Transcript 42_43_Transplantation_immunology_LAx

TRANSPLANTATION
IMMUNOLOGY
ARPAD LANYI PhD
ORGAN, TISSUE OR CELL TRANSPLANT
AUTOLOGOUS
autograft
SYNGENEIC
syngraft
Skin, muscle, dendritic cell, cartilage
Bone marrow-derived haematopoietic cells
(HSC)
ALLOGENEIC
allograft
Kidney, cornea, liver, heart, lung
Bone marrow-derived haematopoietic
cells (HSC)
A FEW MILESTONES IN TRANSPLANTATION
First Human-To-Human Blood Transfusion
1818
James Blundell, a British obstetrician transfuses four ounces of blood from a man to his wife, replacing
the blood she just lost during childbirth--the first well-documented case of person-to-person blood
transfusion. Ten other women suffering from similar blood loss receive transfusions, which help half of
them.
Photo: Blundell's Gravitator Source: NIH/ Pennsylvania State University Libraries
A FEW MILESTONES IN TRANSPLANTATION
18th century: succesful transplant experiments is animals (skin grafts)
First succesful fresh skin allograft 1869 Jacques Louis Reverdin
First Successful Human-To-Human Bone Transplant 1878
This operation, which used bone from a cadaver, remained unusual because
there was no way to process and preserve human tissues.
Thyroid transplantation 1883 Theodor Kocher
Thyroid transplantation became the model for a whole new therapeutic
strategy: organ transplantation. After the example of the thyroid, other
organs were transplanted in the decades around 1900.
First Attempts At Bone Marrow Transplant 1896
First attempts to use bone marrow as treatment for leukemia; patients
receive the marrow orally after meals, but it has no effect. In the next few
years, intravenous injections of bone marrow to treat aplastic anemia have
some success.
Discovery of ABO blood groups 1902 Karl Landsteiner
First human xenotransplant 1906
A French surgeon inserts slices of rabbit kidney into a child suffering from
kidney failure. The immediate results are good, but the child dies after two
weeks. Four years later, a monkey kidney is transplanted into a young girl
suffering from mercury poisoning; it produces a small amount of urine, but
the girl lives for only five days.
A FEW MILESTONES IN TRANSPLANTATION
Discovery of MHC 1940’ George Snell
Human HLA: 1958 Jean Dausset
First successful human kidney transplant (syngraft) 1954
Joseph E. Murray
First Successful Human Heart Transplant 1967
"It's going to work!" Dr. Christiaan Barnard shouts as the heart of Denise Darvall,
a 23-year-old woman who died in an automobile accident, begins to beat in the
chest of 54-year old Louis Washkansky. The transplant was performed at Groote
Schuur Hospital in Cape Town, South Africa. The heart functioned until
Washkansky died of pneumonia eighteen days later because of his suppressed
immune system.
First Successful Bone Marrow Transplant 1973
Doctors at the Memorial Sloan-Kettering Cancer
Center in New York City perform the first successful
bone marrow transplant from an unrelated donor. They
transplant marrow from a person in Denmark into a
five-year-old with severe combined immunodeficiency
disease known as bubble boy syndrome. A few
successful previous bone marrow transplants had all
involved related donors.
PEOPLE IN THE US LIVING WITH FUNCTIONING ORGAN GRAFTS
TISSUE/SOLID ORGAN TRANSPLANTATION
The success of transplantation and maintenance of the donor tissue
depends majorly on histocompatibility
GRAFTS WITH MATCHING MHC ARE TOLERATED
WHILE ALLOGENEIC GRAFTS ARE REJECTED
BOTH IN MICE AND HUMANS
ALLORECOGNITION
DIRECT AND INDIRECT ALLOANTIGEN
RECOGNITION
MOLECULAR BASIS OF DIRECT
RECOGNITION OF
ALLOGENEIC MHC MOLECULES
T-cell responses to directly presented allogeneic MHC molecules are very
strong because there is a high frequency of T-cells (1-2%) that can
directly recognize any single allogeneic MHC. 100-1000X times greater
than that of during infection.
• Many different peptides derived from donor cellular proteins may
combine with a single allogeneic MHC molecule: a different clone of
recipient T- cells can be activated.
• Every APC expresses thousands of copies of different MHC molecules:
if these are foreign MHC molecules, many or all of them can be
recognized by alloreactive T-cells. (infection: 0.1-0.9%)
• Many of the T-cells that respond to an allogeneic MHC molecule, even
on first exposure, are memory T-cells (cross-reaction).
DIRECT AND INDIRECT ALLOANTIGEN
RECOGNITION
It is essentially the same as the
recognition of any foreign (e.g.,
microbial) protein antigen
ACTIVATION OF ALLOREACTIVE
T-CELLS
Antibodies
against
graft
antigens
(most
frequently HLA) also contribute to rejection.
Antibody production follows the same sequence of
events as any helper T-cell-dependent antibody
response (an example of indirect presentation of
alloantigens).
TRANSPLANT REJECTION
BLOOD TRANSFUSION IS THE MOST COMMON FORM
OF TISSUE TRANSPLANTATION
Incompatibility of blood group antigens causes type II hypersensitivity reactions
Incompatibility is tested by the cross-match test.
The patient’s serum is tested against the red cells of the potential donors. The
patient’s red cells are NOT tested against the serum of the donor!!! (no problem).
Organ transplantation requires compatibility of the main blood group antigens
HYPERACUTE TRANSPLANT REJECTION
ANTIBODY-MEDIATED TYPE II HYPERSENSITIVITY REACTION
• The most dramatic form of tissue incompatibility, begins within minutes to hours
• BLOOD GROUP ANTIGENS are expressed by endothelial cells of blood vessels
(solid vascularized organs)
• ANTIBODIES (IgM) – COMPLEMENT FIXATION – NEUTROPHIL, PLATELET
ACTIVATION – INTRAVASCULAR COAGULATION - IRREVERSIBLE ISCHEMIC
NECROSIS
• Cannot be reversed, should be avoided!!!!!
• Today, hyperacute rejection by anti-ABO antibodies is extremely rare BUT
• HLA I is expressed on vascular endothelium cells, preexisting antibodies (IgG)
against HLA class I variants can also cause hyperacute rejection!!
Where do anti-HLA antibodies come from?
THE SOURCES OF ANTI-HLA ANTIBODIES
Pregnancy – during childbirth
With successive pregnancies, increasing levels of anti-HLA
antibodies can develop
Blood transfusions (platelets, leukocytes) - HLA type is not
assessed, the matching being restricted to just the ABO and
Rhesus D types
Previous organ transplant
Cross-match!!!
CROSSMATCH TESTING
Complement-dependent cytotoxicity
• Lymphocytes from the donor are isolated and
separated into T- and B-cells.
• Serum from the recipient is mixed with the
lymphocytes in a multi-well plate.
• Complement is then added (usually derived
from rabbit serum).
• If donor-specific antibody is present and
binds to donor cells, the complement cascade will
be activated via the classical pathway resulting
in lysis of the lymphocytes.
DOI: 10.1111/j.1440-1797.2010.01414.x
CROSSMATCH TESTING
Flow cytometry
• More sensitive
• T- and B-cells can be separated to detect MHC
class I and MHC class II specific antibodies
• Anti – MHC I react with both B and T lymphocytes
• Anti – MHC II react with B lymphocytes only
DOI: 10.1111/j.1440-1797.2010.01414.x
CROSSMATCH TESTING
Panel reactive antibody (PRA)
Beads:
Bb
• The patient’s serum is tested for reactivity with
leukocyte antigens representing the population.
• The number of positive reactions is expressed as a
percentage PRA.
DOI: 10.1111/j.1440-1797.2010.01414.x
ACUTE TRANSPLANT REJECTION
T-CELL-MEDIATED TYPE IV HYPERSENSITIVITY
REACTION
Th1, Th17, CD8+ T-cells, macrophages
Antibodies, complement, neutrophils, platelets
takes days to develop
The rejected graft is swollen and
has deep-red areas of
hemorrhage and gray areas of
necrotic tissue.
ACUTE TRANSPLANT REJECTION
T-CELL-MEDIATED TYPE IV HYPERSENSITIVITY
REACTION
KIDNEY TRANSPLANTATION
HEART TRANSPLANTATION
T CELLS
Plasma cells
Lymphocytes and plasma cells
around renal tubules
T lymphocytes in the myocardium
Labeled with anti-CD3 antibody
Lymphocytes around an arteriole (A)
Lymphocytes surrounding the renal tubules (T)
Staining of T lymphocytes with anti-CD3
(brown staining) in the same section
CHRONIC TRANSPLANT REJECTION
TYPE III HYPERSENSITIVITY REACTION
Thickening of the vessel wall and narrowing of the lumina
Chronic rejection in a kidney
allograft with graft
arteriosclerosis
E: endothel
G: granulocyte
T: alloreactive T
M: macrophage
EL: elastic lamina
SMC: smooth muscle cell
DIFFERENCE IN GRAFT SURVIVAL OF RECIPIENTS
RECEIVING KIDNEYS FROM LIVE OR CADAVERIC
DONORS
10.5812/numonthly.12182
HLA MATCHING IMPROVES
THE SURVIVAL OF TRANSPLANTED ORGANS
(for example kidneys)
Blue
Orange
Red
Dark blue
Green
Black
Brown
HLA-DR, B and A are the most important to match.
HLA-typing is mainly based on DNA.
HLA TYPING
MIXED LYMPHOCYTE REACTION
The response of alloreactive T-cells to foreign MHC
molecules can be analyzed in vitro
HLA TYPING
DNA-BASED TYPING METHODS
PCR-SSOPH: Sequence-specific oligonucleotide probe hybridization
Specimen 1 (Type A*0203)
Specimen 2 (Type A*0501)
TAG C GAT
ATC G CTA
TAG A GAT
ATC TCTA
Amplify, denature, and
spot onto membranes
• Intermediate resolution
• Sreening test to identify potential donors or individuals
who may later require higher resolution testing
Specimen 1 Specimen 2
• High volume, relatively low cost
Probe with allele-specific probes
...TAGCGAT..(A*02)
Specimen 1
Specimen 2
...TAGAGAT…(A*05)
Specimen 1
Specimen 2
HLA TYPING
DNA-BASED TYPING METHODS
SSP-PCR: Sequence-specific PCR (allele-specific primers)
Reagent blank
SSP: Sequence-specific primer
SSP matches allele
SSP
SSP
Amplification
controls
Allele-specific
product
Primers recognizing different
alleles are supplied in a 96-well
plate format
Amplification
Amplification control
No
amplification
SSP does not match allele
Allele-specific product
Agarose gel
Very rapid test that can be performed in 3-4 hours from the time a sample is received.
PCR-SSP is used for typing deceased organ donors when speed is an important consideration.
HLA TYPING
DNA-BASED TYPING METHODS
SBT: Sequence-based typing
Polymorphic regions are amplified by PCR and then sequenced
Isolate DNA
The highest resolution HLA typing
Reverse PCR primer
PCR
Forward PCR primer
Exon 2
clean amplicons
Exon 3
Sequencing primers
sequence
amplicon
Sequences are compared to reference
sequences for previously assigned alleles
EVEN COMPLETE MATCHING AT THE MHC DOES NOT ENSURE
GRAFT SURVIVAL
MINOR HISTOCOMPATIBILTY ANTIGENS
Peptides derived from polymorphic cellular proteins bound to MHC class I molecules
The response to minor H antigens is in many ways analogous to that of viral infection
MINOR HISTOCOMPATIBILITY ANTIGENS
IMMUNOSUPPRESSION
DEPLETING ANTIBODIES
Antibodies that broadly react with white blood cells are given to
patients before and after transplantation to deplete these cells
and generally weaken the immune system.
• Rabbit antithymocyte globulin (rATG)
• Polyclonal mixture of high-affinity antibodies
• Binds to T-, B-, NK, dendritic and endothelial cells
• Fixes human complement well, delivers the cells to be killed
by phagocytes
• Serum sickness
• Alemtuzumab: anti-CD52
• CD52-specific humanized rat monoclonal IgG
• CD52 is expressed on almost all lymphocytes, monocytes
and macrophages
• Alemtuzumab induces a profound, long-lasting lymphopenia
CORTICOSTEROIDS
• Effective immunosuppressive drugs
• Used to treat patients before the transplantation and during episodes of
rejection
• A key immunosuppressive effect: inhibition of NFkB activity (central transcription
factor of the inflammatory response) by enhancing IKB
• Alters lymphocyte homing, lymphocytes are barred
from entering secondary lymphoid tissues
• Many adverse side-effects: fluid retention, weight
gain, diabetes, loss of bone mineral, thinning of the skin
• Prolonged use is avoided wherever possible
• Need novel, milder drugs
T-CELL ACTIVATION CAN BE TARGETED
BY IMMUNOSUPPRESSIVE DRUGS
Immunophilins: cyclosporin, tacrolymus
Calcineurin inhibitors
Side effects: Neurotoxicity, Nephrotoxicity, Diabetes
INFLUENCE OF CYCLOSPORINE ON GRAFT SURVIVAL
INHIBITION OF T-CELL CO-STIMULATION BY BELATACEPT,
A SOLUBLE FORM OF CTLA-4
Belatacept works as well as the best of the older, established drugs and is better than them in preserving
kidney function. On the downside, belatacept is associated with increased incidence of episodes of acute
rejection.
BLOCKING CYTOKINE SIGNALING
CAN PREVENT ALLOREACTIVE
T-CELL ACTIVATION
• Chimeric basiliximab, humanized daclizumab:
monoclonal IgG1 antibodies specific for CD25
• First given just before the transplant is
performed, subsequent infusions given during the
first 2 months when acute rejection is most likely
• A single dose of the antibody will saturate all the
CD25 molecules in the body within 24 hours, the
half-life of the antibody is 2 weeks, and its
suppressive effect can last for more than a month.
• Specific for activated T-cells, no severe T-cell
depletion and immunosuppression associated with
many other drugs
BLOCKING CYTOKINE SIGNALING CAN PREVENT ALLOREACTIVE
T-CELL ACTIVATION
Rapamycin (sirolimus)
• Immunosuppressive macrolide (Streptomyces
hygroscopicus)
• Binds to FK-binding proteins, but does not
interfere with calcineurin
• mTOR
inhibitor,
prevents
signal
transduction from the IL-2 receptor
• More toxic than either cyclosporin A or
tacrolimus but is a useful component of
combination therapy
• Does not impair the survival and functions
of regulatory T-cells as much, which may
promote immune suppression of allograft
rejection
• Dendritic cells, B-cells
doi:10.1016/S0167-7799(98)01239-6
CYTOTOXIC DRUGS TARGET THE REPLICATION AND
PROLIFERATION OF ALLOANTIGEN-ACTIVATED T-CELLS
Cytotoxic drugs are administered only after transplantation
Azathioprine: kidney transplantation
• Prodrug, first converted in vivo to 6-mercaptopurine and then to 6thioinosinic acid
• The latter inhibits the production of inosinic acid, an intermediate in
the biosynthesis of adenine and guanine nucleotides
• No selectivity, worst side-effect: collateral damage to those
tissues that are always engaged in cell division: bone marrow,
intestinal
epithelium,
hair
follicles:
anemia,
leukopenia,
thrombocytopenia, intestinal damage, loss of hair
Mycophenolate mofetil:
• Penicillium stoloniferum
• Similar effects to those of azathioprine
• Metabolized in the liver to mycophenolic acid
• Prevents cell division by inhibiting inosine monophosphate
dehydrogenase, an enzyme necessary for guanine synthesis
CYTOTOXIC DRUGS TARGET THE REPLICATION AND
PROLIFERATION OF ALLOANTIGEN-ACTIVATED T-CELLS
Cytotoxic drugs are administered only after transplantation
Cyclophosphamide:
• Originally developed as a chemical weapon (World War I.)
• A pro-drug that is metabolized to phosphoramide mustard, a compound
that alkylates and cross-links DNA molecules
• Specifically damages the bladder, sometimes causing cancer or
hemorrhagic cystitis.
• Unlike azathioprine, cyclophosphamide is not particularly toxic to
the liver, useful alternative for patients with liver damage
• Cyclophosphamide is most effective when used in short courses of
treatment
Methotrexate:
• Prevents DNA replication by inhibiting dihydrofolate reductase, an
enzyme essential for the cellular synthesis of thymidine.
SUMMARY OF IMMUNOSUPPRESSIVE DRUGS ACTING AT
DIFFERENT STAGES IN THE ACTIVATION OF ALLOREACTIVE TCELLS
METHODS TO INDUCE DONOR-SPECIFIC TOLERANCE
Hematopoietic chimerism
• Dizygotic twins who have had a common blood circulation during gestation
are tolerant of each other’s tissues.
• Combining solid organ transplantation with some mild form of
hematopoietic cell transplant from the same donor could induce a
more robust and stable tolerance and eliminate the necessity for
long-term immunosuppression.
• Promising results: renal/bone marrow allograft
• Family members differing by one HLA haplotype
• Non-myeloablative treatment: cyclophosphamide, cyclosporin, antiCD2 antibody, thymic irradiation
• Immunosuppressive therapy: 9–14 months
• Survival: up to 10 years
Transfer of regulatory T-cells
• Attempts to generate donor-specific regulatory T-cells in culture and to
transfer these into graft recipients are ongoing.
THE NEED FOR HLA MATCHING AND IMMUNOSUPPRESSIVE
THERAPY VARIES WITH THE ORGAN TRANSPLANTED
Anterior chamber-associated immune deviation (ACAID)
• Immunological environment that suppresses inflammation while maintaining
sufficient protection against pathogens.
• Cornea lacks vasculature
• Anterior chamber contains immunomodulatory factors (TGF-β)
• Tolerogenic DCs, Tregs, IL-4, TGF-β.
• Corneal transplants: 90% success in the absence of HLA matching or
immunosuppressive therapy
Liver
• Specialized architecture and vasculature
• Very low levels of HLA class I, no HLA class II
• Daily exposure to the digestion products of a myriad of foreign proteins from the
intestines with their characteristic anti-inflammatory environment
• Liver is relatively refractory to rejection
• HLA type or cross-match are not assessed before liver transplantation; ABO type assessed
• Cyclosporin and tacrolimus has markedly improved the success of liver transplants
At the other end of the spectrum from eye and liver is the bone marrow
HEMATOPOIETIC STEM CELL TRANSPLANTATION
HEMATOPOIETIC STEM CELL TRANSPLANTATION
• Myeloablative therapy: destroy the bone marrow: combination of
cytotoxic drugs and irradiation
• Engraftment: pluripotent stem cells colonize the bones
• When the immune system has been fully reconstituted (can take a
year or more), the patient is a chimera (has a genetically different
immune system)
•
•
•
•
•
HSCs are now obtainable from the blood
G-CSF, GM-CSF: stem cells are mobilized from the bone marrow
Leukapheresis
Isolation: CD34
Between a quarter and half a billion CD34-positive cells are needed
to ensure prompt engraftment
MORE SHARED HLA ALLOTYPES,
MORE ROBUST T-CELL RESPONSE
THE SUCCESS OF HSCT CORRELATES WITH THE EXTENT
OF THE HLA MATCH
GRAFT-VERSUS-HOST DISEASE: ALLOREACTIVE DONOR TCELLS IN THE GRAFT ATTACK THE RECIPIENT’S TISSUES
• The conditioning therapy which destroys bone
marrow cells, also damages other tissues : skin,
intestinal epithelium, hepatocytes
• Cytokine storm, dendritic cell activation
• Mature alloreactive T-cells from the transplant
interact with the recipient’s dendritic cells
• Methotrexate in combination with cyclosporin A
• Markedly reduced GVHD: higher incidence of
graft failure
Minor histocompatibility
antigens trigger
alloreactive T-cells in
recipients of HLAidentical
transplants
UMBILICAL CORD BLOOD AS THE SOURCE OF
HEMATOPOIETIC STEM CELLS
• Umbilical cord blood has the combined benefits of being rich in
stem cells and having fewer alloreactive T-cells
• There is less GVHD, and a greater HLA disparity can be
tolerated.
• Engraftment is slower
• Limited volume: combined stem cells from two unrelated samples
of cord blood
• The patient’s reconstituting hematopoietic system becomes a
chimera but in time the cells from one donor overwhelm the cells
of the second
• First use: 1988; child with Fanconi’s anemia
• More than 25,000 cord blood transplants have been performed
GENETIC DISEASES FOR
WHICH HSCT IS A THERAPY
HSCT is a therapy for many
genetically determined
immunodeficiencies, such as
SCID
HSCT IN TUMOR THERAPY
Autologous HSCT
• A sample of the patient’s bone marrow is taken before the
remainder is ablated
• The hematopoietic stem cells in are purified away from any
tumor cells and are then given back to the patient
• Perfectly histocompatibility: no GVHD, no immunosuppressive
drugs
• Limitation: the rate of relapse is significantly higher for
autologous transplants than for allogeneic transplants
GRAFT-VERSUS-LEUKEMIA (GVL) EFFECT
Alloreactive T-cells in the graft help rid the patient of residual leukemia cells
• Promotion of GVL reaction, less emphasis on tumor elimination by
chemotherapy and irradiation
• Less severe conditioning regimens: hematopoietic system is damaged
but not destroyed, quicker recovery
• One approach: transfusions of donor T- cells at a time after
tranplantation when the inflammation caused by the conditioning
regimen has subsided and the likelihood of GVHD is diminished
NK CELLS ALSO MEDIATE GRAFT-VERSUS-LEUKEMIA EFFECTS
Haploidentical transplant
• GVHD prevention: T-cell depletion and infused antiT-cell antibodies
• No further immunosuppressive
transplantation
treatment
after
• Reconstitution of NK cells occurs more rapidly than
that of T-cells, alloreactive NK cells can emerge
• The occurrence and specificity of the NK-cellmediated alloreactions are determined by the
interactions of inhibitory KIR with HLA-B and HLA-C
ligands and are predictable from the HLA types of
the donor and recipient.
• NK-cell alloreactions occur when the recipient’s HLA
class I allotypes provide ligands for fewer types of
inhibitory KIR than the donor’s HLA class I allotypes.
• AML+, ALL• Alloreacive NK response wanes and is undetectable 4
months after transplantation.
• With full reconstitution of the immune system, NKcells become tolerant of both recipient and donor
cells.
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