Download Anti-tumor immunity

Antitumour immunity
J. Ochotná
Hypothesis of immune control
• tumor cells normally arise in tissues and are eliminated
by T lymphocytes
Malignant transformation
• Failure of cell division regulation and regulation
of cells "social" behavior
• The uncontrollable proliferation, dissemination
to other tissues
• Mutations in protoonkogenes and antionkogenes
Tumour antigens
Tumour specific antigens (TSA)
a) complexes of MHCgp I with abnormal fragments of cellular proteins
chemically induced tumors
leukemia with chromosomal translocation
b) complexes of MHC gp with fragments of oncogenic viruses proteins
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
Tumour associated antigens (TAA)
• expressed also on normal cells
• differences in quantity, time and local of expression
• auxiliary diagnostic markers
a) onkofetal antigens
• on normal embryonic cells and some tumor cells
• -fetoprotein (AFP) - hepatom
• canceroembryonal antigen (CEA) - colon cancer
b) melanoma antigens
• MAGE-1, Melan-A
Tumour associated antigens (TAA)
c) antigen HER2/neu
• receptor for epithelial growth factor
• mammary carcinoma
d) EPCAM
• epithelial cell adhesion molecule
• metastases
e) differentiation antigens of leukemic cells
• present on normal cells of leukocytes linage
• CALLA -acute lymphoblastic leukemia (CD10, pre-B cells)
Defensive immune response
• DC are necessary for activation of antigen specific mechanisms
• cancer-associated antigens are processed by DC and
presented to T lymphocytes in complexes with HLA I and II
• predominance of TH1 (IFN  TNF); IFN support DC
maturation
• tumor cells are eliminated by TC
Defensive immune response
•
tumor cells are also destroyed by NK cells (low HLA I
expression on tumor cells)
•
interferons - antiproliferative, cytotoxic effect on tumor
cells
- INF - DC maturation
• TH2 activation → support B lymphocytes→ tumor specific
antibodies (complement activation, opsonisation, ADCC)
Mechanisms of tumor resistance
 high variability of tumor cells
 low expression of tumor antigens
 sialylation
 tumor cells do not provide costimulatory signals →
T lymphocyte anergy
Mechanisms of tumor resistance
 some antitumor substances have a stimulating effect on tumor
 production of factors inactivating T lymphocytes
 expression of FasL → T lymphocyte apoptosis
 inhibition of the dendritic cell functions and durability
(NO, IL-10, TGF-
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 other species
Allogeneic transplantation
• differences in donor-recipient MHC gp and secondary
histocompatibility Ag
• alloreactivity of T lymphocytes - the risk of rejection and graftversus-host
• direct recognition of alloantigens – recipient T lymphocytes
recognize
the different MHC gp and non-MHC molecules on donor APC
• indirect recognition of alloantigens - APC absorb different
MHC gp from
donor cells and present the fragments to T lymphocytes
Recognition of alloantigens
Tests prior to transplantation
ABO Compatibility -risk of hyperacute or accelerated rejection =
formation of Ab against A or B Ag on graft vascular endothelium)
HLA typing (determination 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 reaction - test for 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
 HLA typing
 Serotyping
 Genotyping (PCR SSP, PCR SSO, PSR SBT)
HLA typing
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 stain 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 typing
Serotyping (phenotyping)
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
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
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 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
Mixed lymphocyte reaction (MRL)
• determination of T lymphocyte alloreactivity
• mixed donor and recipient lymphocytes → T lymphocytes
after recognition allogeneic MHC gp activate and proliferate
• the total proliferation of lymphocytes is measured by adding
[3H]-thymidine to the culture medium and monitoring its
integration to DNA of new cells
One-way MRL
•determination of recipient T lymphocytes reactivity against
donor cells
•donor cells treated with chemotherapy or irradiated lose
the ability of proliferation
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
Hyperacute rejection
• minutes to hours after transplantation
• antibodies 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
Hyperacute rejection
Accelerated rejection
Accelerated rejection
• 3 to 5 days after transplantation
• caused by antibodies that don‘t activate complement
• cytotoxic and inflammatory responses activated
by antibodies which bind 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:
• recipient TH1 and TC cells response against Ag of graft tissue
• infiltration of lymphocytes, mononuclears, granulocytes
around small vessels → destruction of transplant tissue
Chronic rejection
• occures after 2 months from 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 fibrous tissue,
endothelial damage →impaired perfusion of graft → gradual
loss of its function
dominating findings: vascular damage
Bone marrow transplantation
• hematopoetic stem cell collection
• myeloablation
• transplantation
• engraftment
• rejection
• graft versus host disease
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 stem cells transplantation
•
damage of liver, skin and intestinal mucosa
•
Prevention: appropriate donor selection, T lymphocytes
removal from the graft and effective immunosuppression
Chronic GVHD
• months to years after transplantation
• TH2 lymphocytes infiltration of tissues and organs, production
of alloantibodies and production of 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
• mechanism is consistent with acute GVHD
• associated with a certain degree of GVHD (adverse
reactions)
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 IgG and IgM antibodies
Type III reaction - response based on the formation of immune
complexes
Type IV reaction - cell-mediated response
Immunopathological reaction based
on IgG and IgM antibodies (reaction type II)
Cytotoxic antibodies IgG and IgM:
• complement activation
• binding to phagocytes and NK cells (ADCC) 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
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
Immunopathological reactions based on immune
complex formation (reaction type III)
•
caused mainly by IgG antibodies → bind to antigen → creation
of immunecomplexes
•
immunocomplexes - bind to Fc receptors on phagocyte
- 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 cells surface (vasculitis) and in synovial joints
(arthritis)
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
Immunopathological delayed-type reaction
(reaction type IV)
• delayed-type hypersensitivity (DTH)
• local reaction caused by TH1 cells and monocytes / macrophages
(physiologically – elimination of macrophage intracellular parasites)
• antigen immunization → formation of antigen specific TH1 cells
(and memory cells)
• 12-48 hours after next contact with antigen arise local reaction –
granuloma (TH1 and macrophage infiltration)
Tuberculin reaction
Tissue damage in tuberculosis and leprosy
Sarcoidosis
Subtype IV - Cellular cytotoxic response
(Tc activation)
•similar to DTH reaction
•TH1 cells activate CD8 + T lymphocytes
viral rashes
viral hepatitis
acute rejection of transplanted organ
some autoimmune thyroiditis
contact dermatitis
Contact dermatitis
•is a localized rash or irritation of the skin caused
by contact with alergen (nickel , chromium,
ingredients in cosmetic products , plant allergens and other)
•the first is senzitization
•appears in 24 – 48 hours after second contact with alergen
•diagnosis : patch test
Patch test
•patch test is a method used
to determine
if a specific substance causes allergic
inflamation of the skin
• Allergens are applied to special
hypoallergenic patch on the back skin
•Results are evaluated after
48 and 72 hours
•In positive reaction appears eczema
*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
*
the dendritic cell immunotherapy
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
* Dendritic cell vaccine
* dehttps://www.youtube.com/watch?v=Aaho7YV_r10
* Imunotherapy of cancer
* https://www.youtube.com/watch?v=-NNjDjXSJt0
* Immunopathologycal reaction type IV
* https://www.youtube.com/watch?v=e1X_7jqxEzA