity IL-2 receptorIL-2IL-2RIL-2IL-2RResting T cellsSignal 1NFAT binds to the promoter of of thea chain gene of the IL-2 receptor.The a chain converts the IL-2Rto a high affinity formExpress IL-2 receptor- and chains but no a chain or IL-2
70Mechanism of co-stimulation in T cells 2Costimulation1AntigenSignal 2Activates AP-1 and NFk-B to increase IL-2 gene transcription by 3 foldStabilises and increases the half-life of IL-2 mRNA by foldIL-2 production increased by 100 fold overallIL-2IL-2RImmunosuppressive drugs illustrate the importance of IL-2 in immune responsesCyclosporin & FK506 inhibit IL-2 by disrupting TcR signallingRapamycin inhibits IL-2R signalling
711 Anergy Antigen Naïve T cell Signal 1 only IL-2IL-2R1AntigenSignal 1onlyEpithelialcellThe T cell is unable to produce IL-2 and therefore is unable to proliferate or be clonally selected.Unlike immunosupressive drugs that inhibit ALL specificities of T cell, signal 1 in the absence of signal 2 causes antigen specificT cell unresponsiveness.Self peptide epitopes presentedby a non-classical APC e.g. anepithelial cell
72Arming of effector T cells Clonal selection and differentiationAPCTIL-2How can this cell give help to, or kill cells, that express low levels of B7 family costimulators?Activation of NAÏVE T cells by signal 1 and 2 is not sufficient to trigger effector function, but…..the T cell will be activated to proliferate and differentiate under the control of autocrine IL-2 to an effector T cell.These T cells are ARMED
73Effector function or Anergy? Clonally selected,proliferating anddifferentiatedT cell i.e. ARMED sees antigen ona B7 -ve epithelial cellThis contrasts the situation with naïve T cells, which are anergised without costimulationThe effector programmeof the T cell is activatedwithout costimulationArmedEffectorT cellCD28Co-receptorTcRIL-2ArmedEffectorT cellNaïveT cellKillEpithelialcellEpithelialcellEpithelialcell
74Costimulatory molecules also associate with inhibitory receptors CD28T cellCD28loActivated T cellCross-linking of CTLA-4by B7 inhibits co-stimulationand inhibits T cell activation-2Signal 1 +CTLA-4hiB7B7CD28 cross linked by B7Co-stimulationinduces CTLA-4CTLA-4 binds CD28 with a higher affinity than B7 moleculesThe lack of signal 2 to the T cell shuts down the T cell response.
75The danger hypothesis & co-stimulation Full expression of T cell function and self tolerancedepends upon when and where co-stimulatory molecules are expressed.Cell containing onlyself antigensAPCNo dangerApoptotic cell death.A natural, often usefulcell death.Innocuous challenge to the immune system fails to activate APC and failsto activate the immune systemFuchs & Matzinger 1995
76DANGER The danger hypothesis APC Necrotic cell deathe.g. tissue damage,virus infection etcPathogens recognisedby microbial patternsAPCDANGERAPC that detect ‘danger’ signals express costimulatorymolecules, activate T cells and the immune response
77How the danger hypothesis suggests a review of immunological dogma Antigens induce tolerance or immunity depending upon the ability of the immune system to sense them as ‘dangererous’, and not by sensing whether they are self or ‘non-self’.There is no window for tolerance induction in neonates - if a ‘danger signal’ is received, the neonatal immune system will respondNeonatal T cells are not intrinsically tolerisable but the natural anti-inflammatory nature of the neonatal environment predisposes to toleranceApoptosis, the ‘non-dangerous’ death of self cells may prevent autoimmunity when old or surplus cells are disposed of.Suggests that tolerance is the default pathway of the immune system on encountering antigens.Explains why immunisations require adjuvants to stimulate cues of danger such as cytokines or costimulatory molecule expression.Doesn’t exclude self-nonself discrimination, but the danger hypothesis will be very hard to disprove experimentally.