Tumour immunology

J. Ochotná

Tumor antigensa)Antigens specific for tumors (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 proteins of oncogenic viruses - 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

b) Antigens associated with tumors (TAA)

present also on normal cells differences in quantity, time and local expression auxiliary diagnostic markers

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 adhesion molecule metastases

differentiation antigens of leukemic cells present on normal cells of leukocytes linage CALLA -acute lymphoblastic leukemia (CD10 pre-

B cells)

Anti-tumor immune mechanisms

Immune control

tumor cells normally arise in tissues and are eliminated by T lymphocytes

Defensive immune response

tumor cells are weakly immunogenic

occurs when tumor antigens are presented to T lymphocytes by dendritic cells activated in the inflammatory environment

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, antibodies)

DC are necessary for activation of antigen specific mechanisms

cancer-associated antigens are processed by DC and recognized by T lymphocytes in complex with HLA I. and II. class with providing costimulus signals

predominance of TH1 (IFN g, TNF)

specific cell-mediated cytotoxic reactivity – TC

activation of TH2 → support B lymphocytes→ tumor specific antibodies (involved in the ADCC)

tumor cells are destroyed by cytotoxic NK cells (ADCC)

interferons - antiproliferative, cytotoxic effect on tumor cells - INFγ - DC maturation

Regulatory T cells prevents removal of cancer cells and thus

contribute to the development of the tumor.

Mechanisms of tumor resistance to the immune system

- high variability of tumor cells low expression of tumor antigens sialylation tumor cells signals do not provide costimulus → 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-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

● direct detection of alloantigens – 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

Testing before transplantation

● ABO compatibility -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 - lymphocytotoxic test – detection of preformed Ab (after blood transfusions, transplantation, repeated childbirth) ● Mixed lymphocyte reaction - testing of T lymphocytes alloreactivity, monitor for reactivity of lymphocytes to allogeneic HLA

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

1) Serotyping Microlymfocytotoxic test Allospecific serums (obtained from multiple natal to 6

weeks after birth, obtained by vaccination of volunteers, 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 (forces and antileukocyte antibody titre)

Positive reaction is considered more than 10% dead cells (serological typing can be done also by flow cytometry

HLA - serotyping

2) Molecular genetic methods

2a) 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, each combination of alleles has a specific electrophoretic painting

2b) PCR-SSO PCR reaction with sequence-specific

oligonucleotides Multiplication of hypervariable sections of genes coding HLA

Hybridization with enzyme or radiolabeled DNA probes specific for individual alleles 2c) PCR-SBT

Sequencing based typing The most accurate method of HLA typing We get the exact sequence of nucleotides, which

compares with a database of known sequences of HLA alleles

Cross-match testing

● 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

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 don´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 TH2 reactoin, supression of TH1 reaction

• FasL expression• production of TGFb

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 (lysis of cells)

● 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 TH1 and TC cells against Ag of graft tissue● infiltration by lymphocytes, mononuclears, granulocytes around small vessels → destruction of tissue transplant

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)

● 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

Bone Marrow Transplantation

● Removal of hematopoietic stem cells

● Myeloablation

● Transplantation

● Engraftment

● Rejection

● Graft-versus-host reaction

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 (alooreactivity)

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

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

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)

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“ 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 protects against mother alloreactive T lymphocytes - don´t express classical MHC gp, expresses non-classical HLA-E and HLA-G● transfer of small doses of fetal antigens in maternal circulation causes tolerance ... suppressin of TH1 and preference of TH2

immune mechanisms in pregnancy

Rh incompatibility • Complications in pregnancy: production of anti-

RhD antibodies by RhD- mother carrying an RhD+ fetus (hemolytic disease of newborns)

• Fetal erythrocytes penetrate into the maternal bloodstream during pregnancy - a small amount, don´t immunize

• During childbirth or abortion (after 8 weeks of gestation) fetal erythrocytes can penetrate into the bloodstream of mother → immunization, formation of anti-RhD antibodies

• After childbirth, investigate Rh factor of born child, if is child Rh+, mother gets up to 72 hours after birth injection of anti-Rh antibodies (administered after abortion too)

Rh incompatibility

• Anti-Rh(D) injection, this 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 actively inhibit B lymphocytes

• 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

Classification by Coombs and GellImmunopathological 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

Immunopathological reactions based on antibodies IgG and IgM (reaction type II)

Cytotoxic antibodies IgG and IgM:● 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 RhD antigen

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

Immunopathological reactions based on immune complexes formation (reaction type III)

● caused by IgG 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● 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● 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

Thank you for your attention

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

Immunotherapy using antibodies

Antibodies functions - opsonization - activation of complement - induction of ADCC - carriers of drugs or toxins

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

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

4) 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

5) tumor vaccines- in vitro stimulation of TH1 cells and TC 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

8) Anti CTLA-4 antibody- Treg inhibition, longer activation of effector T cells