27. Tumour immunology - tumour antigens, mechanisms of defence.

1. 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. GvH. Principle of materno-foetal tolerance. Rh incompatibility 30. IgG and IgM based immunopathological reaction (reaction of hypersensitivity type II). 31. Immunocomplex based immunopathological reaction (reaction of hypersensitivity type III).

2. Tumour immunology

3. Tumor antigens • Tumor – specific antigens (TSA) • complexes of MHCgp I with abnormal fragments of cellular proteins (chemically induced tumors, leukemia with chromosomal translocation) • complexes of MHCgp with fragments of oncogenic viruses proteins (tumors caused by viruses: EBV, SV40, polyomavirus…) • abnormal forms of glycoproteins (sialylation of surface proteins of tumor cells) • idiotypes of myeloma and lymphoma (clonotyping TCR and BCR)

4. Tumor antigens b) Tumor - associatedantigens (TAA) • presentalso on normalcells • differences in quantity, timeandlocalexpression • auxiliarydiagnosticmarkers

5. 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 HER2/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 (CD10 pre-B cells)

6. Anti-tumor immune mechanisms Immunecontrol • tumor cellsnormallyarise in tissues and are eliminated by Tcells

7. Immune surveillance of tumours

8. 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

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

10. Anti-tumor immune mechanisms If tumor cells are detected, in defense maybeinvolvednon-specificmechanisms(neutrophilicgranulocytes, macrophages, NK cells, complement) andantigen-specificmechanisms(TH1 and TC cells, antibodies).

11. Anti-tumor immune mechanisms • DC are necessary for activation of antigen specific mechanisms • predominance of TH1(IFN g, TNFa) • specific cell-mediated cytotoxic reactivity –TC • activation of TH2 → 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

12. Mechanisms of tumor resistance to the immune system • high variability of tumor cells • lowexpressionof tumor antigens • sialylation • someanticancersubstanceshave a stimulatingeffect • productionoffactorsinactivating T lymphocytes • expressionofFasL → T lymphocyteapoptosis • inhibitionofthefunctionordurabilitydendriticcells (NO, IL-10, TGF-b)

13. Transplantation

14. Transplantation = transfer oftissueor organ • autologous - donor = recipient • syngeneic- geneticallyidentical donor and recipient (identicaltwins) • allogeneic - geneticallynonidentical donor ofthesame species • xenogenic - the donor ofanother species • implant - artificialtissuecompensation

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

16. 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 lymphocytesalloreactivity

17. HLA typing= determminationof HLA antigens on thesurfaceoflymphocytes • Carry out during the testing before transplantation and in determination of paternity • serotyping • genotyping

18. Serotyping (microlymfocytotoxic test)

19. Serotyping (microlymfocytotoxic test) • Allospecificserums(obtainedfrom multiple natal to 6 weeksafterbirth, orcommerciallypreparedsetsoftypingserums (monoclonalantibodies)) • Principle- theincubationoflymphocyteswithtypingserums in the presence ofrabbitcomplement, thenisaddedthevitaldyewhichstaineddeadcells - cellscarryingspecific HLA are killed by cytotoxic Ab againsttheAg, thepercentageofdeadcellsisa measureofserumtoxicity • In positive reactionismore than 10% deadcells(serologicaltypingcanbedonealso by flowcytometry)

20. 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

21. Moleculargeneticmethods - genotyping b) PCR-SSO • PCR reactionwithsequence-specificoligonucleotides • Hybridizationwith enzyme orradiolabeledoligonucleotidesprobesspecificforindividualalleles

22. 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

23. 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

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

26. One-way MRL

27. Immunologically privileged sites and tissues • Transplantationofsometissuesdoesn´t lead to theinductionofallogeneicreactivity • Evolutionarilysignificant- protectionofvitalorgans(brain, eye, gonads) • Factorsprotectingimmunologicallyprivilegedstructures • isolationfromtheimmunesystem (minimalcontentoflecocytes) • preference of TH2 reactoin, supressionof TH1 reaction • FasLexpression • productionofTGFb

28. Rejection • hyperacute • accelerated • acute • chronic

29. Hyperacute rejection • minutes to hours after transplantation • humoral mediated immune response • mechanism: • if in recipients blood are present preformed or natural Ab(IgM 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

30. Acceleratedrejection • 3 to 5 daysaftertransplantation • caused by antibodiesthat don´t activatecomplement • cytotoxicandinflammatoryresponsestriggered by bindingofantibodies to Fc-receptors on phagocytesand NK cells • prevention: • negative crossmatchbeforetransplantation, ABOcompatibility

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

32. 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 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

33. Rejection • Factors: • Thegeneticdifferencebetween donor and recipient, especially in thegenescodingfor MHC gp (HLA) • Type oftissue / organ - thestrongestreactionsagainstvascularizedtissuescontaining many APC (skin) • Theactivityofthe recipientimmunesystem – theimmunodeficiency recipient has a smallerrejectionreaction; immunosuppressivetherapyaftertransplantation – suppressionofrejection • Status oftransplanted organ - thelengthofischemia, themethodofpreservation, traumatization of organ at collection

34. Graft-versus-host (GvH) disease • after bone marrowtransplantation • GvHalsoafterbloodtransfusion to immunodeficiencyrecipients • T-lymphocytes in thegraft bone marrowrecognize recipient tissueAg as foreign (alloreactivity)

35. AcuteGvHdisease • days to weeksafterthetransplantationof stem cells • damageof liver, skin andintestinalmucosa • prevention: appropriate donor selection, theremovalof T lymphocytesfromthegraftandeffectiveimmunosuppression

36. Chonic GvH disease • months to years after transplantation • infiltration of tissues and organs by TH2 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

37. Graft versus leukemia effect (GvL) • donor T lymphocytes react against residual leukemick cells of recipient (setpoint response) • mechanism is consistent with acute GvH • associated with a certain degree of GvH (adverse reactions)

38. Immunologic relationship between mother and allogenic fetus

39. Immunologicrelationshipbetweenmotherandallogenic fetus • fetalcellshave on thesurfacealloantigensinheritedfrom his father • Pregnancy = „semiallogenictransplantation“

40. 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 TH1 and preference of TH2 immune mechanisms in pregnancy • transfer of small doses of fetal antigens in maternal circulation causes tolerance ...

41. Rh incompatibility • Complications in pregnancy: production of anti-RhD antibodies by RhD- mother carrying an RhD+ 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-RhD antibodies

42. Rh incompatibility

43. 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-RhD antibodies (administered after abortion too) • Anti-Rh(D) antibodies bind to RhD 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

44. Duringnextchildbirths, if fetus isRh+ andmotherproduceanti-Rhantibodies, thisAbbdestroyredbloodcellsof fetus, whichcanlead to fetaldeath, or in severe postpartumanemia(anemianeonatorum)andneonataljaundice (icterus gravis neonatorum) ForeachpregnantwomanduringthefirsttrimesterinvestigateblodRhfactorandthe presence ofantibodies, in Rh- womenperformed a test forantibodiesalso in II. and III. trimester Rh incompatibility

45. Immunopathological reactions

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

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

49. Immunopathologicalreactionsbased on antibodiesIgGandIgM (reaction type II) CytotoxicantibodiesIgGandIgMbind to antigens on own cell: • complementactivation • binding to Fcreceptors on phagocytesand NK cells (ADCC)

50. Examples of immunopathological reaction Type II • Transfusionreactionsafteradministrationofincompatibileblood: bindingofantibodies to antigens on erythrocytes → activationoftheclassicalpathwayofcomplement → cell lysis • Hemolyticdiseaseofnewborns: caused by antibodiesagainstRhD antigen