Tolleranza delle cellule T nei tessuti linfoidi periferici

1. Tolleranza delle cellule T nei tessuti linfoidi periferici

2. Vigorous reactivity against pathogens A lack of overt reactivity to self Both are best achieved when lymphocytes express diverse, clonally distributedantigen specific receptors (clonal selection theory) As the organism cannot predict the precise pathogen-derived antigens that will be encountered, the immune system relies on the generation and maintenance of a diverse T-cell receptor (TCR) repertoire GOD, generation of immune-receptor diversity

3. Concept of Immune Regulation • Immune responses are tightly regulated by complex interactions of cells & mediators, and by mechanisms to prevent anti-self reactivity • Failure of regulatory control can occur… • Enhancement of immune responses or infection can generate autoimmune reactions (loss of self–tolerance) • Decrease of immune responses may lead to an immunodeficiency state • Shift in immune responses can lead to allergy

5. Immunological Tolerance • History • Ehrlich, Owen, Burnet • Billingham, Brent and Medawar

6. Owen’s observation 1945

7. DEVELOPMENT MATURITY ClonalDeletion Anti-self Lymphocyte Self Ag Activation Differentiation Anti-non-self Lymphocyte Foreign Ag Burnet’s Clonal Selection Model:Central Tolerance Burnet postulated that there was a temporal window of tolerance such that antigens encountered while the immune system was immature tolerized the relevant lymphocytes.

8. Medawar’s experiment demonstrating neonatal tolerance induction (Nobel Prize) Medewar subsequently investigated the effects of transferring hemopoietic 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.

9. Immunological Tolerance • Definition and Properties • Specific unresponsive state induced by exposure to antigenic epitopes • Tolerance to self is initially induced during embryonic life, and is maintained by antigen • Tolerance occurs in both T and B cells • Multiple mechanisms of tolerance exist

10. Tolerance • Central Tolerance - this occurs during lymphocyte development. • Peripheral Tolerance- occurs after lymphocytes leave the primary lymphoid organs.

11. Central Tolerance

12. The thymus provides a fundamental initial step for the elimination of potentially dangerous self-specific T cells

13. Peripheral tolerance

14. Acquisition and maintenance of peripheral tolerance • Different hypotheses: • Self-nonself discrimination (Bretscher, Cohn) • INS model (Janeway) • Danger model (Matzinger) • Localization dose and time model (Zinkernagel)

15. Self nonself Cohn: associative recognition of antigen Bretscher: two step, two-signal model Signal two is given by a T cell

16. Infectious nonself and non-infectious self model No peripheral tolerance only central tolerance Recognition of a pathogen by PRRs induces a set of endogenous signals, including costimulatory molecules (B7), inflammatory and effector cytokines, and CD1d molecules. Additionally, some cells can phagocytose the pathogen (not shown), process its protein constituents, and present its peptides to T cells. Recognition of peptides derived from a pathogen, along with costimulatory molecules induced by the pathogen, results in T cell activation. Effector cytokines induced by the pathogen instruct the activated T cells to differentiate into a particular effector cell type (T1 or T2). Activated T cells then deliver an inducible signal (usually a member of the TNF family, such as CD40L or FasL) to the target cell in an antigen-specific manner: the target cells of T1 and T2 being macrophages and B cells, respectively. L, ligand.

17. Danger model

18. Danger model Absence of signal two induces tolerance

19. Danger Model Anti-DEC205 Steady state DC

20. Come viene indotta la toleranza allo steady state? Topi transgenici esprimenti OVA sotto il cotrollo di RIP

21. Localization dose and time model

22. Localization dose and time model TCR tg anti-gp33 x RIP-gp33 ignoranza

23. c proliferation CD44 50 100 40 80 %Vb14+ on CD4+ 30 60 % of CD44high 20 40 20 10 50 0 0 40 0 10 20 30 40 0 10 20 30 40 e 30 % of CD69+ CD25 20 100 10 0 60 0 10 20 30 40 % of CD25+ 20 0 40 0 10 20 30 Days 80 high frequency BALB/c low frequency 70 60 50 8 7 40 6 30 5 20 4 10 3 0 0,0 0,01 0,1 1,0 2 1 0 0 10-5 10-4 10-3 10-2 10-1 1 CD69 TCR downregulation IFNg production CD69 upregulation naive 120 High frequency 100 80 IFNg ng/ml % TCR high % CD69 high 60 Low frequency 40 20 0 0,0 0,01 0,1 1,0 Bpep mg/ml Bpep mg/ml Bpep mg/ml Localization dose and time model Anti-IgG2ab TCR transgenic mice Transient peptide presentation by B cells Chronic peptide presentation by B cells

24. Organi immunoprivilegiati

25. Nel 1989 sono stati isolati anticorpi monoclonali citolitici per diverse linee cellulari La proteina riconosciuta da questi anticorpi è stata denominata FAS L’espressione costitutiva di FAS in trasfettanti porta alla morte cellulare in seguito ad incubazione con anticorpi anti-FAS FAS trasduce un segnale apoptotico

26. FAS espresso in : TIMO, FEGATO, CUORE, POLMONI, RENI, OVAIO Timo: FAS espresso in tutte le popolazioni ma non nei doppi negativi Periferia: FAS espresso nei T maturi attivati

27. FASL: 40000 D, induce morte cellulare se interagisce con linee cellulari esprimenti FAS FASL: espresso dalle cellule T attivate, espresso nel TESTICOLO, CORNEA, TIMO (organi immunoprivilegiati) FASL esiste anche solubile in forma trimerica Per indurre morte cellulare è necessario il cross-linking di FAS

29. Gli organi immunoprivilegiati esprimono il ligando di FAS Se cellule T attivate arrivano agli organi immunoprivilegiati sono uccise mediante interazione FAS-FASL Dimostrato direttamente per la cornea

30. Walker and Abbas 2002, 2

31. Walker and Abbas 2002, 2

32. Tolerance and Regulatory T cellsPatrolling and Keeping the periphery in check. • Regulatory T cells patrol periphery and maintain tolerance : • Self-reactive T cells are under the constant control of regulatory T cells. • Variety of regulatory or “suppressor” T cells: • Late 70’s - Early 80’s  Present

33. Regulatory T Cells • CD25+CD4+ thymic origin • Th3 - Induced by oral feeding of antigen • TGF-1 producing (IL-10). • TGF-1 dependent • Tr1 - Induced • Antigen administration in the presence of IL-10 • IL-10 dependent • All induced by Ag X, thus known specificity.

34. Tregs

35. The reincarnation of the suppressor T cell More questions than answers. Questions ? n Number of publications * 1 1995 - Present Answers

36. CD4+CD25+ immunoregulatory T cellsThe rebirth and revival…mid 1990’s. • Naturally-occurring or “professional” regulatory T cells. • Immunosuppressive in vivo : • Day 3 thymectomy • Depletion of CD4+CD25+ T cells • Irradiation • Gastritis, oophritis, orchitis, • thyroiditis, pancreatitis, colitis • Co-transfer with CD4+CD25- T cells suppresses autoimmune disease. • Absence of CD4+CD25+ T cells  Increased immunoreactivity to antigens.

37. CD4+CD25+ regulatory T cellsGeneral characteristics • Unique lineage of CD25+ (IL-2R) T cells • Originate from thymus. Role of periphery ? • “Partially activated” phenotype : • CD62Lhigh, CD69low, CTLA-4, GITR and CD45Rblow • Differ from conventional, activated CD25+ T cell. • No other marker for regulatory T cells. • Thymic developmental pathway is unclear: • Altered negative selection? • TCR specificity is unknown : • Diverse T cell repertoire. • Self-specific • Cross-reactive to foreign? • Hyporesponsive (anergic) : • Unresponsive to TCR stimulation : restored by IL-2. • Do not produce IL-2. Secondary lymphoid tissues 5-10% CD4 CD25

38. T cell activation induces expression of functional T cell surface molecules Activated CD4+ T cell MHC/peptide CD40L TCR TCR CD25 Resting CD4+ T cell APC CD69 CD44 CD25 is not a marker of Treg after T cell activation. Only in the naïve T cell repertoire.

39. CD4+CD25+ regulatory T cells suppress T cell proliferation and cytokine production Proliferation Cytokine Production IL-2 77.6 CD4+ and CD8+ T cells CPM IL-2 CD8+ / CD4+CD25+ CD8+ 69 CD25 Suppress the induction of IL-2 mRNA # CD4+CD25- (  ) or CD4+CD25+ ( ) T cells IFN- (ng/ml) g IFN- CD8+/- CD8+/+ CD8+

40. CD4+CD25+ CELLS REQUIRE ACTIVATION TCR ENGAGEMENT FOR Treg ACIVITY DO11.10 CD4+ Tg WT CD4+CD25+

41. CD4+CD25+ T cell mediated suppressionUnknownmechanism. • CD4+CD25+ T cells inhibit the IL-2/IL-2R system in T cells • Do not suppress by consuming IL-2. • Suppressor molecules are not defined. • Not directly cytolytic. • Cell-contact dependent and cytokine independent.

42. Treg effector functions

43. CD4+CD25+ mediated suppression Requirement for a T:T cell interaction APC CD4+ CD25+ CD8+ Activation CD4+ CD25+ CD8+ Direct evidence for T:T conjugates ? Suppression

44. Role for cytokines? CD4+ CD25+ T cell Responder X IL-10 & TGF-1 Immunosuppressive effects on APC and T cells But the role of differentiated Tregs is not abrogated by inhibiting IL-10 and TGF-b1

45. Tissue-specific CD4+CD25+ mediated disease protection in the absence of IL-10. CD4+CD25- CD4+CD25+ CD4+CD25- IL-10-/- CD4+CD25+ CD4+CD25- Nude Gastritis IBD No Gastritis IBD develops ! No Gastritis No IBD

46. Altered peptide ligands o peptidi antagonisti