Defense against pathogens, tumour immunology, transplantology

1. Defense against pathogens, tumour immunology, transplantology Martin Liška

2. Extracellular microorganisms • Typically bacteria or parasites • For defense against extracellular microbes and their toxins, specific humoral immune response is important

3. Humoral immune response • Recognition of antigen by specific Ig, bound in cell membrane of naive B lymphocyte • The binding of antigen cross-links Ig receptors of specific B cells and then biochemical signal is delivered to the inside B cell; a breakdown product of the complement protein C3 provides necessary „second signal“ • Clonal expansion of B cell and secretion of low levels of IgM

4. Humoral immune response • Protein antigens activate CD4+ T helper cells after presentation of specific antigen • T helper cells exprime CD40L on their surface and secrete cytokines →proliferation and differentiation of antigen-specific B cells, isotype switching, affinity maturation

5. Phases of humoral immune response

6. Effector functions of antibodies • Neutralization of microbes (incl.viruses) and their toxins • Opsonization of microbes (binding to Fcreceptors on phagocytes; at the same time, stimulation of microbicidal activities of phagocytes) • ADCC (Antibody-dependent cell-mediated cytotoxicity) – IgG opsonized microb is destroyed by NK cells after its binding to IC • Activation of the complement system (classical pathway)

7. Defense against extracellular pathogens (bacteria and unicellular parasites) a/ non-specific (innate) immune system - monocytes/macrophages, neutrophils, complement system, acute phase proteins (e.g.CRP) b/ specific (adaptive) immune system - antibodies (opsonization, neutralization)

8. Defense against multicellular parasites • Production of IgE→ coating and opsonization of parasites • Activation of eosinophils - they recognize Fc regions of the bound IgE, then they are activated and release their granule contents (MBP,ECP,EPO), which kill the parasites • Th2-lymphocytes support this type of immune response

9. Intracellular microorganisms • Initially: non-specific immune response (ingestion by phagocytes) • Some microorganisms are able to survive inside phagocytes (e.g.some bacteria, fungi, unicellular parasites, viruses) – they survive inside phagosomes or enter the cytoplasm and multiply in this compartment • The elimination of these microorganisms is the main function of T cells (specific cell-mediated response)

10. Processing and presentation of antigen • Professional antigen-presenting cells: macrophages, dendritic cells, B lymphocytes (they express constitutionally class II MHC) a/ exogenous antigens – e.g.bacterial, parasitic - hydrolysed in endosomes to linear peptides → presentation on the cell surface together with class IIMHC to CD4+ T lymphocytes

11. Processing and presentation of antigen b/ endogenous antigens – e.g.autoantigens, foreign antigens from i.c.parasites or tumorous antigens • hydrolysed to peptides → associated with class I MHC → presentation on the cell surface to CD8+ T lymphocytes

12. T cell-mediated immune response • Presentation of peptides to naive T lymphocytes in peripheral lymphoid organs → recognition of antigen by naive T lymphocytes • At the same time, T lymphocytes receive additional signals from microbe or from innate immune reactions → production of cytokines → clonal expansion → differentiation → effector & memory cells → effector cells die after elimination of infection

13. T cell-mediated immune response • TCR (T cell receptor) – T cell antigen-specific receptor - TCR recognizes (together with co-receptors - CD4 or CD8) the complex of antigen and MHC • a signal is delivered into the cell through molecules associated with TCR and co-receptors (CD4 or CD8) after antigen recognition

14. T cell-mediated immune response • APC exposed to microbes or to cytokines produced as part of innate immune reactions to microbes express costimulators that are recognized by receptors on T cells and delivered necessary „second signals“ for T cell activation • Activated macrophages kill ingested bacteria by reactive oxygen intermediates, NO and lysosomal enzymes

15. Function of Th1and Th2 lymphocytes

16. Activation of T lymphocytes

17. Mechanisms of resistance of intracellular microbes to cell-mediated immune response • Inhibiting phagolysosome fusion • Escaping from the vesicles of phagocytes • Inhibiting the assembly of class I MHC-peptide complexes • Production of inhibitory cytokines • Production of decoy cytokine receptors

18. Defense against intracellular pathogens (bacteria and unicellular parasites) • Intracellular bacteria (Mycobacteria, Listeria monocytogenes), fungi (Cryptococcus neoformans), parasites (Plasmodium falciparum, Leishmania) • Specific immune response is necessary

19. Anti-viral defense • Viruses may bind to receptors on a wide variety of cells and are able to infect and replicate in the cytoplasm of these cells, which do not possess intrinsic mechanisms for destroying the viruses • Some viruses can integrate viral DNA into host genome and viral proteins are produced in the infected cells (e.g. Retroviruses)

20. Tumor immunology Tumor antigens • Mutant proteins (the products of carcinogen- or radiation-induced animal tumors) • Products of oncogenes or mutated tumor suppressor genes (Bcr/Abl fusion protein) • Overexpressed or aberrantly expressed self protein (AFP in hepatomas) • Oncogenic virus products (HPV products in cervicalCA)

21. Tumor immunology Mechanisms of defense • The principle is formation of cytotoxic T lymphocytes clone (CTL) specific for tumor antigens • The cooperation of naive CD8+ T cells and APC (co-stimulation) and T-helper cells of the same antigenic specifity (cytokines) is required • APC enables formation of antigen-specific CD8+ T cells and CD8+ T cells = cross presentation

22. Tumor immunology • Ig, activated macrophages and NK-cells also participate in anti-tumor-defense • Immunotherapy of tumors – aims to enhance anti-tumor immunity passively (by providing immune effectors) or actively (vaccination with tumor antigens or with tumor cells engineered to express co-stimulators and cytokines)

23. Tumor immunology How tumors evade immune responses: a/ lack of T cell recognition of tumor - generation of antigen-loss variant of tumor cells • mutations in MHC genes or genes needed for antigen-processing b/ inhibition of T cell activation - production of immuno-suppressive proteins

24. Transplantation immunity – alloimmune reaction • Recipient’s T cells recognize donor’s allogeneic HLA molecules that resemble foreign peptide-loaded self HLA molecules • Graft antigens are recognized: a/ Directly – donor’s HLA molecules on graft APC bind peptide fragments of allogeneic cellular proteins (different from that ones that recipient’s APC bind)→ they are recognized by recipient’s T cells as foreign b/ Indirectly – graft antigens are presented by recipient’s APC to recipient’s T cells

27. Transplantation immunity - alloimmune reaction Antibodies against alloantigens • They can react with HLA molecules or with another surface polymorphic antigens • Especially the complement binding antibodies have harmful effect (cytotoxic) • Possible presence of preformed antibodies (e.g. after blood transfusion, repeated pregnancy)

28. Immunologically privileged tissue • In allogeneic transplantation, some tissues are rejected less frequently (e.g. CNS, cornea, gonades). • The mechanisms of protection from the immune system: separation from the immune system (haematoencephalic barrier); the preference of Th2- and suppression of Th1-reactions; active protection from effector T cells • The privileged status is not absolute (see MS)

29. Types of transplantation • Autologous – within the same individual (e.g. a skin graft from an individual’s thigh to his chest); that is, they are not foreign • Syngeneic – in genetically identical individuals (e.g. identical twins); that is, they are not foreign • Allogeneic (alloantigens) – in genetically dissimilar members of the same species (e.g. a kidney transplant from mother to daughter); it is foreign • Xenogeneic (heterogeneic) – in different species (e.g. a transplant of monkey kidneys to human); it is foreign

30. Immunological examination before transplantation • HLA is the most important • MHC genes are highly polymorphic = there is a great number of gene variants (alleles) in the population • MHC haplotype = a unique combination of alleles (at multiple loci)encoding HLA molecules, that are transmitted together on the same chromosome

31. HLA typing 1/ Sera typing – identification of specific class I and class II HLA molecules using sera typing - less time-consuming method, however, alsoless accurate 2/ DNA typing – human DNA testing by PCR - low resolution (groups of alleles), highresolution (single alleles) - more time-consuming method, however, also highly accurate

32. Transplantation immunity - tests • Mixed lymphocyte reaction = T cells from one individual are cultured with leukocytes of another individual → the magnitude of this response is proportional to the extent of the MHC differences between these individuals • Cross match = preformed antibodies detection test (donor’s serum is mixed with recipient’s lymphocytes in the presence of complement proteins → if preformed cytolytic antibodies are present in serum thenthe lysis of donor’s leukocytes occurs)

33. Rejection = rejection of the graft by recipient’s immune system, which considers it as non-self • Hyperacute rejection (minutes) – mediated by preformed antibodies (natural or generated after previous immunization) → complement fixation → endothelial injury; activation of haemocoagulation → thrombosis of graft vessels → accumulation neutrophils → amplification of inflammatory reaction • Acute rejection (days or weeks) – mediated by T cells (→ graft cells and endothelial injury) and by antibodies (thes bind to endothelium) • Chronic rejection (months or years) – mediated by alloantigen-specific T cells → cytokines, stimulating growth of vascular endothelial and smooth muscle cells and tissue fibroblasts

35. Graft versus Host reaction (GvH) = donor’s T cells, present in graft recognize recipient’s tissue antigens as non-self and, therefore, they react to them 1/ Acute GvH – days or weeks after transplantation → liver, skin and intestinal injury 2/ Chronic GvH – moths or years after transplantation → chronic vascular, skin, organs or glands inflammation → replacement of functional by fibrous tissue, disorder of graft’s blood circulation → loss of tissue function

36. Therapeutic approaches of GvH prevention and treatment • The selection of an appropriate donor • Immunosupression – the development of coexistence is possible later • Donor’s T cells replacement from the graft

37. Maternal-foetal tolerance • Th2-type responses in mother • Protective effect of hormones – hCG, estrogens • Specific placental(Bohn’s) proteins – immunosuppressive effect • Blocking antibodies • Sialomucinous membrane – between mother and fetus

38. Rh incompatibility Mother Rh-/Foetus Rh+: delivery → senzitization (anti-D antibodies); next pregnancy (Rh incompatibility) → anti-D IgG pass through the placenta into foetal circulation → destruction of foetal erythrocytes → hemolytic anemia Prevention: administration of anti-D to Rh- mother after delivery of Rh+ child