Why is a mechanism for repertoire selection and self tolerance needed? Generation of the TcR repertoire involves many random mechanisms The specificity of TcR in the immature repertoire is also random & will include cells with receptors that are: T T T T T T T T T T T T T T T T T T T T T T T T T 2. Useless T T T APC 3. Useful Foreign antigen recognition T 1. Harmful Self antigen recognition
Self proteins enter the endogenous and exogenous antigen processing pathways Processing pathways do not distinguish self from non-self Self cellular proteins Self serum & cellular proteins
>90% of eluted peptides are derived from self proteins Yet self antigens do not usually activate T cells Self peptides load onto MHC class I & II molecules Purify stable MHC-peptide complexes Fractionate and microsequence peptides Acid elute peptides
TcRs recognise the non-self peptide antigen and the self MHC molecule MHC molecules RESTRICT T cell activation But how do T cells learn how much self recognition is acceptable? The immune system allows a limited degree of self recognition
T cells are only allowed to develop if their TcR recognise parts of self MHC MHC A haplotype T CELL Explains why T cells of MHC haplotype A do not recognise antigen specific presented by MHC haplotype B MHC B haplotype APC MHC A haplotype APC
Harmful Useless Useful Positively select Negatively select Wholly self-reactive and useless T cells are removed MHC-restricted are retained THYMUS Neglect Y T Y Y T T APC Y T ? Y T Y T Y Y Y Y T T Y Y T T Y Y Y T T T Y Y Y Y T Y T T T Random TcR repertoire ensures diversity Y T Y T Y T
The thymus Lobulated structure with a STROMA of epithelial cells & connective tissue Stroma provides a microenvironment for T cell development & selection Lobules differentiated into an outer CORTEX & inner MEDULLA , both filled with bone-marrow-derived THYMOCYTES Cortex Medulla Cortical epithelial cell Medullary epithelial cell Dendritic cell Thymocyte Macrophage
The thymus is required for T cell maturation Athymic mice ( nude ) and humans (DiGeorge syndrome) are immunodeficient due to a lack of T cells Neonatal thymectomy No mature T cells In adult Thymus intact Mature T cells In adult
Roles of the bone marrow and thymus in T cell maturation Defective lymphocyte production Normal thymus scid/scid Thymus defect Normal bone marrow nu/nu No mature T cells In adults No mature T cells In adults
Bone marrow supplies T cells, and they mature in the thymus Thymus colonised by thymocytes from the thymus defective, i.e. orange, mouse Thymus graft Bone marrow transplant Thymus colonised by thymocytes from thymus defective, i.e. orange, mouse Marrow defect Thymus defect
The thymus matures T cells after birth, but early in life Remove Thymus Mature T & B cells No T cells Mature B cells present T cells not yet left thymus The thymus is needed to generate mature T cells Adult Neonate
The thymus is most active in the foetal and neonatal period The thymus is needed for NEONATAL TOLERANCE T cells vs. OVA Adult Neonate No T cells vs.OVA OVA KLH T cells vs. KLH T cells vs. KLH
T cells mature in the thymus but most die there. 98% of cells die in the thymus without inducing any inflammation or any change in the size of the thymus. Thymic macrophages phagocytose apoptotic thymocytes. Constant 1-2 x 10 8 cells Mouse thymus 5 x 10 7 per day 2 x 10 6 per day
T cell development is marked by cell surface molecule changes As 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 maturation 98% CD3/TcR- CD4-, 8- Double negative CD3+ TcR -chain + pre-TcR + (pT CD4+, 8+ Large double positive CD3+ TcR + CD4+ CD8+ Small double positive TcR+ CD3+ CD4-, 8- CD3+ TcR + CD4+ Single positive CD3+ TcR + CD8+ Single positive
Different developmental stages of thymocytes are present in different parts of the thymus Cortex Immature double negative & positive thymocytes Medulla Mature single positive thymocytes DP CD3+ pT : DN CD3+ pT : CD25-, CD44- DP CD3+ TcR + DN CD25+ CD44+ DN CD25- CD44+ DN CD25+, CD44low SP CD3+ TcR + CD8+ CD3+ TcR + CD4+ SP
TcR rearrangement C region spliced to VDJ fusion and -chain protein produced in cytoplasm No TcR at cell surface DN CD25+, CD44low V D J C Germline configuration V D J C D-J fusion DN CD25+ CD44+ DN CD25- CD44+ V D J C V-DJ fusion
Similarities in the development of T and B cells: A B cell reminder Surrogate light chain is transiently expressed when V H D H J H C H is productively rearranged
Triggers entry into cell cycle
Expands pre-B cells with in frame VDJ joins
2. Suppresses further H chain rearrangement
1. Cell proliferates rapidly to yield daughter cells with the same chain Expands only cells with in-frame TcR chains 2. Successful rearrangement shuts off rearrangement on 2nd chromosome Ensures only one specificity of TcR expressed per cell Similarities in the development of T and B cells: Pre T cell receptor DN CD3+ very low pT : CD25- CD44- TcR -chain preTcR -chain TcR -chain preTcR -chain CD8 CD4 DP CD3+ low pT : CD25- CD44- CD4+ CD8+
TcR rearrangement When proliferation stops, the chain starts to rearrange Germline TcR J C V V-J rearranged TcR 1° transcript Spliced TcR mRNA CD3+ TcR + DP T cells can now recognise antigens and interact with MHC class I & II through CD4 & CD8 Selection can now begin DP CD3+ low pT : CD25- CD44- CD4+ CD8+
How does the thymus choose which of the cells entering the thymus are useful, harmful and useless Mouse thymus 5 x 10 7 per day 2 x 10 6 per day
Retention of thymocytes expressing TcR that are RESTRICTED in their recognition of antigen by self MHC i.e. selection of the USEFUL Removal of thymocytes expressing TcR that either recognise self antigens presented by self MHC or that have no affinity for self MHC i.e. selection of the HARMFUL and the USELESS Sorting the useful from the harmful and the useless Positive selection Negative selection
MHC restriction Antigen can be seen by the TcR only in the context of an MHC molecule TcR will not bind to an MHC molecule unless there is an antigen in the groove In the presence of antigen, the TcR must have some affinity for the MHC molecule
Thymus defect Marrow defect Experimental evidence for MHC restriction as a marker of positive selection CHIMERA Orange strain cells in a blue strain mouse Which MHC haplotype will restrict the T cells, Orange or blue ? Bone marrow transplant Transplant reconstitutes marrow defective mouse
Studies in bone marrow chimeras show that MHC restriction is learnt in the thymus T cell response of recipient T cells to antigen The MHC haplotype of the environment in which T cells mature determines their MHC restriction element Irradated bone marrow recipients MHC A MHC (AxB)F1 Bone marrow donor MHC (AxB)F1 MHC haplotype of APC A B A B A B MHC B
Explanation of bone marrow chimera experiment: Mice of a particular MHC haplotype only make T cells restricted by that haplotype MHC (AxB)F1 Able to make T cells restricted by MHC A or B MHC A Able to make T cells restricted by MHC A MHC B Able to make T cells restricted by MHC B Bone marrow must contain potential to make T cells restricted by A and B MHC molecules
Explanation of bone marrow chimera experiment: Irradiation prevents the bone marrow from generating lymphocytes These mice are severely immunodeficient and can only be reconstituted by a bone marrow transplant Irradiation destroys the immune system but has no effect on the epithelial or dendritic cells of the thymus MHC A MHC B MHC A MHC B Normal mice MHC A MHC B Mice now have an intact, functional thymic stroma but have no thymocytes, T cells or bone marrow
Bone marrow contains the potential to make T cells restricted by A and B MHC molecules Explanation of bone marrow chimera experiment: Reconstitution of irradiated mice with (AxB)F1 bone marrow MHC (AxB)F1 MHC A MHC (AxB)F1 MHC B Irradiated bone marrow recipients Transplant bone marrow to reconstitute immune system of immunodeficient mice
Mouse with an MHC A thymus, but A x B bone marrow Mouse with an MHC B thymus, but A x B bone marrow Explanation of bone marrow chimera experiment: MHC restriction is learnt in the thymus by positive selection Mature T cells restricted only by MHC A Mature T cells restricted only by MHC B A x B T cell precursors MHC A Thymus A x B T cell precursors MHC B Thymus
MHC haplotype of antigen presenting cells Explanation of bone marrow chimera experiment: Peripheral T cells are restricted by the MHC type of the thymus that they mature in MHC (AxB)F1 Bone marrow donor T cell response of recipient T cells to antigen A B A B MHC A MHC B Bone marrow recipients
T cells are ‘educated’ in the thymus to recognise antigens only in the context of self MHC The MHC haplotype of the environment in which T cells mature determines their MHC restriction element Bone marrow chimeras show that MHC restriction is learnt in the thymus Summary MHC restriction is learnt in the thymus by positive selection
Removal of thymocytes expressing TcR that either recognise self antigens presented by self MHC or that have no affinity for self MHC i.e. selection of the HARMFUL and the USELESS Negative Selection Superantigens can be used to probe the mechanisms of negative selection
Nominal antigens & superantigens Nominal antigens Require processing to peptides TcR and chains are involved in recognition >1 in 10 5 T cells recognise each peptide Recognition restricted by an MHC class I or II molecule Almost all proteins can be nominal antigens Superantigens Not processed Only TcR chain involved in recognition 2-20% of T cells recognise each superantigen Presented by almost any MHC class II molecule Very few antigens are superantigens Suggests a strikingly different mechanism of antigen presentation & recognition.
Superantigens e.g. Staphylococcal enterotoxins Toxic shock syndrome toxin I (TSST-1) Staphylococcal enterotoxins SEA, SEB, SEC, SED & SEE Do not induce adaptive responses, but trigger a massive burst of cytokines that may cause fever, systemic toxicity & immune suppression Severe food poisoning Toxic shock syndrome Class II from MHC A to Z haplotypes TcR from MHC A haplotype T cell APC V V
Interaction of SEB with MHC Class II molecules and the TcR MHC class II TcR beta chain MHC class II SEB TcR beta chain SEB
Exogenous superantigen-V relationship Superantigen Human V region SEA 1.1, 5.3, 6.3, 6.4 6.9, 7.3, 7.4, 9.1 SEB 3, 12, 14, 15, 17, 20 SEC 1 12 SEC 2 12, 13.1, 13.2 SED 5, 12 SEE 5.1, 6.3, 6.4, 6.9, 8.1 TSST-1 2 Explains why superantigens stimulate so many T cells
Effect of TSST-1 on T cells expressing V 2 Fresh PBMC stained with anti-V 2 PBMC cultured with TSST-1 Stained with anti-V 3 Fresh PBMC unstained Fluorescence intensity (i.e. amount of staining with anti-V antibody) Cell number PBMC cultured with TSST-1 Stained with anti-V 2 Cell number
Other exogenous superantigens Bacterial exoproteins Staphylococcal exfoliative toxins Streptococcus pyogenes erythrogenic toxins A & C (?Streptococcal M protein?) Yersinia enterocolitica superantigen Clostridium perfingens superantigen Mycoplasma arthritidis mitogen
Superantigens Mouse mammary tumour viruses (Mtv) Cell-tethered superantigen encoded by the viral genome TcR from MHC A haplotype T cell APC Class II from MHC A to Z haplotypes Vb V V
Endogenous superantigens Mouse mammary tumour viruses (MMTV) Retroviruses that contain an open reading frame in a 3’ long terminal repeat that encodes a superantigen associated with the cell surface of APC Most mice carry 2-8 integrated MMTV proviruses in their genome Integrated MMTV Mtv-1, 2, 3, 6, 7 (Mls-1 a ), 8, 9, 11, 13 & 43 Infectious and transmitted by milk MMTV (C3H) MMTV (SW) MMTV (GR)
Mtv Murine V region Mtv 8 11 Mtv 11 11 Mtv 9 5.1, 5.2, 11 Mtv 6 3, 5.1, 5.2 Mtv 1 3 Mtv 3 3 Mtv 13 3 Mtv 7 6, 7, 8.1, 9 MMTV SW 6, 7, 8.1, 9 MMTV C3H 14 MMTV GR 14 Endogenous superantigen V -relationship Stimulate T cells in a similar manner to exogenous supernatigens Valuable tools in analysis of self tolerance
Irradiated Mtv-7 superantigen APC T Only T cells with TcR containing V 6, V 8.1 and V 9 proliferate Mtv-7 interacts with V 6, V 8.1 and V 9 and activates only cells bearing those TcR Selective expansion of cells bearing certain V chains Mtv act in a similar manner to exogenous superantigens in vitro T T T T T T T STIMULATOR CELLS Mtv-7 +ve RESPONDING T CELLS Mtv-7 -ve
How do pathogens use superantigens?
Reduces the possibility that effective T cell clonal selection can eliminate the pathogen
Upon resolution, cells activated by the superantigen die, leaving the host immunosuppressed
Unfocussed adaptive immune response activates cells of all specificities as well as those specific for the superantigens Transmission of infection
1. MMTV infected, MHC class II positive B cells Transmission of infection B T 2. Massive T cell response to MMTV superantigen 3. Vigorous T cell help leads to B cell proliferation and differentiation to long-lived B cells 4. Infected cells traffic to mammary gland and infect young via milk
Analysis of negative selection in vivo. Mtv Mtv-7 superantigen binds to V 6, V 8.1 and V 9+ve thymocytes Mtv-7 superantigen positive Negative selection Immature CD4+8+ thymocytes expressing V V 8.1 and V 9 in the thymus No mature CD4+ or CD8+ V V 8.1 and V 9 T cells in periphery Mtv-7 superantigen negative Immature CD4+8+ thymocytes expressing V V 8.1 and V 9 in the thymus Negative selection Mature CD4+ or CD8+ V V 8.1 and V 9 T cells in periphery THYMUS PERIPHERY
Analysis of negative selection in vivo. Milk transmissible superantigens - MMTV (C3H) V 14 present? Yes No Male or female B10.BR Male or female C3H
No X Male C3H Female B10.BR V 14 present? F1 offspring Male B10.BR Female C3H X V 14 present? F1 offspring Yes
No Deletion of V 14 T cells in mice infected with MMTV by milk MMTV transmitted to fostered pups by infected B cells found in milk + Foster female B10.BR Young male or female C3H Yes V 14 present in fostered pups? + Foster female C3H Young male or female C3H Or B10.BR
Are the signals that induce positive & negative selection the same, or different? Negative selection Peripheral T cells SAME specificity DIFFERENT specificity Positive selection Immature thymocytes X T H Y M U S
Hypotheses of self-tolerance Avidity hypothesis Affinity of the interaction between TcR & MHC Density of the MHC:peptide complex on the cell surface Quantitative difference in signal to thymocyte. Differential signalling hypothesis Type of signal that the TcR delivers to the cell Qualitative difference in signal to thymocyte.
Removal of useless cells Peptide is not recognised or irrelevant Thymocyte receives no signal, fails to be positively selected and dies by apoptosis. WEAK OR NO SIGNAL CD8 TcR T cell Thymic epithelial cell MHC Class I
Positive selection Peptide is a partial agonist Thymocyte receives a partial signal and is rescued from apoptosis i.e. the cell is positively selected to survive and mature. PARTIAL SIGNAL Thymic epithelial cell MHC Class I CD8 TcR T cell
Negative selection Peptide is an agonist Thymocyte receives a powerful signal and undergoes apoptosis i.e. the cell is negatively selected and dies. FULL SIGNAL Thymic epithelial cell MHC Class I CD8 CD8 TcR T cell
The thymus accepts T cells that fall into a narrow window of affinity for MHC molecules Number of cells Affinity of TcR/MHC interaction Low High Useless Neglect Useful Positively select Harmful Negatively select
How accurate are these models of positive and negative selection? Positive selection: Relied on very complex chimera experiments Relied on proof of MHC restriction as an outcome which is tested in an ‘unnatural’ response using MHC mismatched presenting cells Negative selection: Relied on exceptionally powerful superantigens operating outside the normal mechanisms of antigen recognition
Illustration of selection using TcR transgenic mice Generation of transgenic mice In TcR transgene-expressing mice almost all thymocytes express the transgenic TcR due to ALLELIC EXCLUSION . T T cell clone with known TcR specificity and MHC restriction Rearranged chain cDNA construct Rearranged chain cDNA construct } Inject into fertilised mouse ovum Re-implant Analyse offspring for transgene expression.
Cells that fail positive selection die in the thymus (neglect) Thymocytes die at the double positive stage after failing +ve selection due to a lack of MHC A In TcR transgenic mice expressing an MHC A restricted TcR, all thymocytes express the MHC A restricted TcR Transgenically express MHC A restricted TcR in an MHC B mouse DP CD3+ TcR + No single +ve cells are present in the periphery SP CD3+ TcR + CD8+ or CD4+ DN CD3- MHC B
Positive selection determines the restriction element of the TcR AND the expression of CD4 or CD8 Restriction element and co-receptor expression are co-ordinated Instructive 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? TcR transgenic mouse TcR from MHC class I- restricted T cell TcR transgenic mouse TcR from MHC class II- restricted T cell Only CD8 cells mature Only CD4 cells mature
Instructive model: Signal from CD4 silences the CD8 expression & vice versa Double positive thymocyte Thymic epithelial cell Double positive to single positive transition Single CD4+ thymocyte X √ CD8 MHC Class I MHC Class II 3 2 TcR TcR CD4 MHC Class II 2 TcR CD4 MHC Class I 3 TcR -ve CD8
Stochastic/selection model: Cells randomly inactivate CD4 or CD8 gene, whilst testing a match of TcR restriction Double positive thymocyte Thymic epithelial cell Single CD4+ thymocyte Double positive to single positive transition X √ MHC Class II 2 TcR MHC Class I 3 TcR CD8 MHC Class I MHC Class II 3 2 TcR TcR CD4 CD4 CD8 CD4 CD8 CD4
Deletion of cells in the thymus: differential effect on the mature and immature repertoire TcR from T cell specific for hen egg lysosyme (HEL) ~100% of T cells/thymocytes express anti-HEL TcR Immunise with HEL Thymocytes activated by antigen in the thymic environment die T cells activated by antigen in the periphery proliferate TcR transgenic mouse Analyse peripheral T cells: All transgenic T cells proliferate Analyse thymus: All transgenic T cells die by apoptosis
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?
Nature Immunology November 2001
Promiscuous expression of tissue-specific genes by medullary thymic epithelial cells
How 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 puberty
Self tolerance can be induced outside the thymus
PERIPHERAL TOLERANCE or ANERGY
A state of immunological inactivity caused by a failure to deliver appropriate signals to T or B cells when stimulated with antigen
i.e. a failure of antigen presenting cells to deliver COSTIMULATION
T helper cells costimulate B cells Two - signal models of activation Y Y Y B T cell antigen receptor Co-receptor (CD4) CD40 Ligand (CD154) Th Signal 2 - T cell help CD40 MHC class II and peptide Signal 1 antigen & antigen receptor ACTIVATION
Antigen presentation - T cells are co-stimulated Costimulatory 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. APC Th Signal 1 antigen & antigen receptor Signal 2 B7 family members (CD80 & CD86) CD28 ACTIVATION
Express IL-2 receptor- and chains but no chain or IL-2 Mechanism of co-stimulation in T cells Signal 1 NFAT binds to the promoter of of the chain gene of the IL-2 receptor. The chain converts the IL-2R to a high affinity form Resting T cells Low affinity IL-2 receptor IL-2 IL-2R IL-2 IL-2R 1 Antigen
Signal 2 Activates AP-1 and NF -B to increase IL-2 gene transcription by 3 fold Stabilises and increases the half-life of IL-2 mRNA by 20-30 fold IL-2 production increased by 100 fold overall Mechanism of co-stimulation in T cells Immunosuppressive drugs illustrate the importance of IL-2 in immune responses Cyclosporin & FK506 inhibit IL-2 by disrupting TcR signalling Rapamycin inhibits IL-2R signalling IL-2 IL-2R 1 Antigen 2 Costimulation
Signal 1 only Anergy The 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 presented by a non-classical APC e.g. an epithelial cell IL-2 IL-2R 1 Antigen Epithelial cell Naïve T cell
Arming of effector T cells 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 APC T IL-2 Effector T cell Clonal selection and differentiation How can this cell give help to, or kill cells, that express low levels of B7 family costimulators?
Clonally selected, proliferating and differentiated T cell i.e. ARMED sees antigen on a B7 -ve epithelial cell The effector programme of the T cell is activated without costimulation This contrasts the situation with naïve T cells, which are anergised without costimulation Effector function or Anergy? Armed Effector T cell CD28 Co-receptor TcR IL-2 Epithelial cell Naïve T cell Epithelial cell Epithelial cell Armed Effector T cell Kill
CD28 cross linked by B7 Costimulatory molecules also associate with inhibitory receptors CTLA-4 binds CD28 with a higher affinity than B7 molecules Co-stimulation induces CTLA-4 The lack of signal 2 to the T cell shuts down the T cell response. CD28lo Activated T cell CTLA-4hi B7 CD28 T cell B7 2 2 Signal 1 + Cross-linking of CTLA-4 by B7 inhibits co-stimulation and inhibits T cell activation - - - - -
The danger hypothesis & co-stimulation Fuchs & Matzinger 1995 Full expression of T cell function and self tolerance depends upon when and where co-stimulatory molecules are expressed. Innocuous challenge to the immune system fails to activate APC and fails to activate the immune system Apoptotic cell death. A natural, often useful cell death. APC APC No danger No danger Cell containing only self antigens
The danger hypothesis APC that detect ‘danger’ signals express costimulatory molecules, activate T cells and the immune response APC APC Necrotic cell death e.g. tissue damage, virus infection etc Pathogens recognised by microbial patterns DANGER
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 respond
Neonatal T cells are not intrinsically tolerisable but the natural anti-inflammatory nature of the neonatal environment predisposes to tolerance
Apoptosis, 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.
How the danger hypothesis suggests a review of immunological dogma Doesn’t exclude self-nonself discrimination, but the danger hypothesis will be very hard to disprove experimentally.