**4. Non-HLA antibodies**

Acute and chronic allograft rejection can occur in HLA-identical sibling transplants implicat‐ ing the importance of immune response against non-HLA targets. Non-HLA anti-bodies may occur as alloantiboides, yet they seem to be predominantly autoantibodies. Antigenic targets of non-HLA antibodies described thus far include various minor histocom-patibility antigens, vascular receptors, adhesion molecules, and intermediate filaments. Non-HLA antibodies may function as complement and non-complement-fixing antibodies and they may induce a wide variety of allograft injuries, reflecting the complexity of their acute and chronic actions.

#### **4.1. The KIR receptor complex**

The adaptive immune response recognises infection through presentation of pathogen-de‐ rived peptides in association with MHC to the host T cells. One of the mechanisms which pathogens use to evade this immune response is to down regulate their MHC cell surface expression. Natural Killer cells are able to detect altered expression of MHC through a number of cell surface receptors leading to target cell lysis [19]. These recep‐ tors include the killer immunoglobulin like receptors (KIR), which are also expressed on some effector T cells. In humans, the KIR gene cluster is located on chromosome 19. KIR genes are both polygenic and polymorphic [20]. The KIR gene cluster codes for 15 ex‐ pressed KIR genes and 2 pseudo genes.

The ligands for KIR receptors are HLA class I molecules [21]. These include HLA-C lo‐ cus antigens with either Asn (Group 1 HLA-C antigens) or Lys (Group 2 HLA-C anti‐ gens) at position 80, the HLA-Bw4 epitope and some HLA-A antigens. KIR receptors binding to HLA class I are either inhibitory or are stimulatory with the overall effect of NK cell interaction with the target cell dependent on the balance between these inhibito‐ ry and stimulatory signals. It is thought that the inhibitory KIR's bind class I with great‐ er affinity than the corresponding activating KIR with the effect that under normal circumstances the inhibitory signal prevails. The 'missing self' hypothesis holds that NK cell alloreactivity occurs when the ligand for inhibitory KIR receptors is down regulated or 'missing', leading to activation. This however requires that KIR receptors engage their cogent HLA class I molecules during maturation to acquire effector function. NK cells that express only inhibitory KIRs for absent HLA class I molecules are hypo responsive in the non transplant setting.

Inhibitory KIR receptors possess long cytoplasmic tails with immunoreceptor tyrosine based inhibitory motifs (ITIMs). Activating KIR receptors have short cytoplasmic tails that pair with adaptor molecules with immunoreceptor tyrosine based activating motif (ITAMs). The nomenclature for KIR receptors therefore includes an 'L' (long tail) for inhibitory KIR's and an 'S' (short tail) for activating KIR's. The nomenclature also includes 'P' for pseudo genes. The inhibitory and activating KIR receptors share sequence and structural similarities in their extracellular domains. KIR's have either 2 or 3 extracellular immunoglobulin domains and this is reflected in their nomenclature as either '2D' or '3D', giving KIR receptors nomenclature such as KIR2DL1, KIR2DS2 and KIR3DL1, where the final digit indicates the order in which the genes were described.

The KIR genes assemble into haplotypes with two haplotypes described, 'A' and 'B'. The 'A' haplotype has only one activating KIR (2DS4), while the 'B' haplotype has a higher number of activating KIRs and generally possess more KIRs than the 'A' haplotype.

#### **4.2. MICA/B**

probes, which are complementary for DNA sequences, characteristic for certain HLA antigens. These probes will then "type" for the presence of specific DNA sequences of HLA genes.

PRA has been used to measure patient HLA sensitisation ever since pre-formed donor specific HLA antibodies were associated with hyperacute rejection in renal transplantation in the 1960's [17]. As traditionally defined, PRA refers to the percentage of an antibody screening panel with which the patient's serum reacts. A kidney patient with a PRA > 85% is considered highly sensitised. This measure of PRA however relies on the composition of the panel which may not necessarily reflect the antigen frequencies in the donor population. This measure of PRA is not therefore a good reflection of the chances of the patient finding a compatible donor. Variations in cell panels, both commercial and in house, result in wide variations in recorded

The calculated PRA (cPRA) was introduced to overcome this problem [18]. The cPRA can be calculated in a number of different ways, but relies on the identification of a potential recipi‐ ent's anti-HLA antibody profile. This has been made much easier by the wide adoption of solid phase assays such as Luminex. Luminex assays, especially those involving the use of single antigen beads (SABs) allow fine specificity definition and allow the strength of the reactions (MFI) to be used to assess immunological risk and help decide whether or not specificity should be listed. The cPRA is then calculated by defining a set of unacceptable mismatches for that recipient, and weighting those mismatches according to the frequency of the antigen in the donor population. This could be based on the frequency of different HLA antigens in the most recent 10,000 deceased donors.The cPRA therefore gives a measure of the chances of a patient

cPRA removes some of the variability between laboratories using different panels and allows

Acute and chronic allograft rejection can occur in HLA-identical sibling transplants implicat‐ ing the importance of immune response against non-HLA targets. Non-HLA anti-bodies may occur as alloantiboides, yet they seem to be predominantly autoantibodies. Antigenic targets of non-HLA antibodies described thus far include various minor histocom-patibility antigens, vascular receptors, adhesion molecules, and intermediate filaments. Non-HLA antibodies may function as complement and non-complement-fixing antibodies and they may induce a wide variety of allograft injuries, reflecting the complexity of their acute and chronic actions.

The adaptive immune response recognises infection through presentation of pathogen-de‐ rived peptides in association with MHC to the host T cells. One of the mechanisms

a PRA value to be assigned which reflects the patients' transplantability.

**3.5. Panel Reactive Antibodies (PRA)**

378 Current Issues and Future Direction in Kidney Transplantation

PRA for patients on the waiting list.

finding a compatible donor in the donor pool.

**4. Non-HLA antibodies**

**4.1. The KIR receptor complex**

The major histocompatibility complex class I related chain was first described in the 1990's [22]. The genes are located centromeric to the HLA class I B gene. The only two MIC genes which are expressed are MICA and MICB. MICA and MICB share a significant amount of sequence homology with HLA class I and have some similarity in their conformation. MICA and MICB antigens have α1, 2 and 3 domains like classical HLA antigens but do not associate with β2 microglobulin and do not bind peptide for presentation to T cells. Instead, MIC antigens serve as ligands for the NKG2D receptor on NK cells and on some T cells.

MICA and MICB genes are polymorphic but not as much as the classical HLA class I genes. Over 70 MICA alleles and over 30 MICB alleles have been described [23]. Unlike HLA class I where the polymorphic residues are located mainly in the region that forms the peptide binding groove, polymorphism in MIC is more dispersed throughout the α2 and α3 domains. There is also polymorphism in the trans-membrane region. Many MIC antigens have the same extracellular domains with the only differences lying in the trans-membrane regions.

**5. Cross-matching techniques**

hence prevent this [17].

the lymphocytes.

tion or rarely as a result of other non-HLA antibodies.

There are different types of crossmatch tests available.

**5.1. Complement-Dependent Cytotoxicity (CDC) crossmatch**

reaction (deemed 'positive') suggests the presence of preformed DSA.

Crossmatching was developed in an attempt to identify recipients who are likely to de‐ velop acute vascular rejection of a graft from a given donor. This phenomenon, hyper‐ acute rejection (HAR) [24], is a result of preformed antibodies against the donor; referred to as donor-specific antibodies (DSA). Such antibodies are usually formed as the result of previous exposure to HLA, generally through pregnancy, blood transfusion or previous transplantation [25]. There are other debated forms of developing anti-HLA Abs such as via microbial exposure but the above three are thought to be the most prevalent. Particu‐ larly relevant is the exposure of women during pregnancy, to their partner's HLA. This commonly results in direct sensitization against the partner, potentially making him an unsuitable living donor. HAR may also occur in blood group incompatible transplanta‐

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Preformed antibodies cause rejection by binding to HLA antigens expressed on the endothe‐ lium of vessels in the transplanted kidney, resulting in activation of the complement cascade with resultant thrombosis and infarction of the graft. HAR can occur immediately upon reperfusion of the donor kidney. This catastrophic outcome necessitates the immediate removal of the graft. Clearly avoiding HAR is desirable and crossmatching helps predict and

A CDC crossmatch involves placing recipient serum (potentially containing donor-specific anti-HLA antibodies) onto donor lymphocytes (containing HLA antigens). A cytotoxic

CDC crossmatching was pioneered by Terasaki and colleagues in the 1960s [13, 17]. It identifies clinically significant donor specific HLA antibody mediated responses for a given recipient. Lymphocytes from the donor are isolated and separated into T and B cells. Serum from the recipient is mixed with the lymphocytes in a multi-well plate. Complement is then added (usually derived from rabbit serum). If donor-specific antibody is present and binds to donor cells, the complement cascade will be activated via the classical pathway resulting in lysis of

The read-out of the test is the percentage of dead cells relative to live cells as determined by microscopy. The result can thus be scored on the percentage of dead cells, with 0 correlating to no dead cells; scores of 2, 4 and 6 represent increasing levels of lysis. On this basis, a score of 2 is positive at a low level, consistent with approximately 20% lysis (generally taken as the cut-off for a positive result). A score of 8 represents all cells having lysed and indicates the strongest possible reaction. The use of a scoring system allows a semi-quantitative analysis of the strength of reaction. Another way to determine the strength of the reaction is to repeat the crossmatch using serial doubling dilutions of the recipient serum (often known as a 'titred crossmatch'). In this way, dilutions are usually performed to 1 in 2, 4, 8, 16, 32, 64 and so on.

MICA and MICB antigens are constitutively expressed on epithelial cells, especially those of the gastrointestinal tract and on fibroblasts, monocytes, dendritic cells and on endothelial cells. They are not constitutively expressed on lymphocytes. They are however up regulated in stressed cells.

The structure of MICA is similar to that of HLA class I but has some striking differen‐ ces. Like HLA class I, MICA has three extracellular domains (α1, 2 and 3), a transmem‐ brane region and a cytoplasmic domain. Unlike HLA class I, the MICA protein does not associate with β2 microglobulin. The MICA α1 and 2 domains form a platform that is analogous to the platform formed by HLA class I α1 and 2 domains. In HLA class I, this platform forms the peptide binding groove. The MICA molecule however has extensive disordering of sections of the alpha helix in the α2 domain resulting in a very shallow groove, incapable of binding peptide. The MICA α1 and 2 platform domains do not in‐ teract with the α3 domain except for being linked together through a short linker chain. This allows for some flexibility in the structure.

The NKG2D receptor forms a complex with MICA by binding orthogonal to the alpha helices of the platform α1 and 2 domains.

#### **4.3. Minor histocompatibility antigens**

HLA presents the major genetic barrier to stem cell transplantation. However, evidence that other genetic systems are involved includes GvHD and some degree of rejection even when transplanting with HLA identical siblings. A non HLA system which is thought to contribute to this is the minor histocompatibility antigen (mHA) system. Mi‐ nor histocompatibility antigens comprise of peptides derived from proteins in which some degree of polymorphism exists such there may be differences between the patient and donor repertoires. These peptides can be presented to the immune system by both HLA class I and II antigens.

The best characterised minor antigens are the Y chromosome derived HY peptide and the autosomal HA1 to HA5 peptides. Minor histocompatibility antigens such as HA1 and HA2 have restricted tissue distribution and are present normally only on haematopoietic cells. Others such as HY are more ubiquitously distributed, expressed for instance on gut epithelium. HA1 and HA2 are expressed on leukaemic cells and some tumour cells, making them potential targets for cellular therapy. Minor HLA antigens are restricted by certain HLA types such as HLA-A2 for instance.
