Introduction

Organ transplantation has become a therapeutic modality to treat patients with end-stage disease. While many types of organs have been successfully transplanted, the histocompatibility barrier between recipient and donor remains a problem in that it will activate immune responses leading to graft rejection. Although immunosuppressive drugs such as tacrolimus (FK-506) and cyclosporine will reduce rejection, the successful management of the transplant patient requires an understanding of the Major Histocompatibility Complex (MHC) also referred to in humans as the HLA (or Human Leukocyte Antigen) system. This overview addresses some biological aspects of HLA in relation to issues of histocompatibility testing in transplantation.

Role of HLA in Transplant Immunity
In order to appreciate the role of HLA in transplantation, we must first consider the structural and functional aspects of HLA molecules. HLA antigens are controlled by a series of highly polymorphic genes on the short arm of chromosome 6, referred to as the human MHC (Figure 1).

These genes have been classified into major categories. HLA-A, HLA-B and HLA-C encode for Class I molecules consisting of a 45kD glycopeptide chain complexed to a 12kD b2-microglobulin chain encoded by a nonpolymorphic gene on chromosome 15 (Figure 2). The genes in the HLA-DR, HLA-DQ and HLA-DP regions encode for Class II molecules consisting of a ~30kD a-chain and a ~28kD b-chain. These HLA class I and class II alloantigens can induce transplant immunity at both humoral (antibody) and cellular (T lymphocyte) immune levels. The human MHC contains many other Class I and Class II genes (e.g. HLA-G and HLA-DM, respectively) whose products do not seem important as transplantation antigens. A third set of so-called Class III genes controls a heterogeneous group of proteins that include Complement components C2, C4 and Factor B, Tumor Necrosis Factor, 21-Hydroxylase and Heat Shock Protein-70.

Structurally, HLA molecules are cell surface-bound glycoproteins that contain four immuno-globulin-like domains. The two external domains form a peptide-binding groove consisting of two parallel a-helix amino acid chains on an eight b-pleated polypeptide sheet (Figure 4). The peptide-binding grooves of Class I and Class II molecules are structurally analogous. Their primary role is to bind small antigenic peptides for presentation to the T-Cell Receptor (TCR) which then may lead to specific T-Cell activation. Many antigenic peptides are generated or "processed" by so-called antigen-presenting cells (APC) and they can vary in length: from 8-9 amino acid residues bound to Class I molecules to 10-25 residues bound to Class II molecules.

The considerable polymorphism of HLA is well-known. By serology for instance, at least 25 alleles have been reported for HLA-A, 60 alleles for HLA-B and 18 alleles for HLA-DR. Molecular typing has resulted in even much greater numbers of HLA alleles and it is often very difficult to find HLA-matched unrelated donors for transplant recipients. The polymorphism of HLA is reflected by allelic substitutions of many amino acid residues in the polypeptide chains, especially the external domains which contain the peptide-binding site. This affects the spectrum of antigenic peptides presented by the different allelic types of HLA molecules and the repertoire of responding T-cells.

Functionally, HLA molecules play a crucial role in T-cell activation by APC. This antigenic recognition depends on the interaction between the antigenic peptide-binding HLA molecule and the T-Cell Receptor (TCR), an immunoglobulin-like heterodimeric protein expressed on T-lymphocytes. Two general pathways of T-cell alloactivation have been recognized in transplant immunity (Figure 4). The direct pathway refers to the alloreactive responses of recipient T-cells to donor APC expressing incompatible HLA antigens. It provides a powerful mechanism of T-cell alloactivation. In the indirect pathway, allogeneic HLA antigens are taken up and processed by recipient APC and presented in context with autologous HLA molecules to recipient T-cells.

The transplanted organ represents a continuous source of HLA alloantigens that can induce a rejection response at any time post-transplant. Both alloactivation pathways are important in the generation of donor-specific cell-mediated cytotoxicity and delayed-type hypersensitivity (DTH)-like mechanisms of allograft rejection. Conversely a continuous presence of donor MHC antigens is also needed for the maintenance of allograft tolerance.

HLA is also involved in other cellular immune mechanisms that affect transplant recipients. In Graft-Versus-Host (GVH) disease, donor-derived immuno-competent lymphocytes react with HLA-incompatible recipient cells and induce inflammatory responses in host tissues such as the skin and gastrointestinal tract. This complication is frequent after bone marrow transplantation, but may also affect recipients of liver and other organ transplants and even blood transfusions. GVH disease seems more likely in situations whereby the donor is well matched for the patient but not the other way around.

During infection, microbial antigens are processed by APC and presented via HLA molecules to T-cells that elicit cytotoxic and DTH-like inflammatory reactions in the allograft. The so-called HLA-restricted mechanisms are more effective if the relevant HLA antigens are shared between recipient T-cells and donor APC or target cells. Recurrent autoimmune disease represents another potential problem. Although end-stage organ failure due to autoimmune disease can be successfully treated with transplantation, HLA-restricted T-cell autoimmunity can be expected to persist in the patient. This may promote recurrent disease especially, if the donor organ shares the relevant HLA antigens with the recipient. Thus, HLA compatibility can under certain conditions, promote certain non-alloimmune mechanisms of allograft injury. The table below summarizes the HLA-related cellular immune mechanisms that might affect organ allograft outcome.

Direct and indirect HLA allorecognition mediate rejection and conversely, GVH reactions if immunocompetent donor cells recognize recipient incompatibilities. HLA-restricted immune responses to microbial (viral) antigens and autoantigens can mediate non-rejection-related inflammatory mechanisms. HLA matching can have a dualistic effect on transplant outcome: on one hand, it reduces rejection but conversely, it may promote other HLA-restricted mechanisms of allograft injury. For most transplants, the HLA type of the donor can be expected to have incompatibility for many HLA loci but compatibility for some HLA loci. This would mean that both allospecific and self-HLA-restricted immune mechanisms can occur simultaneously and may even synergize in mediating inflammatory injury of the allograft..

Histocompatibility in Organ Transplantation

HLA matching clearly improves the survivals of transplanted kidneys, hearts and lungs, but an HLA-based donor organ allocation has been implemented only for kidney transplantation. This system considers ABO and HLA compatibility of the donor and the results of the lymphocytotoxicity crossmatch test between patient serum and donor cells. Time constraints regarding the preservation of donor hearts and lungs do not permit prospective HLA matching for these organs. In some instances, crossmatching is done for cardiothoracic transplantation especially if the patient is HLA sensitized.

In kidney transplantation, current criteria for HLA matching consider three loci: HLA-A, HLA-B and HLA-DR; each allele is defined as a single antigen assigned by serological typing criteria, e.g. HLA-A1, HLA-B44, HLA-DR7, etc. Each donor and recipient can type for up to six different HLA antigens encoded by these loci and HLA compatibility is usually assessed by the number of HLA mismatches (or matches) of the donor. Many studies have shown a stepwise decrease in graft survival of cadaver kidneys with increasing numbers of HLA. The superior results with zero HLA-A,B,DR mismatches have led to a system of mandatory sharing of such donor kidneys. Nevertheless, a rather small proportion of recipients, especially African-American patients, benefit from this system.

During recent years, alternative strategies for HLA matching have been considered in kidney transplantation. They are referred to as CREG (Cross-Reactive Group) matching, or "public" epitope matching (the conventional HLA antigens are called "private" epitopes) or, residue matching (determined from amino acid residue sequence information of HLA antigens). All are based on the concept that HLA molecules contain multiple antigenic determinants many of them are shared and, that some are more important for matching than others. CREG matching strategies are now being implemented in kidney transplantation.

Histocompatibility testing for liver transplantation remains somewhat of an enigma. Many investigators have noted that HLA compatibility does not seem to benefit the overall group of liver transplant recipients. In fact, several studies have shown lower survivals of HLA-DR matched livers. HLA matching seems to has a dualistic effect on liver transplant outcome: it reduces graft rejection but it promotes other immune mechanisms of graft injury related to viral infection (e.g. cytomegalovirus and hepatitis viruses) and recurrent disease. Moreover, a liver allograft has a distinguished feature of promoting a hematolymphoid chimeric state associated with transplant tolerance but liver graft-derived immunocompetent cells may also induce GVH disease. Donor-specific crossmatching has limited relevance to liver transplantation because the liver allograft is relatively resistant to humoral rejection . In some sensitized patients, a liver allograft may even protect a subsequent kidney transplant from hyperacute rejection.

Tissue Typing for Clinical Transplantation

The main purpose of tissue typing in transplantation is (1) to assess donor-recipient compatibility for HLA and ABO and (2) to analyze patient serum for antibodies that react with transplant donor tissues. Most relevant is the crossmatch assay whereby patient sera are tested for their reactivity with donor lymphocytes. This is usually done by lymphocytotoxicity testing whereby donor lymphocytes are first incubated with patient serum, then with rabbit complement and lysis of lymphocytes is assessed by the uptake of an extravital dye like trypan blue or eosin red. A positive crossmatch is a contraindication for organ transplantation because of the risk for hyperacute rejection and the higher incidence of vascular rejection during the early post-transplant period. This applies particularly for kidney and heart transplants whereas the liver allograft is more resistant to antibody-mediated injury.

In kidney transplantation, several modifications of the crossmatch assay have been used to increase its sensitivity including anti-human globulin (AHG) augmentation, flow cytometry and enzyme-linked immunoassays (ELISA) and B-cell crossmatches. Serum treatment with dithiothreitol (DTT) is used to distinguish clinically irrelevant IgM type antibodies.

Transplant candidates may become sensitized following a prior transplant, blood transfusions and previous pregnancies. Serum screening for alloreactive antibodies against a random cell panel will provide an assessment of the degree of sensitization expressed as the percentage Panel Reactive Antibody (PRA). The PRA can vary between 0% (non-sensitized) to 80-100% indicating a high degree of sensitization. Patients with high PRA values are less likely to have crossmatch-negative donors. They must wait much longer for a transplant and some may never receive a kidney.

Several techniques have been used to screen patient sera for alloreactive antibodies. The complement-dependent lymphocytotoxicity technique has two versions, one is a direct assay of patient antibodies exert a lymphocytotoxic effect through complement-dependent mechanisms of cell membrane lysis. Examples are the NIH (or "standard") and the Amos modified techniques. The indirect technique utilizes an extra step with a goat or rabbit anti-human IgG antibody-mediated augmentation of complement-dependent lymphocytoxicity. This antiglobulin (or AHG) modification is more sensitive than the direct lymphocytotoxicity but is also technically more demanding. The source of lymphocytes is an important consideration, several laboratories utilize B-cell screening to detect HLA class II-specific antibodies.

Recently, additional serological methods have been developed that do not utilize lymphocytotoxicity as an endpoint. One is based on flow cytometric analysis of alloantibodies binding to panel donor lymphocytes with different HLA types. Serum screening is also now being done with an ELISA assays using solubilized HLA antigens immobilized on a solid surface.

An important goal of any serum screening procedure is to gain insight about the spectrum of HLA-specific antibodies, especially in highly sensitized patients. This is based on the concept that each HLA molecule has multiple antigenic determinants (epitopes) which can be grouped as private (e.g. HLA-A1, HLA-B7, etc.) and public determinants (sometimes referred to as crossreactive groups, CREGs). A public determinant is an epitope shared by molecules with different private specificities (e.g. A1+A3+A11, A2+A9+A28, B5+15+35, B7+22+27+40, etc.). Most highly sensitized patients show a persistence of the same pattern of antibody specificity against one or a few public epitopes. A better understanding of the antibody specificity improves the selection of transplant donors with acceptable HLA mismatches.

Alloreactive T-lymphocytes are the primary mediators of cellular rejection, and a considerable research effort has emphasized the Mixed Leukocyte Culture (MLC) as an in vitro system the determine T-cell alloreactivity between donor and recipient. These MLC assays measure proliferative responses of alloactivated lymphocytes and offer also opportunities to test for cell-mediated cytotoxicity, cytokine production and the quantitation and functional characterization of primed T-cells. Since these MLC procedures are technically demanding and time-consuming, they are not routinely used for prospective histocompatibility testing in a clinical service laboratory.

Considerable evidence has been obtained that matching for HLA will reduce cellular rejection thereby promoting survival of kidney and heart transplant patients. While the HLA system comprises multiple class I and especially class II genes, most matching strategies consider only three loci: HLA-A, HLA-B and HLA-DR. Although it makes sense to find a perfect match for each transplant patient, the reality of clinical practice dictates the selection of less well-matched donors. As noted above, current strategies are directed towards the identification of donors permissible or acceptable HLA mismatches.

Conclusions

Because HLA plays such a dominant role in transplant immunity, pre-transplant histocompatibility testing seems important for organ transplantation. The table below summarizes the most commonly used tissue typing procedures for clinical transplantation.. The practical problem remains whether tissue typing can be applied on a prospective basis for all types of transplants. Organ preservation time remains an important limitation for an HLA-based tissue allocation strategy especially hearts, lungs and intestines. Nevertheless, histocompatibility testing yields relevant information for the clinical management of any type of transplant recipient.