27 Tumour immunology tumour antigens mechanisms of defence

27. Tumour immunology - tumour antigens, mechanisms of defence. 28. Alloimmune reaction. Types of transplantations and immunological examination before transplantation. Immunologically privileged tissues. 29. Types of graft rejection and their mechanisms. Gv. H. Principle of maternofoetal tolerance. Rh incompatibility 30. Ig. G and Ig. M based immunopathological reaction (reaction of hypersensitivity type II). 31. Immunocomplex based immunopathological reaction (reaction of hypersensitivity type III).

Tumour immunology

Tumor antigens a) Tumor – specific antigens (TSA) § complexes of MHCgp I with abnormal fragments of cellular proteins (chemically induced tumors, leukemia with chromosomal translocation) § complexes of MHC gp with fragments of oncogenic viruses proteins (tumors caused by viruses: EBV, SV 40, polyomavirus…) § abnormal forms of glycoproteins (sialylation of surface proteins of tumor cells) § idiotypes of myeloma and lymphoma (clonotyping TCR and BCR)

Tumor antigens b) Tumor - associated antigens (TAA) § present also on normal cells § differences in quantity, time and local expression § auxiliary diagnostic markers

Tumor - associated antigens § onkofetal antigens -on normal embryonic cells and some tumor cells § -fetoprotein (AFP) - hepatom § carcinoembryonic antigen (CEA) - colon cancer § melanoma antigens - MAGE-1, Melan-A § antigen HER 2/neu -receptor for epithelial growth factor, mammary carcinoma § EPCAM – epithelial cell adhesion molecule, metastases § differentiation antigens of leukemic cells - present on normal cells of leukocytes linage § CALLA -acute lymphoblastic leukemia (CD 10 pre-B cells)

Anti-tumor immune mechanisms Immune control § tumor cells normally arise in tissues and are eliminated by T cells

Immune surveillance of tumours

Anti – tumor immune response § tumor cells are weakly immunogenic § occurs when tumor antigens are presented to T cells by dendritic cells activated in the inflammatory environment

Regulatory T cells prevents removal of cancer cells and thus contribute to the development of the tumor.

Anti-tumor immune mechanisms If tumor cells are detected, in defense may be involved non-specific mechanisms (neutrophilic granulocytes, macrophages, NK cells, complement) and antigen-specific mechanisms (TH 1 and TC cells, antibodies).

Anti-tumor immune mechanisms § DC are necessary for activation of antigen specific mechanisms § predominance of TH 1 (IFN g, TNFa) § specific cell-mediated cytotoxic reactivity – TC § activation of TH 2 → stimulation of B cells→ tumor specific antibodies production (involved in the ADCC) § tumor cells are destroyed by cytotoxic NK cells (ADCC) § interferons - antiproliferative, cytotoxic effect on tumor cells - INFg - DC maturation

Mechanisms of tumor resistance to the immune system § high variability of tumor cells § low expression of tumor antigens § sialylation § some anticancer substances have a stimulating effect § production of factors inactivating T lymphocytes § expression of Fas. L → T lymphocyte apoptosis § inhibition of the function or durability dendritic cells (NO, IL-10, TGF -b)

Transplantation

Transplantation = transfer of tissue or organ § autologous - donor = recipient § syngeneic - genetically identical donor and recipient (identical twins) § allogeneic - genetically nonidentical donor of the same species § xenogenic - the donor of another species § implant - artificial tissue compensation

Allotransplantation § differences in donor-recipient MHC gp and secondary histocompatibility Ag § alloreactivity of T lymphocytes - the risk of rejection and graft-versus-host disease

Tests prior to transplantation § ABO compatibility (matching blood group) -risk of hyperacute or accelerated rejection (= formation of Ab against A or B Ag on graft vascular endothelium) § HLA typing (matching tissue type) - determining of HLA alelic forms by phenotyping or genotyping § Cross-match - detection of preformed alloantibodies (after blood transfusions, transplantation, repeated childbirth) § Mixed lymphocyte reaction - testing of T lymphocytes alloreactivity

HLA typing = determmination of HLA antigens on the surface of lymphocytes § Carry out during the testing before transplantation and in determination of paternity § serotyping § genotyping

Serotyping (microlymfocytotoxic test)

Serotyping (microlymfocytotoxic test) § Allospecific serums (obtained from multiple natal to 6 weeks after birth, or commercially prepared sets of typing serums (monoclonal antibodies)) § Principle - the incubation of lymphocytes with typing serums in the presence of rabbit complement, then is added the vital dye which stained dead cells - cells carrying specific HLA are killed by cytotoxic Ab against the Ag, the percentage of dead cells is a measure of serum toxicity § In positive reaction is more than 10% dead cells (serological typing can be done also by flow cytometry)

Molecular genetic methods - genotyping a) PCR-SSP (Polymerase chain reaction with sequential specific primers) § Extracted DNA is used as a substrate in a set of PCR reactions § Each PCR reaction contains primers pair specific for a certain allele (or group of alleles) § Positive and negative reactions are evaluated by electrophoresis

Molecular genetic methods genotyping b) PCR-SSO § PCR reaction with sequence-specific oligonucleotides § Hybridization with enzyme or radiolabeled oligonucleotides probes specific for individual alleles

Molecular genetic methods - genotyping c) PCR-SBT § Sequencing based typing § We get the exact sequence of nucleotides, which compares with a database of known sequences of HLA alleles

Tests prior to transplantation Cross-match testing § determination of preformed alloantibodies § recipient serum + donor lymphocytes + rabbit complement → if cytotoxic Ab against donor HLA Ag are present in recipient serum , Ab activate complement → lysis of donor lymphocytes. Dye penetration into lysis cells. § positive test = the presence of preformed Ab → risk of hyperacute rejection! → contraindication to transplantation

Tests prior to transplantation Mixed lymphocyte reaction (MRL) § determination of T lymphocytes alloreactivity § mixed donor and recipient lymphocytes → T lymphocytes after recognition of allogeneic MHC gp activate and proliferate One-way MRL § determination of recipient T lymphocytes reactivity against donor cells § donor cells treated with chemotherapy or irradiated lose the ability of proliferation

One-way MRL

Immunologically privileged sites and tissues § Transplantation of some tissues doesn´t lead to the induction of allogeneic reactivity § Evolutionarily significant- protection of vital organs (brain, eye, gonads) § Factors protecting immunologically privileged structures § isolation from the immune system (minimal content of lecocytes) § preference of TH 2 reactoin, supression of TH 1 reaction § Fas. L expression § production of TGFb

Rejection § hyperacute § accelerated § acute § chronic

Hyperacute rejection § minutes to hours after transplantation § humoral mediated immune response mechanism: § if in recipients blood are present preformed or natural Ab (Ig. M anti- carbohydrate Ag) before transplantation → Ab + Ag of graft (MHC gp or endothelial Ag) → graft damage by activated complement § the graft endothelium: activation of coagulation factors and platelets, formation thrombi, accumulation of neutrophil granulocytes prevention: § negative cross match before transplantation, ABO compatibility

Accelerated rejection § 3 to 5 days after transplantation § caused by antibodies that don´t activate complement § cytotoxic and inflammatory responses triggered by binding of antibodies to Fc-receptors on phagocytes and NK cells prevention: § negative cross match before transplantation, ABO compatibility

Acute rejection § days to weeks after the transplantation or after a lack of immunosuppressive treatment § cell-mediated immune response mechanism: § reaction of recipient TH 1 and TC cells against Ag of graft tissue § infiltration by lymphocytes, monocytes, granulocytes around small vessels → destruction of the transplant tissue

Chronic rejection § from 2 months after transplantation § the most common cause of graft failure mechanism is not fully understood: § non-immunological factors (tissue ischemia) and TH 2 response with production alloantibodies, pathogenetic role of cytokines and growth factors (TGFβ) § fibrosis of the internal blood vessels of the transplanted tissue, endothelial damage →impaired perfusion of graft → gradual loss of its function § dominating findings: vascular damage

Rejection Factors: § The genetic difference between donor and recipient, especially in the genes coding for MHC gp (HLA) § Type of tissue / organ - the strongest reactions against vascularized tissues containing many APC (skin) § The activity of the recipient immune system – the immunodeficiency recipient has a smaller rejection reaction; immunosuppressive therapy after transplantation – suppression of rejection § Status of transplanted organ - the length of ischemia, the method of preservation, traumatization of organ at collection

Graft-versus-host (Gv. H) disease § after bone marrow transplantation § Gv. H also after blood transfusion to immunodeficiency recipients § T-lymphocytes in the graft bone marrow recognize recipient tissue Ag as foreign (alloreactivity)

Acute Gv. H disease § days to weeks after the transplantation of stem cells § damage of liver, skin and intestinal mucosa § prevention: appropriate donor selection, the removal of T lymphocytes from the graft and effective immunosuppression

Chonic Gv. H disease § months to years after transplantation § infiltration of tissues and organs by TH 2 lymphocytes, production of alloantibodies and cytokines → fibrosis § process like autoimmune disease: vasculitis, scleroderma, sicca-syndrome § chronic inflammation of blood vessels, skin, internal organs and glands, which leads to fibrosis, blood circulation disorders and loss of function

Graft versus leukemia effect (Gv. L) § donor T lymphocytes react against residual leukemick cells of recipient (setpoint response) § mechanism is consistent with acute Gv. H § associated with a certain degree of Gv. H (adverse reactions)

Immunologic relationship between mother and allogenic fetus

Immunologic relationship between mother and allogenic fetus §fetal cells have on the surface alloantigens inherited from his father §Pregnancy = „semiallogenic transplantation“

Immunologic relationship between mother and allogenic fetus Tolerance of fetus by mother: § the relative isolation of the fetus from maternal immune system (no mixing of blood circulation) § trophoblast - immune barrier witch protects against mother alloreactive T lymphocytes (don´t express classical MHC gp, expresses non-classical HLA-E and HLA-G) § suppressin of TH 1 and preference of TH 2 immune mechanisms in pregnancy • transfer of small doses of fetal antigens in maternal circulation causes tolerance. . .

Rh incompatibility § Complications in pregnancy: production of anti-Rh. D antibodies by Rh. D- mother carrying an Rh. D+ fetus (hemolytic disease of newborns) § During childbirth or abortion (after 8 weeks of gestation) fetal erythrocytes can penetrate into the bloodstream of mother → immunization, formation of anti-Rh. D antibodies

Rh incompatibility

Rh incompatibility § After childbirth, investigate Rh factor of born child, if is child Rh+, mother (Rh-) gets up to 72 hours after birth injection of anti-Rh. D antibodies (administered after abortion too) § Anti-Rh(D) antibodies bind to Rh. D Ag on baby´s red blood cells, this Ag than can´t bind to BCR and can´t activate B lymphocytes, this immune comlexes also inhibit B lymphocytes

Rh incompatibility § During next childbirths, if fetus is Rh+ and mother produce anti-Rh antibodies, this Abb destroy red blood cells of fetus, which can lead to fetal death, or in severe postpartum anemia (anemia neonatorum) and neonatal jaundice (icterus gravis neonatorum) § For each pregnant woman during the first trimester investigate blod Rh factor and the presence of antibodies, in Rh- women performed a test for antibodies also in II. and III. trimester

Immunopathological reactions

Immunopathological reactions § Immune response which caused damage to the body (Consequence of immune response against pathogens, inappropriate responses to harmless antigens; autoimmunity)

Immunopathological reactions Classification by Coombs and Gell Immunopathological reactions: immune response, which caused damage to the body (secondary consequence of defense responses against pathogens, inappropriate responses to harmless antigens, autoimmunity) IV types of immunopathological reactions: Type I reaction - response based on Ig. E antibodies Type II reaction - response based on antibodies, Ig. G and Ig. M Type III reaction - response based on the formation of immune complexes Type IV reaction - cell-mediated response

Immunopathological reactions based on antibodies Ig. G and Ig. M (reaction type II) Cytotoxic antibodies Ig. G and Ig. M bind to antigens on own cell: § complement activation § binding to Fc receptors on phagocytes and NK cells (ADCC)

Examples of immunopathological reaction Type II § Transfusion reactions after administration of incompatibile blood: binding of antibodies to antigens on erythrocytes → activation of the classical pathway of complement → cell lysis § Hemolytic disease of newborns: caused by antibodies against Rh. D antigen

Examples of immunopathological reaction Type II Autoimmune diseases: § organ-specific cytotoxic antibodies (antibodies against erythrocytes, neutrophils, thrombocytes, glomerular basement membrane. . . ) § blocking or stimulating antibodies Graves - Basedow's disease - stimulating antibodies against the receptor for TSH Myasthenia gravis - blocking of acetylcholin receptor→ blocking of neuromuscular transmission Pernicious anemia - blocking the absorption of vitamin B 12 Antiphospholipid syndrome - antibodies against fosfolipids Fertility disorder - antibodies against sperms or oocytes

Immunopathological reactions based on immune complexes formation (reaction type III) § caused by Ig. G antibodies → bind to antigen → creation of immune complexes § immunocomplexes - bind to Fc receptors on phagocytes - activate complement § immune complexes, depending on the quantity and structure, are eliminated by phagocytes or stored in tissues

Immunopathological reactions type III § pathological immunocomplexes response arises when is a large dose of antigen, or antigen in the body remains; arise 10 -14 days after aplication of Ag and induced inflamation (can get to chronic state) § immune complexes are deposited in the kidneys (glomerulonephritis), on the surface of endothelial cells (vasculitis) and in synovie joint (arthritis)

Serum sickness § therapeutic application of xenogeneic serum (antiserum to snake venom) § creation of immune complexes and their storage in the vessel walls of different organs § clinical manifestations: urticaria, arthralgia, myalgia Systemic lupus erythematosus § antibodies against nuclear antigens, ANA, anti-ds. DNA Farmer's lung § Ig. G antibody against inhaled antigens (molds, hay) Post-streptococcal glomerulonephritis, cryoglobulinemia, revmatoid arthritis, post-infectious arthritis

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