1. Mgr. Ochotná Jitka 23. Defence against extracellular pathogens (bacteria and unicellular parasites). 24. Defence against intracellular pathogens (bacteria, fungi and unicellular parasites. 25. Anti-viral defence. 26. Defence against multicellular parasites. 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 inkompatibility 30. IgG and IgM based immunopathological reaction (reaction of hypersensitivity type II). 31. Immunocomplex based immunopathological reaction (reaction of hypersensitivity type III).

2. Defense against extracellular pathogens

3. Defence against extracellular pathogens
Bacteria, unicellular parasites
Pathogen induce inflammation
Neutrophil granulocytes phagocytosis, degranulation, NETs
Opsonization (C3b, IgG and IgA antibodies, lectins, CRP...)

4. Defence against extracellular pathogens
Complement
IgM - complement activation
IgG - complement activation, opsonization
IgA - opsonization sIgA prevents against infection by intestinal and respiratory bacteria
Neutralizing antibodies apply in the defense against bacterial toxins (Clostridium tetani and botulinum …)

5. Defence against extracellular pathogens
„Indirect toxins“ - bacterial Lipopolysaccharide (LPS) stimulates big number of monocytes to release TNF, which can cause septic shock
Individuals with immunodeficiency of phagocytes, complement and antibodies production are especially at risk of infections with extracellular bacterial

6. Defense against intracellular pathogens

7. Defense against intracellular pathogens
Bacteria, fungi and unicellular parasites
Intracellular parasites are resistant to the microbicidal mechanisms of phagocytes
Macrophage which engulf pathogen produce IL-12 → TH1 differentiation, production of IFN and membrane TNF → activation of macrophage and NO production
NO kills intracellular pathogens

8. Defense against intracellular pathogens
TC lymphocytes apply in the defense against intracelular parasites, which escape from phagolysosomes
Individuals with certain disorders of phagocytes and defects of T lymphocytes are at risk of infections with intracellular microorganisms

9. Anti-viral defense

10. Anti-viral defence
interferons - production of IFN and IFN is induced in infected cells; IFN activates macrophages (iNOS)
IFN and IFN- prevents viral replication induce proliferation ofNK cells increase the expression of HLA-I (Ag presentation for Tc)

11. Anti-viral defence NK cells ADCC
ADCC (Antibody-dependent cell-mediated cytotoxicity); NK cell bind with CD16 (Fcreceptor) to IgG which has bound to the surface of infected cell and then NK cell release perforins and granzymes

12. Anti-viral defence
Effector TC lymphocytes destroy infected cells in direct contact (granzym/perforin; FasL) and by produced cytokines (lymfotoxin)

13. Anti-viral defence
IgM neutralization, complement activation (lysis of some viruses)
IgG neutralization, complement activation, opsonization
sIgA – neutralization, pathogen aglutination, opsonization (defense against respiratory viruses and enteroviruses)
IgA and IgG have preventive effect in secondary viral infection

14. Defense against multicellular parasites

15. Defense against multicellular parasites
IgE, mast cells, basophils and eosinophils
TH2 stimulation under the influence of IL-4 (mast cells and other APC stimulated by parasite)
TH2 with IL-4 production stimulate isotype switching to IgE
IgE bind to FcRI on mast cells and basophils

16. Mast cell activation by cross-linking of IgE Fc receptors
Binding of IgE to highaffinnity Fc receptor for IgE (FcRI)
Binding of multivalent antigen (multicellular parasite) to IgE
Aggregation of several molecules of FcRI

17. Mast cell activation
Cytoplasmic granules: hydrolytic enzymes, histamine, serotonin, heparin
Activation of arachidonic acid metabolism (leukotriene C4, prostaglandin D2)
Start of cytokines production (TNF, TGF, IL-4, 5,6 ...)

18. Defense against multicellular parasites
Histamine
Vasodilatation, increase vascular permeability (erythema, edema, itching)
Bronchoconstriction (cough)
Increases intestinal peristalsis (diarrhea)
Increased mucus secretion This helps eliminate the parasite.

19. Defense against multicellular parasites
Eosinophils release from granules (eosinophilic katoin protein, proteases ...)
Eosinophils phagocyte complexes of parasitic particles with IgE via its IgE receptors
IL-5 activation of eosinophils

20. Tumor immunology 20

21. Tumor antigens a) Tumor – specific antigens (TSA)
are present only on tumor cells
b) Tumor - associated antigens (TAA)
present also on normal cells
differences in quantity, time and local expression
auxiliary diagnostic markers 21

22. 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, SV40, polyomavirus…)
abnormal forms of glycoproteins (sialylation of surface proteins of tumor cells)
idiotypes of myeloma and lymphoma (clonotyping TCR and BCR) 22

23. Tumor - associated antigens (TAA)
onkofetal antigens -on normal embryonic cells and some tumor cells
-fetoprotein (AFP) - hepatom
carcinoembryonic antigen (CEA) - colon cancer
melanoma antigens - MAGE-1, Melan-A
prostate-specific antigen (PSA) – prostate cancer 23

24. Tumor - associated antigens (TAA)
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) 24

25. Immune surveillance of tumors25

26. 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 (TH1 and TC cells, antibodies).
tumor cells are weakly immunogenic
regulatory T cells promote progression of cancer
occurs when tumor antigens are presented to T cells by dendritic cells activated in the inflammatory environment 26

27. Anti – tumor defense
DC are necessary for activation of antigen specific mechanisms
predominance of TH1 (IFN , TNF)
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 INF - DC maturation 27

28. 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 FasL → T lymphocyte apoptosis
inhibition of the function or durability dendritic cells (NO, IL-10, TGF-) 28

29. Transplantation 29 29

30. 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 30 30

31. Allotransplantation
differences in donor-recipient MHC gp and secondary histocompatibility Ag
alloantigens – nonself antigens from members of the same species
alloreactivity of T lymphocytes - the risk of rejection and graft-versus-host disease 31 31

32. Tests prior to transplantation
ABO compatibility (matching blood group) -risk of hyperacute 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 32 32

33. 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

34. HLA typing
1) Serotyping (microlymfocytotoxic test) 34

35. HLA typing 2) Molecular genetic methods- genotyping 2a) PCR-SSP
2b) PCR-SSO
2c) PCR-SBT 35

36. 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 36 36

37. Mixed lymphocyte reaction (MLR)
Two – way MLR
determination of T lymphocytes alloreactivity
mixed donor and recipient lymphocytes → T lymphocytes after recognition of allogeneic MHC gp activate and proliferate
One – way MLR
determination of recipient T cell reactivity against donor cells
donor cells treated with chemotherapy or irradiated lose the ability of proliferation 37 37

38. Rejection
hyperacute
accelerated
acute
chronic 38

39. 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 39 39

40. Accelerated rejection
3 to 5 days after transplantation
caused by reactivation of T lymphocytes (secondary response)

41. 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 with lymphocytes, monocytes, granulocytes around small vessels → destruction of tissue transplant 41 41

42. 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 42 42

43. 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 43 43

44. Graft-versus-host (GvH) disease
after bone marrow transplantation
GvH also after blood transfusion to immunodeficiency recipients
T-lymphocytes in the graft bone marrow recognize recipient tissue Ag as foreign (alloreactivity) 44 44

45. Acute GvH 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 45

46. 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 46 46

47. 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) 47 47

48. Immunologic relationship between mother and allogenic fetus

49. Immunologic relationship between mother and allogenic fetus
Tolerance of the 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 (doesn´t express classical MHC gp, expresses non-classical HLA-E and HLA-G)
suppressin of TH1 and preference of TH2 immune mechanisms in pregnancy
Treg
transfer of small doses of fetal antigens in maternal circulation causes tolerance ...

50. Rh incompatibility

51. Rh incompatibility
Complications in second 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

52. Rh incompatibility
After childbirth, investigate Rh factor of born child, if is child Rh+, mother 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

53. 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

54. Immunopathological (hypersensitivity) reactions54 54

55. Immunopathological reactions
Immune response which caused damage to the body (Consequence of immune response against pathogens, inappropriate responses to harmless antigens; autoimmunity) 55

56. Immunopathological reactions
Classification by Coombs and Gell
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 56 56

57. Immunopathological reaction type II (cytotoxic hypersensitivity reactions)
Cytotoxic antibodies IgG and IgM bind to cell surface antigens on own cells:
complement activation
binding to Fc receptors on phagocytes and NK cells (ADCC) 57 57

58. 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 RhD antigen 58

59. 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 B12
Antiphospholipid syndrome - antibodies against fosfolipids
Fertility disorder - antibodies against sperms or oocytes 59 59

60. Immunopathological reaction type III (immune-complex reactions)
circulating antigen- IgG antibody immune complexes that deposit in tissues
immunocomplexes - activate complement
- bind to Fc receptors on phagocytes
immune complexes, depending on the quantity and structure, are eliminated by phagocytes or stored in tissues 60 60

61. Immunopathological reactions type III
pathological immunocomplexes response arises when is a large dose of antigen, or antigen in the body remains; arise 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) 61

62. clinical manifestations: urticaria, arthralgia, myalgia
Serum sickness
the 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 antibody against inhaled antigens (molds, hay)
Post-streptococcal glomerulonephritis, cryoglobulinemia, revmatoid arthritis, post-infectious arthritis 62 62

63. Thank you for your attention63

64. HLA typing

65. 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)

66. 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

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

68. 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