Essential histocompatibility for the renal clinician—Part 1

INTRODUCTION

Immune responses to foreign tissue

The human immune system has evolved as a highly sophisticated defence system that functions to distinguish between self and non-self. From an evolutional perspective, this was essential to survive attacks by pathogens, but inconvenient penalties are occasional reactivity to self, manifest by autoimmune disease and reactivity to genetically disparate members of the species (alloreactivity). This latter phenomenon came to light when pioneers attempted to transplant organs between individuals.

Immune reactivity occurs because, unlike laboratory mice, human beings are highly outbred and are therefore genetically heterogeneous. Only identical twins truly share the same genetic material and are thus syngeneic. It is for this reason that the first successful human kidney transplant was carried out between identical twins in 1954 with minimal immunosuppression [1]. The vast majority of the population have many differences in their genetic composition, called polymorphisms.

The area of the human genome with the greatest genetic variation between individuals is located on the short arm of chromosome 6 in the major histocompatibility complex (MHC). In humans, the glycoprotein products derived from these genes are called human leukocyte antigens (HLA) and they are widely expressed on the surface of human cells. These molecules clearly did not evolve to mediate alloreactivity. In fact, they are intimately involved in antigen presentation, and heterogeneity ensures a broad repertoire of responses to pathogens.

The study of histocompatibility and immunogenetics (H&I) involves, on one hand, determining the potential target antigens in individuals, principally focused on the highly polymorphic HLA molecules (HLA typing) and, on the other hand, characterizing the immune receptor reactivity to allogeneic antigens in potential transplant recipients (assessing sensitization). H&I characterization of alloreactivity is largely restricted to measuring the responses of the adaptive immune system through antibody analysis.

Characteristics of HLA molecules

HLA are grouped into classes depending on their structural similarities, function and distribution patterns (see Figure 1). Class I HLA (HLA -A, -B and C) are expressed on all nucleated cells and consist of an α chain that is non-covalently associated with β₂ microglobulin. The α chain is made up of three domains. α₁ and α₂ domains fold to form the peptide-binding groove, which can present endogenous peptides of up to 8–10 amino acids in length. The α₃ domain associates with β₂ microglobulin and binds to the CD8⁺ co-receptor on cytotoxic T cells during antigen presentation. Exons 2 and 3, which code for α₁ and α₂, are highly polymorphic, and it is the variation in α₁ and α₂ that dictates which peptides can be presented.

Class II HLA (DR, DQ and DP) are made up of two non-covalently linked chains (α and β chain). In the case of HLA-DR, the α chain is relatively conserved, with polymorphism exhibited in the β chain. However, both the α and β chains in HLA-DP and HLA-DQ are highly polymorphic, and biodiversity is increased further by combinations between different α and β chains. The peptide-binding groove, which is comprised of the interaction between the α₁ and β₁ domains, can present peptides of 15–24 amino acids in length to a corresponding T-cell receptor. Class II HLA is primarily found on professional antigen-presenting cells but in an inflammatory environment can be upregulated on other cells including vascular endothelial cells (e.g. glomerular and peritubular capillaries). Class II HLA present exogenous peptides to CD4⁺ T cells, thus forming signal 1, or the activating signal to a corresponding cognate T cell. Successful activation of CD4⁺ T cells (with costimulation or signal 2) results in expansion and cytokine release. If the presenting cell is a B cell, CD4⁺ T cell activation results in downstream B-cell activation, antibody class switching with increased affinity of antibody production towards the activating peptides. Importantly, the generation of donor-specific IgG antibodies is dependent on the prior generation of a corresponding anti-donor T-cell response.

The MHC is inherited from each parent in a Mendelian fashion as a haplotype, and gene expression is codominant. Therefore, in heterozygotes, up to 12 different classical HLA alleles can be expressed on a cell surface, each allele able to bind a different repertoire of peptides. The number of different reported alleles at each locus is shown in Table 1, and underpins the molecular basis of human genetic diversity.

Allorecognition

Recipient T cells may encounter alloantigens by three pathways: direct, indirect and semidirect. Direct allorecognition is thought to be an important mechanism associated with acute rejection, specifically CD8⁺-mediated cytotoxic tubular damage (tubulitis). During graft reperfusion, donor leukocytes, expressing donor-derived HLA molecules, enter the recipient circulation and encounter naïve alloreactive recipient T cells within the secondary lymphoid tissue, which form a strong immune response directly against allogeneic HLA molecules. Once primed these cells mature, acquire the trafficking molecules necessary to access the transplanted graft, enter the circulation and travel to the transplanted organ.

In indirect allorecognition (the normal mechanism of foreign antigen processing), recipient APCs take up and process allogeneic proteins for presentation to T cells in peptide fragments. This mechanism, emerging as dominant after the disappearance of donor-derived passenger leukocytes, is thought to be instrumental in the development of chronic antibody-mediated rejection.

Finally, with semidirect allorecognition, dendritic cells acquire intact donor HLA molecules and present these intact proteins to recipient T cells for activation. The physiological importance of this pathway remains to be determined.

HLA typing

The methods of HLA typing have progressed from serological typing (determining which HLA was expressed on the cell surface by antibodies), to molecular, or DNA level typing, and as such, the HLA nomenclature has evolved. The minimal level of resolution required for renal transplantation is at the level of the allele group (e.g. HLA-A*02) for HLA -A, -B and -DR, and can be achieved using PCR amplified sequence-specific primers or through hybridization of sequence-specific oligonucleotide probes. Typing to a higher resolution (required for bone marrow transplantation) can be achieved through next-generation sequencing.

Assessing sensitization

A patient is considered to be sensitized when they develop reactivity to alloantigens. The molecular correlate of sensitization is the formation of highly specific immune receptors by an iterative process of genetic recombination. The risk of developing antibodies has been shown to increase with the amount of exposure to foreign HLA [3]. This exposure occurs through three principle routes, paternal antigens in utero during pregnancy, blood transfusion (contaminated by leukocytes) and previous solid organ transplants. A minority of cases may be attributable to vaccination, infections or sexual exposure. Certain sensitizing events, for example, pregnancy, can result in a stronger and more durable immune response [4].

The current gold standard test utilizes Luminex technology where HLA is purified from cell lines and coated onto polystyrene microbeads. The differing ratio of internal dyes within each bead allows hundreds of antigen specificities to be tested in a single assay, resulting in a rapid and a highly sensitive screening tool.

The degree of sensitization is quantified in the UK using a calculated reaction frequency (cRF) score, which compares the profile of antibody specificities (also known as panel reactive antibodies score) from a recipient with the HLA types of the last 10 000 UK donors. A cRF of 99% indicates that the antibody profile of the recipient will react to and therefore be incompatible with 99% of the donor pool. It goes on to follow that a patient with a higher %cRF will inevitably experience a longer wait time to receive a compatible kidney offer. It is therefore imperative to limit any avoidable sensitizing events to facilitate future compatible transplant offers. This involves limiting the mismatches between donor and recipient during organ allocation, whilst balancing the increased time associated with waiting for a favourably matched tissue type in a limited donor pool. The avoidance of blood transfusion has been greatly facilitated by the use of erythrocyte-stimulating agents and intravenous iron.

The median life expectancy for a deceased donor graft remains at ∼20 years, and it is likely that a patient will receive more than one graft in their renal journey. A close working relationship is therefore required between the laboratory and the transplant team to allow individualized decisions to be made at listing and at transplantation to ensure that each recipient receives a compatible kidney based on tissue type, sensitization history and immunological risk while balancing the wait time required to achieve this match.

CONFLICT OF INTEREST STATEMENT

None declared.

REFERENCES