 Immunogenetics and transplantation.

Major Histocompatibility Complex.

Rejection

 Prof. Ileana Constantinescu

 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.

 The structure of HLA class I molecules

 The structure of HLA class II molecules

 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.

 That is APCs in the graft directly present alloantigens (the foreign MHC molecules) for recognition by
alloreactive T cells.

 A recipient’s T cells can also react to donor MHC peptides presented on the recipient’s 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
 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

Prof. Ileana Constantinescu

 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

ALLELE GROUP TYPING:

B*18

B*35

 Assay Report

Sample ID: 456FI38

Patient Name: F.I.

Entered on: 1/24/2002

Account: admin LiPA HLA-B/v.1.4/001102

 AssayResult
ALLELE GROUP TYPING:

B*18

B*39

Assay Report

Sample ID: 455FM59

Patient Name: F.M.

Entered on: 1/21/2002

Account: admin LiPA HLA-DRB/v.5.4/001102

AssayResult

ALLELE GROUP TYPING:

DRB1*
07

DRB1*
11


ALLELE GROUP TYPING:

DRB1*
11

DRB1*
13

Assay Report

Sample ID: 455FM59

Patient Name: F.M.

Entered on: 1/21/2002

Account: admin LiPA HLA-DQB/v.2.6/001102

AssayResult

DQB1*
03
DQB1*
03

 Assay Report

Sample ID: 456FI38

Patient Name: F.I.

Entered on: 1/21/2002

Account: admin LiPA HLA-DQB/v.2.6/001102

AssayResult
DQB1*
03
DQB1*
06

 Importance of DNA Quality

100 ng Genomic DNA 1% Agarose Gel

 Patient result

 HLA SBT

Resolve heterozygous sequence ambiguities

- Separate alleles by SSP-PCR

- 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)

• 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

 Kidney – pancreas transplantation

whole organ transplantation

• 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
 Kidney transplantation in children

 Usualy the donor is one of the parents

 AB0 matched

 Tissue typing: A, B, DRB1

 Cytotoxic antibodies

 crossmatch