Selhání imunitní tolerance: alergie a autoimunita Jan Živný Ústav patologické fyziologie Universita Karlova, 1. lékařská fakulta Seminar 4 hodiny medici

Role imunitního systému Obrana organismu proti „nebezpečným“ antigenům Imunitní dohled Obranné imunitní reakce Tolerance „vlastních“ a „bezpečných“ antigenů Imunitní tolerance

Obranná imunitní reakce Rozpoznání a reakce na „nebezpečí“ imunitním systémem „Nebezpečí“ Antigeny spojené s infekcí Cizí MHC molekuly Poškozené vlastní buňky Je výsledkem interakce a rozpoznání antigenu imunitním systémem

Obranná imunitní odpověď Stimulace přirozené imunity Stimulace adaptivní (specifické) imunity Eliminace „nebezpečí“ (např. infekce) Vytvoření protektivní immunity SELF-NonSELF vs. Nebezpečné

Molekuly zajišťující vztah přirozené a získané imunity The Receptors Involved in the Interplay of the Innate and Adaptive Immune Systems. Recognition of the pathogen-associated molecular pattern (PAMP) by pattern-recognition receptors, such as the toll-like receptors, generates signals that activate the adaptive immune system. Endocytic pattern-recognition receptors, such as the macrophage mannose receptor, bind to components of microbial cell walls and mediate the uptake and phagocytosis of pathogens by antigen-presenting cells (macrophages and dendritic cells). Proteins derived from the microorganisms are processed in the lysosomes to generate antigenic peptides, which form a complex with major-histocompatibility-complex (MHC) class II molecules on the surface of the macrophage. These peptides are recognized by T-cell receptors. In the case of the signaling class of pattern-recognition receptors, the recognition of pathogen-associated molecular patterns by toll-like receptors leads to the activation of signaling pathways that induce the expression of cytokines, chemokines, and costimulatory molecules. Therefore, pattern-recognition receptors have a role in the generation of both the peptide–MHC-molecule complex and the costimulation required for the activation of T cells. K imunitní odpovědi je potřeba aktivace dvou signálních drah Medzhitov, R. et. al. N Engl J Med 2000;343:338-344

Imunitní tolerance Snášenlivost „bezpečných“ antigenů imunitním systémem = nevzniká patologická imunitní reakce (autoimunita nebo alergie) Vlastní antigeny a běžné antigeny okolního prostředí (např. antigeny potravy) Antigen specifická a je výsledkem interakce a rozpoznání antigenu imunitním systémem

Centralní tolerance: Thymus / Kostní dřeň Negativní selekce: eliminace „autoreaktivních“ lymfocytů nebezpečných pro organismus Cortex thymu Pozitivní selekce Medula thymu Negativní selekce Central Mechanisms of the Induction of Tolerance. Immature T cells migrate to the thymus, where they encounter antigen presented by thymic epithelial cells. Cells whose T-cell receptors have a low affinity for the complex of self peptide and a self major-histocompatibility-complex (MHC) molecule do not receive a signal to switch off the process of spontaneous apoptosis and therefore die in the thymus. Cells whose T-cell receptors have a high affinity for such complexes are also eliminated by means of apoptosis. The remaining T cells have an intermediate affinity for these complexes, and these mature in the thymus and migrate to the periphery, where they can become activated. selection of T-cells that are functional (positive selection), and elimination of T-cells that are autoreactive (negative selection). Central tolerance - negative selection (thymus, BM) Susceptible for central immune tolerance induction are immature and maturating lymphocytes CD4+/CD8+/TCRLo thymocytes IgM+ immature B-lymphocytes Přiměřená „autoreaktivita“ Kamradt, T. et. al. N Engl J Med 2001;344:655-664

Negativní selekce Apoptoza (smrt) příliš reaktivních lymfocytů (Clonal deletion) Klonální přesměrování (Clonal diversion) změna funkce autoreaktivních buněk (T reg) Vytvoření anergie Editace receptoru (TCR / BCR) In 1957, Sir Macfarlane Burnet first proposed the concept of “repertoire purging” as a mechanism of lymphocyte tolerance [10]. This process was first described experimentally for thymocytes by studying clonal deletion in response to superantigens Conclusions Recent studies facilitated the dissection of the requirements for T-reg cell differentiation. In the thymus, T-reg cells seem to develop from thymocytes displaying TCR with a higher affinity for self-antigen in the context of MHC class II. However, TCR signal of a particular strength and duration alone is probably not sufficient for the induction of Foxp3 expression. The cofactors such as CD28 co-stimulation, IL-2 and other cytokines are also necessary for commitment to T-reg cell lineage to occur. The mechanisms by which signals mediated by TCR and cofactors involved in Foxp3 induction interact, and whether they coincide or can be can be temporally segregated, is unknown to date. Once thymocytes acquire Foxp3 expression, the regulatory cell fate is reinforced through a positive feedback loop, increasing Foxp3 expression and cell survival including a partial protection from negative selection. Requirements for peripheral T-reg cell maintenance and for their differentiation from non-T-reg cells in the periphery seem to differ from those in the thymus. Involvement of T-reg cells in control of immunoreactivity to self- and tumor antigens, pathogens and commensal flora warrant further research of molecular and cellular mechanisms of T-reg cell differentiation and homeostasis.

Eliminace zralých lymfocytů nebezpečných pro Periferní tolerance Eliminace zralých lymfocytů nebezpečných pro organismus Mechanismus: Rozpoznání antigenu bez patřičné ko-stimulace (2. signál) (B7/CD28; CD40/CD40L; ICOS) Extra-thymic ‘conversion’ of naı¨ve T cells to T-reg cells The peripheral pool of T-reg cells not only includes those differentiated in the thymus but also might include Foxp3+ T-reg cells generated extra-thymically through the ‘conversion’ of naı¨ve T cells on chronic encounter with antigen present in suboptimal dose or in non-immunogenic form [56,57]. In vitro experiments have demonstrated that peripheral T cells retain the ability to turn on Foxp3 expression and suppressive function on TCR cross-linking in the presence of high concentrations of TGFb or in the presence of suboptimal TCR stimulation [56,58–61]. However, the overall numerical contribution of peripheral ‘conversion’ to the peripheral T-reg pool and its functional significance are not clear. The high level of similarity in the TCR repertoire between thymic and peripheral T-reg cells indicates that thymic T-reg cell differentiation accounts for most T-reg cells present in secondary lymphoid organs [17]. In agreement with these results, Belkaid and co-workers [62] have reported that Leishmania-specific T-reg cells present in the skin of infected animals are derived from CD25+ T-reg cells present before infection. Nevertheless, further detailed examination of peripheral T-reg differentiation at different anatomical sites under various conditions is warranted.

Molekuly zajišťující vztah přirozené a získané imunity Tolerance The Receptors Involved in the Interplay of the Innate and Adaptive Immune Systems. Recognition of the pathogen-associated molecular pattern (PAMP) by pattern-recognition receptors, such as the toll-like receptors, generates signals that activate the adaptive immune system. Endocytic pattern-recognition receptors, such as the macrophage mannose receptor, bind to components of microbial cell walls and mediate the uptake and phagocytosis of pathogens by antigen-presenting cells (macrophages and dendritic cells). Proteins derived from the microorganisms are processed in the lysosomes to generate antigenic peptides, which form a complex with major-histocompatibility-complex (MHC) class II molecules on the surface of the macrophage. These peptides are recognized by T-cell receptors. In the case of the signaling class of pattern-recognition receptors, the recognition of pathogen-associated molecular patterns by toll-like receptors leads to the activation of signaling pathways that induce the expression of cytokines, chemokines, and costimulatory molecules. Therefore, pattern-recognition receptors have a role in the generation of both the peptide–MHC-molecule complex and the costimulation required for the activation of T cells. K imunitní odpovědi je potřeba aktivace dvou signálních drah Medzhitov, R. et. al. N Engl J Med 2000;343:338-344

Periferní tolerance - mechanismus Apoptoza reaktivních lymfocytů Anergie = funkční inaktivace Indukce antigen specifických regulačních T lymfocytů (Treg) produkce imuno-inhibičních cytokinů (IL-10, TGF-b, IL-4) kompetice o růstové faktory (IL-2) kompetice o MHC ligandy cytotolytický effect

Selhání imunitní tolerance V organizmu jsou přítomné auto-protilátky i potenciálně auto-reaktivní T lymfocyty Centrální roli hrají Th (CD4+) a Treg (CD4+ CD25+) lymfocyty Více mechanismů selhání i tkáňového poškození Na vzniku se podílí mnohočetné faktory Může být buď systémové nebo orgánově specifické

Poruchy imunity způsobené selháním imunitní tolerance Autoimunitní choroby Alergie Asociované s terapií (GVHD, rejekce štěpu) The first two humans to be discovered with a hereditary deficiency of complement were healthy immunologists.

Teorie rozvoje a progrese autoimunitního onemocnění „Epitope spreading / Antigenic determinant spreading“ postupné rozšiřování spektra patologické imunitní odpovědi na několik antigenních epitopům primárního (auto)antigenu a posléze i ostatních (auto)antigenů společného mikroprostředí, která vede ke klinické manifestaci autoimunitního onemocnění a jeho progresi.

Koncepce rozvoje autoimunitních a alergických onemocnění Centrální tolerance (negativní selekce) Neeliminované auto-reaktivní lymfocyty a lymfocyty reaktivní vůči běžným antigenům okolního prostředí Periferní tolerance okolní prostředí geny How tolerance is established and may fail. Generation of immune repertoires in central lymphoid organs, thymus, and bone marrow is accompanied by deletion of self reactive lymphocytes by apoptosis. The “leakiness” of this process requires back up by peripheral tolerance. Tolerance fails because of the interaction of a wrong environment with the wrong genes, resulting in autoimmune disease. Options for treatment will increasingly include new selective immunotherapies in place of present global immunosuppression Selhání tolerance autoimunita /alergie

Mechanismy autoimunitního poškození Cirkulující protilátky Lýza buněk komplementem (hemolytické anemie) Interakce s buněčným receptorem aktivace cílového receptoru (thyrotoxikoza, např. Gravesova choroba ) zablokování receptoru (Myastenia gravis) Ukládání imunokomplexů (SLE) Na protilátkách závislá buněčná cytotoxická reakce (ADCC), potenciálně možná u orgánově specifických AI chorob Penetrace do živých buněk (?) receptoru pro TSH zablokování a řetězce acerylcholinového receptoru = porucha neuromuskulárního přenosu signálu

Mechanismy autoimunitního poškození T lymfocyty CD4+ buňky zprostředkovaná Th1 imunitní odpověď (RA, MS, DM-I) Aktivované CD8+ T lymfocyty - antigen specifická cytolýza Nespecifické mechanismy Migrace zánětlivých leukocytů do AI leze (synovitis)

Roztroušená skleroza Normální: barvení na myelin RS: barvení na myelin Slide 147. Multiple sclerosis, perivascular cuffing of lymphocytes: When multiple sclerosis is in its active phase and neurologic function is worsening, an inflammatory component such as exhibited in this slide can be seen. The photomicrograph contains a venule with numerous lymphocytes in the Virchow-Robin space. The tissue around the venule is a portion of a plaque. This inflammatory component is one of the major reasons that multiple sclerosis has been considered to be either a disease with a strong immune basis, or of infectious etiology (or both). Slide 149. Myelin stain of normal spinal cord: This is a normal spinal cord at the cervical level stained for myelin. Use this slide for comparison with the next, Slide 150. Slide 150. Multiple sclerosis, myelin stain of spinal cord: There is a large lesion of multiple sclerosis in this section of cervical spinal cord, forming a broad band of demyelination extending from the lateral column on the left through the major portion of both posterior columns into the lateral column on the right. Note three things in particular: 1 ) myelin in the areas affected is virtually totally destroyed; 2) involvement is asymmetrical; and 3) gray matter is involved (myelin in gray matter is destroyed, while neurons remain intact). RS: perivaskulární lymfocytární infiltrace

Perorální podání insulinu: experimentální therapie virovou infekcí indukovaného DM I. typu myší Figure 2. Photomicrographs of histological sections obtained from islets of RIP-NP tg mice receiving oral insulin indicate that such mice have less T cell infiltration and less MHC class I and II expression than similar sections obtained from tg mice not receiving oral insulin. A-D show pancreatic sections from tg mice that received oral insulin and did not develop IDDM 2 mo after infection with 1 × 105 pfu LCMV intraperitoneally. E-H show sections from virus-infected tg mice that developed IDDM and did not receive oral insulin. Sections from tg mice receiving oral insulin and developing IDDM were identical to those shown in E-H and are not displayed. Hematoxylin and eosin (HE) staining and immunohistological staining for MHC class I and II molecules and for insulin were carried out as described (28). A and E compare lymphocyte infiltration into or around islets, B and F relative levels of insulin produced by cells, C and G expression of MHC class II molecules, and D and H expression of MHC class I molecules (see Methods). Generation and characterization of RIP-LCMV tg mice with rapid (8-14 d) or slow-onset (1-6 mo) IDDM after LCMV infection have been described (9). RIP-GP 34-20 (H-2b) tg mice which express the viral GP only in the cells of the islets were used as a model for rapid-onset IDDM. For slow-onset IDDM, RIP-NP 25-3 (H-2d) tg mice were used. RIP-NP 25-3 mice express the viral NP in the pancreas and the thymus but not in any other tissues (9). Abstract Oral administration of self-antigens has been proposed as a therapy to prevent and treat autoimmune diseases. Here we report that oral treatment with insulin prevents virus-induced insulin-dependent diabetes mellitus (IDDM) in a transgenic (tg) mouse model. Such mice express the viral nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) under control of the rat insulin promoter in their pancreatic cells and < 2% spontaneously develop diabetes. However, 2 mo after challenge with LCMV, IDDM occurs in > 95% of tg mice but not in controls. Oral treatment with 1 mg of insulin twice per week for 2 mo starting either 1 wk before or 10 d after initiating LCMV infection prevents IDDM in > 50% of the tg mice (observation time 8 mo). Thus, insulin therapy is effective in preventing progression to overt IDDM in prediabetic tg mice with ongoing islet infiltration. Oral administration of insulin does not affect the generation of LCMV-NP-specific anti-self cytotoxic T lymphocytes nor the infiltration of lymphocytes into the pancreas. However, less cells are destroyed in insulin- treated mice, upregulation of MHC class I and II molecules does not occur, and antiviral (self) cytotoxic T lymphocytes are not found in the islets, events present in tg mice developing IDDM. The majority of lymphocytes in the islets of insulin-treated tg mice without IDDM produces IL-4, IL-10, and TGF-. In contrast, lymphocytes from islets of tg mice developing IDDM mainly make -IFN. (J. Clin. Invest. 1996. 98:1324-1331.) von Herrath MG., et al., J. Clin. Invest. 1996. 98:1324-1331

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