Anti-tumor immunity

1. Anti-tumor immunity

2. Malignant transformation Failure of regulation of cell division and regulation of "social" behavior of the cells The uncontrollable proliferation, dissemination to other tissues Mutations in protoonkogenes and antionkogenes

3. Mutagens(carcinogens) • - physical (eg various forms of radiation) • - chemical (eg aromatic hydrocarbons) • - biological (mainly various oncogenic viruses)

4. Protoonkogens promitotic (promoting cell division) for the malignant transformation is enough mutation in one copy of the gene protoonkogen (dominant oncogenes)

5. Antionkogens • tumor-suppressor genes • regulation of cell cycle • for the malignant transformation should be excluded from • function both copies of the gene (recessive oncogenes) • TP53, RB1

6. Anti-tumor immune mechanisms Hypothesis of immune control tumor cells normally arise in tissues and are eliminated by T lymphocytes (probably wrong hypothesis) Defensive immune response tumor cells are weakly immunogenic occurs when tumor antigens are presented to T lymphocytes by activated dendritic cells in defense may be involved: non-specific mechanisms (neutrophilic granulocytes, macrophages, NK cells, interferons) and antigen-specific mechanisms (complement activating antibodies or ADCC, TH1 and TC)

7. cancer-associated antigens are processed by APC and recognized by T lymphocytes in complex with HLA I. and II. class with providing costimulus signals • predominance of TH1 (IFN  TNF) • specific cell-mediated cytotoxic reactivity – TC • activation TH2 → support B lymphocytes→ tumor specific antibodies (involved in the ADCC) • tumor cells are destroyed by cytotoxic NK cells (low MHC gpI expression on tumor cells) • interferons - antiproliferative, cytotoxic effect on tumor cells - INFγ - DC maturation

8. Cytotoxic mechanisms of NK cells

9. Tumor antigens Antigens specific for tumors (TSA) a) complexes of MHCgp I with abnormal fragments of cellular proteins - chemically induced tumors - leukemia with chromosomal translocation b) complexes of MHCgp with fragments of proteins of oncogenic viruses - tumors caused by viruses (EBV, SV40, polyomavirus) c) abnormal forms of glycoproteins - Sialylation of surface proteins of tumor cells d) idiotypes of myeloma and lymphoma - clonotyping TCR and BCR

10. Antigens associated with tumors (TAA) - also on normal cells - differences in quantity, time and local expression - auxiliary diagnostic markers a) onkofetal antigens - on normal embryonic cells and some tumor cells - -fetoprotein (AFP) - hepatom - canceroembryonalantigen (CEA) - colon cancer b) melanoma antigens - MAGE-1, Melan-A

11. c) antigen HER2/neu - receptor for epithelial growth factor - mammary carcinoma d) EPCAM - epithelial adhesion molecule - metastases e) differentiation antigens of leukemic cells - present on normal cells of leukocytes linage - CALLA -acute lymphoblastic leukemia (CD10 pre-B cells)

12. Mechanisms of tumors resistance to immune system high variability of tumor cells low expression of tumor antigens sialylation tumor cells do not provide costimulus signals → T lymphocyte anergy some anticancer substances have a stimulating effect production of factors inactivating T lymphocytes expression of FasL → T lymphocyte apoptosis inhibition of the function or durability dendritic cells (NO, IL-10, TGF-

13. Tumor immunotherapy Therapy - surgical removal of tumor - chemotherapy or radiotherapy - immunotherapy Immunotherapy - induction of anti-tumor immunity, or the use of immune mechanisms to targeting drugs to the tumor site

14. Immunotherapy using antibodies Antibodies functions - opsonization - activation of complement - induction of ADCC - carriers of drugs or toxins

15. 1) Monoclonal antibodies - against TAA - mouse and humanised antibodies - imunotoxins, radioimunotoxins - the possibility of damage surrounding tissues - HERCEPIN - Ab against HER2/neu, breast cancer - RITUXIMAB - Ab against CD20, lymphoma 2) Bispecific antibodies - bind a tumor antigen and the T lymphocyte or NK cell - Fc fragment of antibody binds to Fc receptors on phagocytes and NK cells 3) Elimination of tumor cells from the suspension of bone marrow cells using monoclonal antibodies for autologous transplantation

16. Immunotherapy using cell-mediated mechanisms 1) stimulation of inflammation at the tumor site 2) stimulation of LAK and TIL - isolation of T and NK cells, stimulation by cytokines, and return to the patient - LAK (lymphokine activated killers) - TIL (tumor infiltrating lymphocytes) 3) improving of tumor cells antigenpresenting function - genetic modification of tumor cells - expression of CD80, CD86 - production of IL-2, GM-CSF - modified cells are irradiated and returned to the patient

17. 4) tumor vaccines - in vitro stimulation of TH1 cells and TC with tumor antigens 5) the dendritic cell immunotherapy - in vitro cultivation of monocytes in an appropriate cytokine environment (GM-CSF, IL-4) → transformation into dendritic cells - cultivation of dendritic cells with tumor antigens 6) immunotherapy by donor T lymphocytes - after allogeneic transplantation - causing graft-versus-host disease 7) immunotherapy by immune system products - IL-2 - renal cell carcinoma - IFN  - hematoonkology

18. Transplantation

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

20. Allogeneic - differences in donor-recipient MHC gp and secondary histocompatibility Ag - alloreactivityof T lymphocytes - the risk of rejection and graft-versus-host - direct detection ofalloantigens – recipient T lymphocytes recognize the different MHC gp and non-MHC molecules on donor cells - indirect recognition of alloantigens - APC absorb different MHC gp from donor cells and present the fragments to T lymphocytes - CD8+ T cells recognize MHC gp I. - CD4+ T cells recognize MHC gp II.

21. Testing before transplantation Compatibility in the system ABO -risk of hyperacute or accelerated rejection = formation of Ab against A or B Ag on graft vascular endothelium) HLA typing (determining of MHC gp alelic forms) phenotyping and genotyping by PCR Cross-match - lymfocytotoxic test - testing preformed Ab (after blood transfusions, transplantation, repeated childbirth) Mixed lymphocyte test - testing of alloreactivity T lymphocytes monitor for reactivity of lymphocytes to allogeneic HLA

22. HLA typing a) phenotyping: Evaluation of HLA molecules using typing serums Typing antiserums = alloantiserums of multipar (created cytotoxic Ab against paternal HLA Ag of their children), serum of patients after repeated blood transfusions, monoclonal Ab - molecules HLA class I: separated T lymphocytes - molecules HLA class II: separated B lymphocytes b) genotyping: evaluation of specific alleles DNA typing of HLA class II: DR, DP, DQ by PCR.

23. Cross-match test ● determination of preformed antibodies ● recipient serum + donor lymphocytes + rabbit complement → if cytotoxic Ab against donor HLA Ag are present in recipient serum (called alloantibodies = Ab activating complement) → lysis of donor lymphocytes. Visualization of dye penetration into lysis cells. ● positive test = the presence of preformed Ab → risk of hyperacute rejection! → contraindication to transplantation

24. Mixed lymphocyte reaction (MRL) ● determination of alloreactivity T lymphocytes ● mixed donor and recipient lymphocytes → T lymphocytes after recognition allogeneic MHC gp activate and proliferate One-way MRL ● determination of recipient T lymphocytes reactivityagainst donor cells ● donor cells treated with chemotherapy or irradiated lose the ability of proliferation

25. 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 much APC (skin) The activity of the immune system of the recipient - the immunodeficiency recipient has a smaller rejection reaction; immunosuppressive therapy after transplantation – suppression of rejection Status transplanted organ - the length of ischemia, the method of preservation, traumatization of organ at collection

26. Hyperacute rejection ● minutes to hours after transplantation ● antibodies type of immune response mechanism: ● in recipients blood are present before transplantation preformed or natural Ab (IgM anti-carbohydrate Ag) → Ab + Ag of graft (MHC gp or endothelial Ag) → graft damage by activated complement (lysis of cells) ● the graft endothelium: activation of coagulation factors and platelets, formation thrombi, accumulation of neutrophil granulocytes prevention: ● negat. cross match before transplantation, ABO compatibility

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

28. Acute rejection ● days to weeks after the transplantation or after a lack of immunosuppressive treatment ● cell-mediated immune response mechanism: ● recipient TH1 and TC cells response against Ag of graft tissue ● infiltration of lymphocytes, mononuclears, granulocytes around smallvessels → destruction of transplant tissue

29. 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 TH2 responses with production alloantibodies, pathogenetic role of cytokines and growth factors (TGF β) ●replacement of functional tissue by connective tissue, endothelial damage →impaired perfusion of graft → gradual loss of its function dominating findings: vascular damage

30. Graft-versus-host disease (GVHD) ● after bone marrow transplantation ● GVHD also after blood transfusion to immunodeficiency recipients ● T-lymphocytes in the graft bone marrow recognize recipient tissue Ag as foreign (alloreactivity) Acute GVHD ● days to weeks after the transplantation of stem cells ● damage of liver, skin and intestinal mucosa ● Prevention: appropriate donor selection, T lymphocytes removal from the graft and effective immunosuppression

31. Chronic GVHD ● months to years after transplantation ● TH2 lymphocytesinfiltration of tissues and organs, production of alloantibodies and production of cytokines → fibrotization ● process like autoimmune disease: vasculitis, scleroderma, sicca-syndrome ● chronic inflammation of blood vessels, skin, internal organs and glands, which leads to fibrotization, blood circulation disorders and loss of function

32. Graft versus leukemia effect (GVL) ● donor T lymphocytes react against residual leukemick cells of recipient ● mechanism is consistent with acute GVHD ● associated with a certain degree of GVHD (adverse reactions)

33. Immunologic relationship of mother and allogenic fetus ● fetal cells have on the surface alloantigens inherited from his father Tolerance of fetus by mother allow the following mechanisms: ●the relative isolation of the fetus from maternal immune system (no mixing of blood circulation) ●trophoblast - immune barrier witch protect against mother alloreaktive T lymphocytes - don‘t express classical MHC gp, expresses non-classical HLA-E and HLA-G ● depression of TH1 immune mechanisms in pregnancy Complications in pregnancy: production of anti-RhD antibodies by RhD- mother carrying RhD+ fetus (hemolytic disease of newborns)

34. Immunopathological reactions

35. 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 IgE antibodies Type II reaction - response based on IgG and IgMantibodies Type III reaction - response based on the formation of immune complexes Type IV reaction - cell-mediated response

36. Immunopathological reaction based on IgG and IgM antibodies (reaction type II) Cytotoxic antibodies IgG and IgM: ● complement activation ● ADCC ● binding to phagocytes and NK cells Fc receptors Haemolytic reactions after transfusion of ABO incompatible blood: Binding of antibodies to antigens of erythrocytes → activation of the classical way of complement → cell lysis Hemolytic disease of newborns: Caused by antibodies against RhD antigen

37. Autoimmune diseases: ● organ-specific cytotoxic antibodies (antibodies against erythrocytes, neutrophils, thrombocytes, glomerular basement membrane ...) ●blocking or stimulating antibodies Graves - Basedow disease - stimulating antibodies against the TSH receptor Myasthenia gravis - blocking of acetylcholin receptor→ blocking of neuromuscular transmission Pernicious anemia - blocking of vitamin B12 absorption Antiphospholipid syndrome - antibodies against fosfolipids Fertility disorders - antibodies against sperms or oocytes

38. Immunopathological reactions based on immune complex formation (reaction type III) ●caused by IgG antibodies → bind to antigen → creation of immunecomplexes ●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 ● pathological immunocomplexes response arises when is a large dose of antigen, or antigen in the body remains ● immune complexes are deposited in the kidneys (glomerulonephritis), on the endothelial cellssurface (vasculitis) and in synovial joints (arthritis)

39. Serum sickness ● after 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-dsDNA Farmer's lung ● IgG antibodies against inhaled antigens (molds, pollens) Poststreptococcal glomerulonephritis

40. Immunopathological delayed-type reaction (reaction type IV) ● delayed-type hypersensitivity (DTH) ● local reaction caused by TH1 cells and monocytes / macrophages Experimental model (testing of cellular immunity): ● intradermal immunization by antigen → creation of antigen-specificTH1 cells ● after a few weeks intradermal administration of antigen → creates local reaction (granuloma) - TH1 cells and macrophages Tuberculin reaction Tissue damage in tuberculosis and leprosy Sarcoidosis

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