Imunitate de transplant

Transplant
• Definitii
– Autogrefa: transfer de tesuturi de la – la
acelasi organism (piele)
– Singenic (isogrefa): transplant intre gemeni
identici (univitelini)
– Allogrefa: transplant intre indivizi diferiti ai
aceleiasi specii
– Xenogrefa: transplant de la o specie la alta
 Compatibilitate de Transplant
• Pentru a creste sansa de supravietuire a
transplantului:
– Cel mai important: compatibilitate ABO
– Absenta Ac citotoxici preformati impotriva
Ag HLA ale donatorului
– Compatibilitate HLA, in particular pentru
locii D
 Disorders of MHC
Major Histocompatibility Complex
• Transplantation workups
– Transplant recipient must be blood group antigen (ABO)
compatible with the donor and not have any preformed
anti-HLA (cytotoxic) antibodies in the blood
• Compatibility in both areas prevents hyperacute
transplantation reactions
• Normally, these antibodies should not be present unless the
recipient has had a blood transfusion in the past or has been
pregnant and had a fetal-maternal bleed during delivery
and been exposed to paternal antigens on the fetal cells
 VIROLOGICAL ASSESSMENT

Both donor and recipient are tested for: VHB, VHD, VHC,
HIV 1/2, CMV, EBV, HSV 1 si 2, VZV, HTLV 1/2 ,
rubella virus, toxoplasma gondii and chlamydia.
 

 Methods
Indirect diagnostic tests (serological)
Direct diagnostic tests, molecular biology tests (PCR, RT-
PCR).
 HLA

• Determines the fate of transplantation.

• Plays a role in the control of cellular

interactions resposible for both cellular and
humoral immune responses.

• Is associated with a variety of diseases.

HLA
 IMUNOGENETICA 
 
 1. Cross- match
- CDC
- ELISA

2. HLA Typing by molecular biology methods – PCR

 
SSOP- sequence-specific oligonucleotide probe hybridization
(medium resolution )
  SSP – sequence-specific primers (high resolution)
SBT – allele SEQR (the highest available resolution) 

3. Anti-HLA antibody detection and identification

- AHG CDC
- ELISA 
 Disorders of MHC
Major Histocompatibility Complex
• Lymphocyte crossmatch
– Used to screen recipient serum for anti-HLA
antibodies
• Recipient’s serum, complement and donor B lymphocytes
are mixed together in a test tube. Lysis of donor
lymphocytes is indicative of cytotoxic antibodies in the
recipient’s serum directed against donor lymphocytes
• The identity of these antibodies must then be determined
in order to find a suitable donor who is negative for the
corresponding HLA antigen(s).
Sample of cells or tissue

Combine DNA with sequence-

specific primer fix for each
allele

Amplify by
PCR
DNA
80ng for Class I
40 ng for Class II
 Importance of DNA Quality

100 ng Genomic DNA 1% Agarose Gel

 SBT for ALL HLA Typing Requirements:
Resolving Heterozygous Ambiguities HR Typing

R C TG G T C AT R
A C T G G T C A T A Allele 1
G C T G G T C A T G Allele 2
R C T G G T C A T R Allele 1+2

G C T G G T C A T A Allele 3
A C T G G T C A T G Allele 4
R C T G G T C A T R Allele 3+4

Allele 1+2 = 3+4

How are heterozygous ambiguities identified by Assign-SBT ?

The sequence of the test sample

(R040901046[SDRB1) is identical
(MM=0) to the sequences
of:
DRB1*030101+150101
DRB1*0319+1505
Assign-SBT Resolves Ambiguities
Sequences are arranged in “layers”
…Master sequence
Patient result
 Types of Transplants
• Corneal
– Best graft survival rate since the cornea is
avascular and the lymphatic drainage from the
eye is not as well developed as in other tissues
– Associated with transmission of prions-
Creukfeld-Jacob disease-Transmissible
spongiform encephalopathy; also has been
associated with amoebic transmission
(granulomatous amebic encephalitis)
 Types of Transplants
• Renal
– Between living donors with a 2 haplotype match = 90-
95% 5 year survival
– With a 1 haplotype match = 80% 5 year survival
– Cadaver transplants between unrelated donors is the
most common type of transplantation. Similar statistics
to 1 haplotype match when the recipient receives
multiple blood transfusions prior to the surgery
(induces tolerance to the allograft) and is placed on
immunosuppressive therapy
 Types of Transplants
• Liver
– In adults with chronic active hepatitis or
cirrhosis
– In children with biliary atresia
– 1 year survival rate is slightly greater than 90%
 Types of Transplants
• Cardiac transplantation
– In adults, used in patients with chronic ischemic
heart disease and congestive cardiomyopathy
– In children, endocardial fibroelastosis is the usual
indication
– Endomyocardial biopsies are the best means of
diagnosing allograft rejection
– Approximately 80% of transplants survive 1 year
 Types of Transplants
• Bone marrow transplants
– Used in the treatment of aplastic anemia, leukemia and
immunodeficiencies
– Goal is to infuse donor marrow containing
pluripotential hematopoietic stem cells that will
eventually repopulate the lymphoid, erythroid, myeloid,
and megakaryocytic series in the recipient.
– GVH occurs in almost 2/3rds of cases
– Increased incidence of CMV pneumonitis
 Transplant Rejection

• The chance of a sibling in a family having another sibling with

0, 1, or 2 haplotype match is:
– 25% - 0 haplotype match
– 25% - 2 haplotype match
– 50% - 1 haplotype match
– However, a 2 haplotype match is rarely achieved due to
crossovers between the individual loci during meiosis when
homologous chromosomes line up close to each other
 Transplant Rejection
• Three types of transplant rejections
– Hyperacute rejection
– Acute rejection
– Chronic rejection
 Transplant Rejection
• Hyperacute rejection:
– occurs within minutes of attaching the allograft to the
recipient’s blood supply
– Due to the presence of an ABO mismatch or preformed
cytotoxic antibodies in the host against foreign HLA antigens
in the donor tissue (example; a blood group A recipient would
have anti-B IgM antibodies and would react against a group B
donor heart)
• Hyperacute rejection is rare because ABO and anti-HLA
cytotoxic antibody screening is performed prior to the
surgery
 Transplant Rejection

• Acute rejection
– Most common type of rejection encountered
– Usually occurs within the first 3 months of the transplantation
– Involves cell-mediated and antibody-mediated reactions. Cell-
mediated has the greatest role in rejection
– The type II antibody-mediated hypersensitivity produces a
necrotizing vasculitis with subsequent vessel damage and
intravascular thrombosis
 Transplant Rejection
• Acute rejection
– Vessel events can occur over a period of time
leading to fibrosis and vessel lumen obliteration
– The cell-mediated component involves cytotoxic T
cells producing extensive interstitial infiltrate in
the graft with edema and damage to the tissue
(Type IV hypersensitivity)
– Can be reversible with immunosuppressive drugs
such as cyclosporin A, corticosteroids, and OKT3.
 Transplant Rejection

• Chronic rejection
– Irreversible
– Occur over a period of months to years
– Extensive fibrosis and loss of organ structure characterize
the histologic findings in the transplant
– Activated macrophages release growth factors that
stimulate fibroblasts to deposit collagen
– There is also chronic ischemia secondary to antibody-
mediated damage to the vessels
 Type IV Hypersensitivity
– Cytotoxic T cells interact with class I antigens on nucleated cells
– If the antigens are altered (virally infected cells, neoplastic cells) or the cell is
foreign to the host (transplant), the cytotoxic T cells will attach to the cell
membrane, release perforins and destroy the cell.
• Examples: Acute and chronic transplant rejections; destruction of
hepatocytes infected by hepatitis B virus
 Transplant Rejection
• Cyclosporin A inhibits CD4 helper T cell release of
interleukin-2 (blocks calcineurin) which stimulates
the proliferation of cytotoxic and helper T cells
• Corticosteroids inhibit macrophage production of
interleukin-1 and tumor necrosis factor and are
cytotoxic to immature cortical derived thymocytes
• OKT3 is a monoclonal antibody preparation that
attaches to the CD3 antigen receptor of T cells,
blocking their reaction with the graft
ID/CC A 45 year old male with refractory acute myeloid
leukemia is brought to the emergency room with
fever, a generalized rash, jaundice, right upper
quadrant pain, severe diarrhea, and dyspnea; two
months ago, he underwent an apparently
uncomplicated bone marrow transplantation.
HPI Prior to the transplant, he received radiotherapy and
chemotherapy as well as broad-spectrum antibiotics
PE VS: normal blood pressure. PE: cachexia;
moderate dehydration; 2+ jaundice; violaceous and
erythematous macules as well as papules and bullae
with scale formation over extremities
Labs Elevated IgE level. CBC/PBS: falling blood counts;
relative eosinophilia. Elevated direct serum bilirubin
and transaminases, no infectious agents on stool
exam
 Graft versus Host Reactions
• Potential complication in bone marrow and liver
transplants and in blood transfusions administered
to patients with T cell immunodeficiency
• Donor lymphocytes produce interleukin-2
• -->activation of NK cells (primary effector cells in
acute GVH reactions)-->lymphokine-activated NK
cells are called LAKs and produce extensive
epithelial cell necrosis in the biliary tract (jaundice),
skin (maculopapular rash), and GI tract (diarrhea)
 Graft versus Host Reactions
• May progress into chronic GVH which is
marked by the presence of extensive
fibrosis
• To lessen the risk of GVH, donor tissue is
pretreated with anti-thymocyte globulin to
remove donor T cells.
• Cyclosporin A is used also
 Transplant complications
• Immunosuppressive therapy has increased the
incidence of:
– Cervical cancer
– Malignant lymphomas (immunoblastic)
– Basal and squamous cell carcinomas of the skin
• Squamous cell CA is the most common overall
malignancy
• Other complications include infection and bone
marrow suppression
 Antibody Monitoring System

ELISA assay designed to detect

donor reactive IgG antibodies in
recipient sera
 Used for Immunological
monitoring of donor-specific
HLA alloantibodies in
transplant patients that may
lead to early graft loss or
chronic rejection
• Retrospective Crossmatch
• Prospective Crossmatch
• Post-transplant
Immunological Monitoring
• Detects only HLA donor
specific antibodies
• IgG specific - will not detect
IgM (autolymphocytotoxic)
antibodies
• Detects non-complement
binding antibodies
• Detects Class II specific
HLA antibodies in presence
of strong Class I antibody
1st step: lysate preparation

takes about 15 minutes after

isolation of cells
LYSATE PREPARATION
LYSATE PREPARATION
LYSATE PREPARATION
LYSATE PREPARATION
LYSATE PREPARATION
LYSATE PREPARATION
 2nd step: ELISA

takes about 3 to 4 hrs -

depending on number of donors
 Class I Class II

Negative
Control

Lysate
Control
Recipient
Positive Samples
Control
 Disorders of MHC
Major Histocompatibility Complex
• Mixed Lymphocyte Reaction (MLR)
– Utilized to test for class II antigen (D loci) match
between the recipient and donor
– Functional lymphocytes from the recipient and
previously irradiated (killed) donor lymphocytes
are mixed together in a test tube with tritiated
thymidine.
– A baseline radioactive count is obtained before
the donor lymphocytes are added to the tube.
 Disorders of MHC
Major Histocompatibility Complex
• Mixed Lymphocyte reaction (MLR)
– If the recipient’s lymphocytes have different D
antigens than those located on the donor
lymphocytes, they become activated which increases
the radioactive count in the test tube over the
baseline reading
– This reaction evaluates the potential for recipient
rejection of the donor graft, but does not provide
information on whether the graft will reject the host
 Disorders of MHC
Major Histocompatibility Complex
• Mixed lymphocyte Reaction (MLR)
– A modified test to evaluate the risk of a GVH
reaction is to irradiate (kill) the recipient’s
lymphocytes and to allow the functional donor
lymphocytes an opportunity to react against the
host’s HLA D loci