Role OF HLA Antibodies in the Stages of Graft Rejection

HLA alloantibodies have been implicated in all three stages of allograft rejection – Hyperacute (including Accelerated), Acute and Chronic rejection. Hyperacute rejection occurs within minutes of transplant, acute rejection occurs within days to weeks, while chronic rejection occurs months to years post transplant. Antibody mediated rejection has a different pathology to T cell mediated rejection. Antibody mediated rejection preferentially attacks the peritubular and glomerular capillaries in contrast to T Cells which typically infiltrate the tubules and arterial endothelium.

Hyperacute rejection occurs almost immediately after the kidney is transplanted and is usually due to the presence of pre-formed donor specific anti-HLA antibodies. The mechanism of hyperacute rejection involves deposition of antibodies against HLA antigens expressed on the endothelium of the glomeruli and the graft microvasculature. This leads to activation of the classical complement cascade causing endothelial necrosis, platelet deposition and local coagulation. Hyperacute rejection is usually accompanied by C4d deposition, though this may be negative early on. Hyperacute rejection is not typically reversible and requires the immediate removal of the graft. Accelerated rejection usually occurs within days and is due to a memory or anamnestic response to pre-formed antibodies directed against mismatched donor antigen which are absent at the time of transplantation. Improved antibody screening and identification techniques as well as improvements in crossmatch techniques have significantly reduced the incidence of hyperacute and accelerated rejection.

Acute rejection occurs days to weeks (or months in the case of late acute rejection) post transplant and may contain a cellular as well as a humoral component. Acute humoral rejection is caused by pre-existing donor specific anti-HLA antibodies which are negative at the time of the pre-transplant crossmatch and/or de-novo donor specific antibodies. Diagnosis of Acute antibody mediated rejection involves identification of rapid graft dysfunction, accompanied by the presence of circulating anti-donor HLA antibodies and biopsy evidence of C4d deposition in the peritubular capillaries. C4d may also be deposited in the glomeruli though this is variable. Acute antibody mediated rejection is reversible with treatment such as plasmapheresis and intravenous immunoglobulin plus increased immunosuppression (with for example tacrolimus and mycophenolate mofetil). The Anti-CD20 antibody Rituximab is also used in some cases.

The role of alloantibodies in chronic rejection is increasingly being recognised. Recent data from the 14th international histocompatibility workshop demonstrated that four year deceased donor kidney allograft survival was 20% less in patients with donor specific antibodies compared to donors with no HLA antibodies. Chronic rejection is characterised by slow progressive loss of renal function with endothelial antibody deposition leading to endothelial injury and glomerular basement membrane duplication characteristic of transplant glomerulopathy.

H&I laboratory tests carried out in support of the prevention or management of antibody mediated rejection in renal transplantation include HLA antibody screening and identification pre-transplant, crossmatching at the time of at the time of transplant as well as post transplant antibody monitoring either as part of a routine and regular program or at least at the time of suspected rejection episodes.

Current UK standards (BSHI/BTS 2010) require that HLA antibody screening and identification techniques used must be sufficient to identify antibodies to HLA-A, B, C, DR, DQ and DP. In addition, the testing must be able to distinguish IgG allo and IgM allo and auto antibodies. HLA antibody screening and identification has historically been by cell based techniques such as the complement dependent cytotoxicity (CDC) assay or a cell based flowcytometric assays. Currently however, most HLA antibody testing in the UK is by the solid phase Luminex bead based assays. These assays are much more sensitive than the traditional CDC techniques and require careful consideration of the clinical significance of Luminex positive, CDC negative tests.  Pre-transplant, this level of testing helps provide a list of unacceptable mismatches for renal patients, eliminating positive crossmatches and the impact this has in extending cold ischaemia times.

Once a patient is listed for transplantation, HLA antibody testing must be undertaken every three months and 2 – 4 weeks after potentially sensitising events such as blood transfusion. This helps build up a full antibody profile for the patient, avoiding the risk of an unexpected positive crossmatch. HLA antibody investigation is to the same level of detail as described for pre listing. i.e. Antibodies to HLA-A, B, C, DR, DQ and DP must be identified and the testing must be able to distinguish IgG allo and IgM allo and auto antibodies. The unacceptable mismatches listed with UK transplant must be updated.

In the live donor transplant setting initial crossmatches may be undertaken well in advance of the actual transplant as part of the donor selection process, with a final crossmatch at the time of transplantation. The purpose of any crossmatch test is to determine whether the patient has any preformed HLA antibodies which will react with antigens expressed by the donor and therefore helps to inform the immunological risk. In the UK, crossmatching is generally undertaken by CDC and/or flowcytometric assays. Cell separation techniques are used to ensure that reactivity to T and B cells can be distinguished. Tests used are also able to distinguish between IgG and IgM antibodies. In some centres, a virtual crossmatch is undertaken as the initial crossmatch in certain carefully selected cases.

At the time of transplant, antibody screening of the ‘current’ patient sample as well as pre-transplant crossmatching by CDC and flowcytometric techniques have significantly reduced the incidence of hyperacute rejection.

Post transplant, donor specific HLA antibodies contribute to acute humoral rejection characterised by rapid graft dysfunction, accompanied by the presence of circulating anti-donor HLA antibodies and biopsy evidence of C4d deposition in the peritubular capillaries. C4d may also be deposited in the glomeruli though this is variable. In addition, the role of HLA antibodies in chronic rejection is also increasingly recognised. Laboratory HLA antibody screening can be used to monitor patients with suspected rejection for circulating anti-donor HLA antibodies.

In HLA incompatible transplant, the role of H&I laboratory tests is crucial in monitoring the desensitisation to achieve a pre-transplant negative crossmatch as well as to closely monitor the patient post transplant for a rebound in antibody levels to help clinicians respond with therapies to reduce humoral rejection.

Mechanisms for HLA Antibody Removal

Plasma Exchange

Plasma exchange protocols aim to physically remove HLA antibodies to a negative crossmatch before transplantation. Some plasma exchange protocols also incorporate antithymocyte globulin (ATG) and anti-CD20 antibodies such as Rituximab. Depending on the strength of the DSA present, plasma exchange protocol can involve several sessions of whole volume exchanges. Patients need careful assessment as to whether or not they are healthy enough to withstand several rounds of plasma exchange. The H&I laboratory can help determine the number of rounds that may potentially be needed by undertaking HLA antibody titre tests.

HLA antibody removal is effective with the most commonly reported side effect being hypocalcaemia. Antibody removal by these protocols is however not specific to HLA IgG, removing other protective antibodies and clotting factor, potentially increasing the risk of infection or bleeding. This risk is reduced by the use of low dose IVIg and by transfusion if required.

Filtration

Filtration or Immunoadsorption involves an apheresis procedure in which the patients’ blood is ‘filtered’ by passing it through a column containing beads coated with an IgG absorber such as Protein A which selectively filters out IgG molecules. This has few of the side effects of traditional whole volume plasma exchange. A single filtration session typically reduces HLA antibody MFI levels to half their pre-filtration values. Several rounds of filtration are required to reduce antibody levels to achieve a negative crossmatch. HLA antibody levels rebound to some degree after each session as antibodies equilibrate between intravascular and extravascular spaces. The H&I laboratory can help determine the number of rounds that may potentially be needed by undertaking HLA antibody titre tests.

IVIg

IVIg is typically given over the course of several months until a negative crossmatch is achieved. A common dose is 1g/kg patient weight. The exact mechanism of action of high dose IVIg is unknown. Proposed mechanisms include an anti-idiotypic effect of IVIg on HLA antibodies, saturation of the neonatal Fc receptor which would normally protect endogenous HLA IgG molecules and induction of apoptosis in B cells.

One disadvantage of high dose IVIg protocols is that they have been associated with thrombotic complications.

Rituximab

Rituximab is an anti-CD20 recombinant chimeric murine-human monoclonal antibody. It binds to CD-20 which is expressed on precursors and mature B cells but not on plasma cells. Binding triggers a series of cytotoxic immune response resulting in the elimination of B cells and the reduction in antibody formation. As B cells play an important antigen presentation role to helper T cells, their reduction through Rituximab also reduces other immune responses.

Bortezomib

Bortezomib is proteasome inhibitor used mainly in the treatment of relapsed multiple myeloma. Proteasomes are thought to support the immortal phenotype of multiple myeloma by rapidly degrading pro-apoptotic factors. Bortezomib binds to the catalytic site of proteasome with high affinity and specificity, inhibiting its action and permitting the activation of programmed cell death. Bortezomib induces apoptosis in plasma cells, which are immune to Rituximab and is therefore very useful in desensitisation protocols in solid organ transplantation.