Immunogenetics and transplantation. Major Histocompatibility Complex.

Rejection

Immunogenetics a part of genetics which studies the complexity of immune

processes, concerning defense and integrity of the host.
MHC encodes a group of highly diverse cell surface proteins. There are two types of
MHC molecules: class I and class II molecules.
Polymorphism each MHC locus can express any one of hundreds of different
molecules.
Its genes are codominantly expressed.
TRANSPLANT REJECTION
Transplant rejection is graft failure resulting from recipient antibodies and cells
directed against donor cells.
Transplantation, for the purpose of replacing a diseased organ with a healthy donor
organ, represents an increasingly active field in modern medical practice, as our
understanding of the immunological aspects of transplantation continues to grow.
In transplantation, we have to deal with adverse side effects of an immune system
that has evolved to recognize and protect us from nonself, harmful pathogens. In
transplantation we have the responsibility to make the nonself (the allograft) to be
accepted and function normally within the recipient.

LAWS OF TRANSPLANTATION
The laws of transplantation are as follows:
Transplants are accepted between members of a highly inbred genetic strain
(haplotype) or animals that are genetically identical (identical twins).
Transplants are rejected between members of different haplotype or animals that are
genetically nonidentical.
Transplants are accepted from parental haplotype A or B to an F, (AXB) progeny, but
transplants in the reverse direction are rejected.

HISTOCOMPATIBILITY ANTIGENS
Regarding the issue of genetic similarity between transplant donor and recipient
(histocompatibility), early observations on transplant outcome led to the identification
of a set of genes whose codominant expression elicits vigorous rejection responses in
the case of allogeneic transplants. That set of genes has come to be characterized as
the major histocompatibility complex or MHC.
It is important to realize that many target antigens exist in cases of graft rejection. The
MHC represents the most critical set of genes encoding such cell surface antigens;
however, several genetic loci or areas of the MHC have yet to be mapped and defined.
Furthermore, another set of genes, encoding the minor histocompatibility antigens,
may play a very important role in transplant outcome as well. These genes have been
less well characterized compared with those encoded by the MHC and generally are
believed to play a weaker role in graft rejection events.
In order for a graft to be accepted, the recipient must share an identical complement of
donor genes. For example, donor strain A or B into a strain AXB recipient would result
in graft acceptance, since both strain A and B genes would be shared by the AXB
recipient. In the case of donor tissue from strain AXB transplanted into either strain A
or B recipient, the outcome would be graft rejection, since recipient A has donor A
genes but not B genes and recipient B has donor B but not A genes.

HLA Genetics
HLA alleles set found on one chromosome represents a haplotype.
Each individual inherits two MHC haplotypes from each parent and thus he has two
alleles for each gene.
These alleles are codominantly expressed.
The inheritence of MHC genes follows the Mendel segregation rules.

Host immune response against allograft

Direct antigen presentation
The response of recipient T cells to intact MHC/peptide complexes on APCs from a
graft is called direct allorecognition.
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 That is APCs in the graft directly present alloantigens (the foreign MHC molecules)
for recognition by alloreactive T cells.
A recipients T cells can also react to donor MHC peptides presented on the recipients
own APCs. This pathway is called indirect allorecognition.

MEDIATORS OF
REJECTION
Several components of the
immune system are known
to mediate graft rejection.
First, we will consider
antibody-mediated graft cell
destruction, where
antibodies specific for graft
cell MHC antigens may be
elicited with CD4+ T cell
help generated in response
to foreign graft MHC class
II molecules.
In the presence of
complement, these antigraft
antibodies would be capable of lysing (killing) graft target cells.
In the case of cell-mediated graft destruction, at least two scenarios may be
considered.
In the first, foreign graft MHC class II molecules stimulate host T-helper cells to
provide "help" to host CD8+ T cytotoxic cells, which then may exert lytic action
directly via recognition of foreign graft MHC class I molecules. Alternatively,
stimulated host T helper cells may aid macrophages in an MHC-independent fashion
to produce molecules capable of destroying graft cells.
Graft rejection has come to be regarded as being largely cell-mediated, with the T
lymphocyte the primary effector cell. This fact is not surprising if one considers again
and observes the central role occupied by the T cell.
In addition to the cytotoxic T cell, both antibody-mediated and macrophage mediated
graft destruction rely upon T cell help. Antibody-mediated graft destruction, a
feature of hyperacute rejection events, also plays an important role in a special
situation termed second set rejection.
In this case, a transplant recipient is retransplanted due to primary graft failure, and
upon encounter with the graft's foreign antigens or alloantigens to which the host was
previously exposed, the host's immune system generates a hyperacute rejection
response. This response consists of preformed cytotoxic antibodies formed during
the host's first encounter with graft antigens.

INFLUENCES ON TRANSPLANT SUCCESS

Several factors affect the outcome of tissue transplantation, notably the degree of
histocompatibility between donor and recipient.
Fortunately, the availability of reagents specific for cell surface proteins encoded by
the MHC has permitted a high degree of cross-matching of donor and recipient where
possible prior to transplantation.
Tissue typing/cross-matching is the identification of the MHC type of transplant
donor and recipient to optimize genetic similarity or match prior to transplant.
This method and consequently transplant outcome will continue to improve as more
MHC loci are characterized and reagents recognizing the products of these gene loci
are prepared. Equally important in cross-matching transplant donor and recipient is the
knowledge of prior sensitization or foreign antigen encounter by a prospective
transplant recipient. Events such as blood transfusion and pregnancy prior to
transplant, as well as previous transplants, all may affect transplant success and result
in responses similar to hyperacute or second set rejection, a rapid and vigorous
reaction.
Of critical importance to transplant success and viability is the degree of tissue or
organ preservation prior to grafting. Ischemia time, defined as the time during which
the donor organ has been deprived of its proper blood supply, drastically affects
transplant outcome and should be kept to a minimum. Ischemia time is particularly
important for the proper functioning of kidney, heart, and liver transplants, where
organ preservation is an issue. Indeed, considerations of organ preservation in the case
of kidney transplants are compounded by the availability of cadaveric kidney donors,
where ischemia time and longer term organ preservation affect transplant viability.
Interestingly, there exist a few sites in the body (central nervous system, reproductive
tract) that are considered relatively "immune privileged' in terms of their vulnerability
to an immune response. These sites generally lack lymphatic drainage and express few
MHC antigens and are therefore weakly immunogenic.

Clinical signification
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 Successful transplantation relies upon the immunologic compatibility of recipients and

their organ donor.
This compatibility depends on both, the extent of matching of the tissue types (HLA
types) of donor and recipient and the absence of any pre-existing antibody reactivity of
the recipient with the donor.
Both these factors influence the degree of immunosuppression required to prevent
rejection of the graft by the recipient.
The key element to successful transplantation is the ability to correctly identify the
tissue types of recipients and donors and to predict whether a graft is likely to be
rejected ( host versus graft disease) or in the case of a BMT whether the graft will
attack the recipient ( graft versus host disease GvHD)

HLA typing: Bone Marrow

transplantation
Liver
transplantation
Renal transplantation
Pancreatic transplantation
Heart transplantation
National Bone Marrow Donor Registry
Waiting list for - Bone Marrow transplantation
- Liver and Renal transplantation
- Heart transplantation

Transplantat immunology in renal transplantation

Blood group matching

Both donor and recipient have to
matched for ABO

IMMUNOGENETICS
The purpose of tissue typing is to
identify the expression of MHC on cells.
More than one method may be required
to give a complete picture.
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)
Anti-HLA antibody detection and identification
- AHG CDC
- ELISA
Cross- match
- CDC
- ELISA
- LUMINEX
Assay Report
Sample ID: 455FM59
Patient Name: F.M. Kidney donor(mother) for recipient F.I.
Entered on: 1/22/2002
Account: admin

LiPA HLA-A/v.1.4/001102

AssayResult
ALLELE GROUP TYPING:
A*02
A*24

Assay Report
Sample ID: 456FI38
Patient Name: F.I. Kidney recipient
Entered on: 1/22/2002
Account: admin

LiPA HLA-A/v.1.4/001102

AssayResult
ALLELE GROUP TYPING:
A*02
A*24
Assay Report
Sample ID: 455FM59
Patient Name: F.M.
Entered on: 1/24/2002
Account: admin

LiPA HLA-B/v.1.4/001102

AssayResult

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ALLELE GROUP TYPING:

B*18
B*35

HLA SBT
Resolve heterozygous
sequence ambiguities
- Separate alleles by SSPPCR
- Sequence hemizygous PCR product
- Resolve ambiguity
- High throughput
- Uniform Protocols
- Pre-formulated reagents
- All Sequencing platforms
HLA Antibody Detection
HLA antiserum screening is an important work effort in clinical HLA laboratories.
The result is used to determine the degree of humoral alloimmunization,expressed as
percent panel reactive antibody (%PRA).
The antibody specificity can accurately predict donor incompatibility and the
development of chronic allograft rejection.
Methods: AHG CDC
ELISA screening Class I and Class II
- identification Class I and Class II
Luminex
Class I HLA Antibody Analysis
GTI QuikScreen
HLA Class I Ab Screen
Pooled platelets (minimum of 300 donors)
Highly specific (no Class II interference)
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 Flexible formats, easy to use

Screen up to 40 samples per tray in 2.5 hrs
WinScreen software
GTI Quik-ID Class I
HLA Class I antibody specificity
Percent Panel Reactive (%PRA)
Panel of 40 donors
Solubilized Class I antigen from platelets
Sensitive capture assay
Software analysis package including CREG analysis
Class II HLA Antibody Analysis
GTI B-Screen
HLA Class II Ab Screen
Soluble HLA from EBV Transformed cells
Affinity purified
Flexible format - strip wells
Highly specific (no Class I interference)
Screen 40+ samples per tray in 2.5 hrs
WinScreen software
GTI Quik-ID Class II
HLA Class II Ab specificity
Percent panel reactive (%PRA)
Panel of 30 cell lines
Affinity purified Class II HLA from EBV transformed cell lines
Sensitive capture assay
Software analysis package
Antibody Screening Algorithm
New patients full work-up
Flow specificity and PRA
ELISA specificity and PRA
Current patients Negative or Positive

Negatives screened monthly or quarterly

Any neg-pos refluxed to Ab ID

Positives screened monthly by ELISA

Specificity and PRA tracked

Ambiguous specificity refluxed to Flow

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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
2nd step: ELISA
takes about 3 to 4 hrs depending on number of donors
No interference with therapeutic rescue immunosuppresents
Distinguish between Donor and non-Donor HLA Abs
No interference with IvIG/pheresis protocols
Up to 44 patient sera per plate with one donor
Up to 6 donors with 4 recipients each per plate
Flexibility versatile snap-in strips
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Convenience screening on a single tray for a variety of antibodies

Format fits most commercially available microplate readers

Kidney pancreas
transplantation
whole organ
Pancreas
transplantation
alone (PTA)
Simultaneous
pancreas-kidney
(SPK)
transplantation
Pancreas after
kidney (PAK) transplantation
Immunological algorithm:
HLA typing: A, B, DRB1
Cytotoxic antibodies
Crossmatch
Transplantation of pancreatic
islets
Langerhans cells are targeted
Virological assessment of both , donor and recipient
HLA typing: A, B, DRB1
Cytotoxic antibodies
crossmatch
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transplantation

Kidney transplantation in children

Usualy the donor is one of the parents
AB0 matched
Tissue typing: A, B, DRB1
Cytotoxic antibodies
crossmatch

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