Defense against pathogens, tumour immunology, transplantology Martin Liška

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

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

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

Phases of humoral immune response

Effector functions of antibodies Neutralization of microbes (incl.viruses) and their toxins Opsonization of microbes (binding to Fc receptors 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)

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)

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

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)

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 II MHC to CD4+ T lymphocytes

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

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

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

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

Function of Th1and Th2 lymphocytes

Activation of T lymphocytes

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

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

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)

Anti-viral defense a/ non-specific (innate) immune system - monocytes/macrophages, NK cells, interferons (IFN-  → activation of macrophages, IFN-  → antiviral activity, activation of NK cells) b/ specific (adaptive) immune system - T cells, antibodies (e.g. neutralization of viruses)

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 cervical CA)

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

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)

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

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

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)

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)

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

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

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

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 then the lysis of donor’s leukocytes occurs)

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

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

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

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

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