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 REJECTIONTransplant 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.*TISSUE COMPATIBILITY The concept of tissue compatibility (the need for donor and recipient tissues to be "compatible" in order for a transplant to be accepted) was appreciated as early as the beginning of the 20th century. In a series of studies involving the transplantation of tumors and later skin grafts between mice, it became apparent that successful tissue transplantation depended upon genetic similarity of donor and recipient. Tissues transplanted between genetically identical animals, also known as syngeneic transplants or isografts, are virtually always accepted. In contrast, transplants performed between two subjects belonging to the same species who are genetically nonidentical, termed allogeneic transplants or allografts, invariably undergo rejection. Transplants performed between donor and recipient who belong to different species are known as xenogeneic transplants or xenografts and are promptly rejected. Allogeneic transplants or allografts bear allogeneic or foreign major and minor histocompatibility antigens; whereas syngeneic transplants or isografts bear genetically identical or self major and minor histocompatibility antigens.

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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.

*Transplant outcome

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.

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The structure of HLA class I molecules.*

The structure of HLA class II molecules*

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HLA alleles set found on one chromosome represents a haplotype.

HLA GeneticsEach 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.FatherMother*

2 digitsHLA-DRB1*13A group of alleles which encode the DR13 antigen, i.e. there 62 DRB1*13 alleles: 1301 to 1362*

4 digitsHLA-DRB1*1301A specific allele*

8 digitsHLA-DRB1*13010102An allele which contains a mutation outside the coding region*

4 digitsHLA-DRB1*1301NA null allele N*

8 digitsHLA-DRB1*13010102NA null allele N which contains a mutation outside the coding region*

4 digitsHLA-DRB1*1301LL indicated that the allele is low expressed at the cell surface*

4 digitsHLA-DRB1*1301SS indicated that the alleles is secreted but not stable at the cell surface, find at soluble form*

4 digitsHLA-DRB1*1301CC indicated that the allele is present in the cytoplasm but not at the cell surface*

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 recipients T cells can also react to donor MHC peptides presented on the recipients own APCs. This pathway is called indirect allorecognition.*

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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.

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Mediators of rejection*

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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.

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Clinical significationSuccessful 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)*

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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

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.

MethodsIndirect diagnostic tests (serological) Direct diagnostic tests, molecular biology tests (PCR, RT-PCR).

Blood group matchingBoth donor and recipient have to matched for ABO

Sample of cells or tissueCombine DNA with sequence-specific primer fix for each alleleAmplify by PCR

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 recipientEntered on: 1/22/2002

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

AssayResultALLELE GROUP TYPING:

A*02

A*24

ALLELE GROUP TYPING:

B*18

B*35

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*39 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:

DRB1*07

DRB1*11

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*11

DRB1*13 Assay Report Sample ID: 456FI38 Patient Name: F.I.Entered on: 1/21/2002 Account: admin LiPA HLA-DRB/v.5.4/001102 AssayResult

DQB1*03DQB1*03

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*03DQB1*06 Assay Report

Sample ID: 456FI38

Patient Name: F.I. Entered on: 1/21/2002

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

AssayResult

DNA 80 ng for Class I40 ng for Class II

Importance of DNA Quality100 ng Genomic DNA 1% Agarose Gel

Assign-SBT Resolves AmbiguitiesSequences are arranged in layersMaster sequence

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

Add resolution to typings obtained by lower resolution methods (e.g. SSP, SSOP)Take advantage of low resolution data to select appropriate reagents

HLA Antibody DetectionHLA 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 AnalysisGTI QuikScreen

HLA Class I Ab ScreenPooled platelets (minimum of 300 donors)Highly specific (no Class II interference)Flexible formats, easy to useScreen up to 40 samples per tray in 2.5 hrsWinScreen software

GTI Quik-ID Class I

HLA Class I antibody specificityPercent Panel Reactive (%PRA)Panel of 40 donorsSolubilized Class I antigen from plateletsSensitive capture assaySoftware analysis package including CREG analysis

Class II HLA Antibody AnalysisGTI B-Screen

HLA Class II Ab ScreenSoluble HLA from EBV Transformed cellsAffinity purifiedFlexible format - strip wellsHighly specific (no Class I interference)Screen 40+ samples per tray in 2.5 hrsWinScreen softwareGTI Quik-ID Class II

HLA Class II Ab specificityPercent panel reactive (%PRA)Panel of 30 cell linesAffinity purified Class II HLA from EBV transformed cell linesSensitive capture assaySoftware analysis package

Patient sample 1Patient sample 2Patient sample 3Patient sample 4Patient sample 5Negative controlPositive controlBlank WellIgG IgM B-Screen NAW

Antibody Screening Algorithm

New patients full work-upFlow specificity and PRAELISA specificity and PRA

Current patients Negative or PositiveNegatives screened monthly or quarterlyAny neg-pos refluxed to Ab IDPositives screened monthly by ELISASpecificity and PRA tracked Ambiguous specificity refluxed to Flow

Antibody Monitoring SystemELISA 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

2nd step: ELISA

takes about 3 to 4 hrs - depending on number of donors

Negative ControlLysateControlPositiveControlClass IClass IIRecipient Samples

Antibody Monitoring SystemWhat are its benefits?

Employs three sets of Controls Reagent Control Negative Control Lysate Control Lysates can be frozen at 80 C for future use

Antibody Monitoring SystemNo interference with therapeutic rescue immunosuppresents

Distinguish between Donor and non-Donor HLA AbsAntibody Monitoring System

No interference with IvIG/pheresis protocolsAntibody Monitoring System

Antibody Monitoring SystemUp to 44 patient sera per plate with one donorUp to 6 donors with 4 recipients each per plate

Antibody Monitoring System Microtiter plate based ELISAFlexibility versatile snap-in stripsConvenience screening on a single tray for a variety of antibodiesFormat fits most commercially available microplate readers

Luminex technology of antibody identification

Kidney pancreas transplantationwhole organ transplantationPancreas transplantation alone (PTA)Simultaneous pancreas-kidney (SPK) transplantationPancreas after kidney (PAK) transplantationImmunological algorithm:HLA typing: A, B, DRB1Cytotoxic antibodiesCrossmatch

Transplantation of pancreatic isletsLangerhans cells are targetedVirological assessment of both , donor and recipientHLA typing: A, B, DRB1Cytotoxic antibodiescrossmatch

Kidney transplantation in childrenUsualy the donor is one of the parentsAB0 matchedTissue typing: A, B, DRB1Cytotoxic antibodiescrossmatch

5.52010

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************21The general approach to resolve ambiguous typings due to heterozygous sequence is to

1) Separate the two alleles by the specificity of a PCR reaction

2) Sequence the hemizygous PCR product and analyze to resolve the ambiguity*Finally, we also give you the flexibility to include a No Antigen Well for every sample if you wish to do so. No antigen well strips allow the investigator to judge the amount of background from a patient sample that is binding non-specifically in the wells allowing a more accurate picture of the sample.

Lets take a little more specific look at each of the kits