Major technological advances will enhance Australian donor-recipient matching and improve transplant outcomes.

In recent times, numerous and significant technological and supportive changes have taken place in Australian transplantation. These changes are often deployed without the wider clinical community having a full understanding of what has brought about these changes and the impacts they have. Here, we aim to clarify the reasoning behind these changes and shed light on potential future endeavours to improve patient outcomes.

Improve in-depth immunological risk assessment to optimize genetic-compatibility and clinical outcomes in child and adolescent recipients of parental donor kidney transplants: protocol for the INCEPTION study.

BACKGROUND: Parental donor kidney transplantation is the most common treatment option for children and adolescents with kidney failure. Emerging data from observational studies have reported improved short- and medium-term allograft outcomes in recipients of paternal compared to maternal donors. The INCEPTION study aims to identify potential differences in immunological compatibility between maternal and paternal donor kidneys and ascertain how this affects kidney allograft outcomes in children and adolescents with kidney failure. METHODS: This longitudinal observational study will recruit kidney transplant recipients aged ≤18 years who have received a parental donor kidney transplant across 4 countries (Australia, New Zealand, United Kingdom and the Netherlands) between 1990 and 2020. High resolution human leukocyte antigen (HLA) typing of both recipients and corresponding parental donors will be undertaken, to provide an in-depth assessment of immunological compatibility. The primary outcome is a composite of de novo donor-specific anti-HLA antibody (DSA), biopsy-proven acute rejection or allograft loss up to 60-months post-transplantation. Secondary outcomes are de novo DSA, biopsy-proven acute rejection, acute or chronic antibody mediated rejection or Chronic Allograft Damage Index (CADI) score of > 1 on allograft biopsy post-transplant, allograft function, proteinuria and allograft loss. Using principal component analysis and Cox proportional hazards regression modelling, we will determine the associations between defined sets of immunological and clinical parameters that may identify risk stratification for the primary and secondary outcome measures among young people accepting a parental donor kidney for transplantation. This study design will allow us to specifically investigate the relative importance of accepting a maternal compared to paternal donor, for families deciding on the best option for donation. DISCUSSION: The INCEPTION study findings will explore potentially differential immunological risks of maternal and paternal donor kidneys for transplantation among children and adolescents. Our study will provide the evidence base underpinning the selection of parental donor in order to achieve the best projected long-term kidney transplant and overall health outcomes for children and adolescents, a recognized vulnerable population. TRIAL REGISTRATION: The INCEPTION study has been registered with the Australian New Zealand Clinical Trials Registry, with the trial registration number of ACTRN12620000911998 (14th September 2020).

Insights into the labeling effect of Kidney Donor Performance Index reporting: The Australian experience.

In 2016, Australia began reporting the Kidney Donor Performance Index (KDPI) with all deceased donor kidney transplant offers despite this not being used in organ allocation rules, offering a unique opportunity to explore the "labeling effect" of KDPI reporting. We reviewed all kidneys retrieved for transplant in Australia from 2015 to 2018 and analyzed the association of KDPI reporting with organ nonutilization, number of offer declines, and donor/recipient age and longevity matching. Analyses were stratified by organ failure risk: higher risk (KDPI > 80%), standard risk (KDPI 20% to 79%), and lower risk (KDPI 0% to 20%). There was no significant difference in organ nonutilization post KDPI reporting either overall or for higher-risk kidneys. KDPI reporting was associated with an increase in offer declines for both higher-risk (incidence risk ratio 1.45, P = .007) and standard-risk (incidence risk ratio 1.22, P = .021) kidneys but not for lower-risk organs. There was a significant increase in recipient age and expected posttransplant survival score for higher-risk kidneys but no differences among other groups. We conclude that although KDPI reporting in Australia has been associated with an increased number of offer declines for higher-risk kidneys, this has not resulted in increased nonutilization and may have contributed to more appropriate use of these organs.

Tissue typing for kidney transplantation for the general nephrologist.

Tissue typing is the process by which an individual's human leukocyte antigens (HLA) are determined. In transplantation, this vital process allows the immunologic or rejection risk of a donor-recipient pairing to be assessed through reviewing their HLA matching and whether any anti-HLA antibodies present in recipient serum are donor specific. Tissue typing has increased in sophistication over time which has allowed a deeper appreciation of the antigenically important parts of HLA and increased the complexity of determining immunologic risk.

Human leukocyte antigen class I-restricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity.

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

Recurrent HLA-B56 mediated neonatal alloimmune thrombocytopenia with fatal outcomes.

Neonatal alloimmune thrombocytopenia is caused by maternal antibodies against paternally-inherited alloantigens present on foetal platelets and is a significant cause of morbidity and mortality in neonates. While generally thought of as a human platelet antibody mediated phenomenon, cases of HLA mediated NAIT have been reported. We document the investigation of a patient with two pregnancies affected by HLA-B56 mediated NAIT and a proposal for management of future pregnancies.

Four years of experience with the Australian kidney paired donation programme.

New approaches to increase kidney transplantation rates through expansion of live donor kidney transplantation have become necessary due to ongoing shortage of deceased donor organs. These strategies include desensitization in antibody-incompatible transplants to overcome the barrier of blood group incompatibility or human leucocyte antigen antibodies between recipient and donor and kidney paired donation (KPD) programmes. In KPD, a kidney transplant candidate with an incompatible live donor joins a registry of other incompatible pairs in order to find potentially compatible transplant solutions. To match the largest possible number of donor-recipient pairs while minimizing immunologic risk, KPD programmes use sophisticated algorithms to identify suitable matches with simultaneous two-way or more complex multi-way exchanges as well as including non-directed anonymous donors to start a chain of compatible transplantations. Because of the significant immunologic barriers when fewer donor options are available, the optimal solution for difficult-to-match, highly sensitized patients is access to more potential donors using large multi-centre or national KPD registries. This review focuses on the first 4 years of experience with the Australian multi-centre KPD programme that was established in October 2010.

C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

AIMS: Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. METHODS: We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. RESULTS: All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. CONCLUSIONS: These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers.

HLA-C mismatching improves outcomes following lung transplantation.

HLA (HLA) are a major barrier to transplant success, as HLA-A and -B molecules are principal ligands for T-cells, and HLA-C for Killer cell Immunoglobulin-like Receptors (KIR), directing Natural Killer (NK) cell function. HLA-C molecules are designated "C1" or "C2" ligands based on residues 77 and 80, which determine the NK cell responses. Here, we investigated donor/recipient HLA-C mismatch associations with the development of chronic lung allograft dysfunction (CLAD) following lung transplantation (LTx). 310 LTx donor/recipient pairs were Next Generation Sequenced and assessed for C1 and C2 allotypes. PIRCHE scores were used to quantify HLA mismatching between donor/recipients at amino acid level and stratify recipients into low, moderate or highly mismatched groups (n = 103-104). Associations between C ligands and freedom from CLAD was assessed with Cox regression models and survival curves. C2/C2 recipients (n = 42) had less CLAD than those with C1/C1 (n = 138) or C1/C2 genotypes (n = 130) (p < 0.05). Incidence of CLAD was lower in C2/C2 recipients receiving a mismatched C1/C1 allograft (n = 14), compared to matched (n = 8) or heterozygous (n = 20) allografts. Furthermore, ~80% of these recipients (C2/C2 recipients receiving C1/C1 transplants) remained CLAD-free for 10 years post-LTx. Recipients with higher HLA-C mismatching had less CLAD (p < 0.05) an observation not explained by linkage disequilibrium with other HLA loci. Our data implicates a role for HLA-C in CLAD development. HLA-C mismatching was not detrimental to LTx outcome, but potentially beneficial, representing a paradigm shift in assessing donor/recipient matching. This may inform better selection of donor/recipient pairs and potentially more targeted approaches to treating CLAD.

The effect of folinic acid on methylenetetrahydrofolate reductase polymorphisms in methotrexate-treated allogeneic hematopoietic stem cell transplants.

This study examined the contribution single nucleotide polymorphisms (SNPs) of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene have on clinical outcomes in hematopoietic stem cell transplant patients treated with the antiproliferative drug methotrexate. Two common SNPs, 677C>T and 1298A>C, were genotyped by polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) from samples obtained from patient DNA samples. Eleven clinical outcomes including survival and graft-versus-host disease (GVHD) were assessed against donor and recipient MTHFR genotypes against pretransplantation variables. Folinic acid (FA) as treatment for oral mucositis toxicity was used at investigator discretion in 72 of 140. Donor MTHFR 1298AA genotype was associated with decreased 5-year survival (P = .03) and event-free survival (EFS) (P = .02) in patients withheld FA. Donor MTHFR 677CC genotype was associated with earlier GVHD (P = .003), and more severe acute GVHD (P = 0.02). FA was significantly associated with decreased survival (P = 0.02) in patients given a donor MTHFR 677CT transplant. FA was significantly associated with decreased survival (P = .04), EFS (P = .009) in patients given a donor MTHFR 1298AC transplant MTHFR gene polymorphisms indicate a potentially useful gene for donor selection where more than one donor is available. Use of FA following transplantation should be reconsidered in the context of patient and donor MTHFR genotypes.

A new strategy for systematically classifying HLA alleles into serological specificities.

HLA serological specificities were defined by the reactivity of HLA molecules with sets of sera and monoclonal antibodies. Many recently identified alleles defined by molecular typing lack their serotype assignment. We surveyed the literature describing the correlation of the reactivity of serologic reagents with AA residues. 20 - 25 AA residues determining epitopes (DEP) that correlated with 82 WHO serologic specificities were identified for HLA class I loci. Thirteen DEP each located in the beta-1 domains that correlated with 24 WHO serologic specificities were identified for HLA-DRB1 and -DQB1 loci. The designation of possible HLA-DPB1, -DQA1, -DPA1, and additional serological specificities that result from epitopes defined by residues located at both -DQA1 and -DQB1 subunits were also examined. HATS software was developed for automated serotype assignments to HLA alleles in one of the three hierarchical matching criteria: (1) all DEP (FULL); (2) selected DEP specific to each serological specificity (SEROTYPE); (3) one AA mismatch with one or more SEROTYPES (INCOMPLETE). Results were validated by evaluating the alleles whose serotypes do not correspond to the first field of the allele name listed in the HLA dictionary. Additional 85 and 21 DEP patterns that do not correspond to any WHO serologic specificities for common HLA class I and DRB1 alleles were identified, respectively. A comprehensive antibody identification panel would allow for accurate unacceptable antigen listing and compatibility predictions in solid organ transplantation. We propose that antibody-screening panels should include all serologic specificities identified in this study.

Transfusion-related acute lung injury (TRALI): a retrospective review of reported cases in Queensland, Australia over 20 years.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a rare but potentially fatal transfusion reaction. An effective haemovigilance programme is important in implementing successful and targeted risk reduction strategies. We aim to provide a summary of TRALI cases referred for investigation in Queensland (QLD) Australia from 1999 to 2019, describing the epidemiological and laboratory features of local TRALI cases. MATERIALS AND METHODS: A retrospective audit evaluated all cases reported to the QLD Australian Red Cross Lifeblood over the 20-year study period. Cases were categorised according to the 2004 Canadian consensus criteria. RESULTS: Of the 91 cases referred for investigation, expert review confirmed 30 of TRALI and 18 of possible TRALI. A total of 238 donors and 110 blood products were assessed in confirmed cases. TRALI affected patients of all ages. Most patients had underlying haematological malignancies (25%), surgery (15%) or liver disease (13%). TRALI incidence was measured at 1 in 130,000 per issued product in QLD. Red cells were transfused in 32 cases, platelets in 18 and plasma products in 21, with 16 cases involving multiple products. Following laboratory assessment, 23% of cases had findings supportive of antibody mediated TRALI and 21% as likely non-antibody mediated. Possible TRALI was identified in 37.5% of cases of which 25% were antibody mediated and 12.5% non-antibody mediated. Nine (18.5%) cases were uncategorised due to insufficient immunologic investigations. DISCUSSION: Rates of TRALI incidence measured are lower than those seen in many international studies. A reduction in confirmed cases has been noted over recent years, supporting the implementation of risk-reduction strategies. We report a relatively higher proportion of non-antibody mediated TRALI and possible TRALI cases in more recent years, suggesting the need to further understand the role of product age and biological risk modifiers.

Identification and Re-consent of Existing Cord Blood Donors for Creation of Induced Pluripotent Stem Cell Lines for Potential Clinical Applications.

We aim to create a bank of clinical grade cord blood-derived induced pluripotent stem cell lines in order to facilitate clinical research leading to the development of new cellular therapies. Here we present a clear pathway toward the creation of such a resource, within a strong quality framework, and with the appropriate regulatory, government and ethics approvals, along with a dynamic follow-up and re-consent process of cord blood donors from the public BMDI Cord Blood Bank. Interrogation of the cord blood bank inventory and next generation sequencing was used to identify and confirm 18 donors with suitable HLA homozygous haplotypes. Regulatory challenges that may affect global acceptance of the cell lines, along with the quality standards required to operate as part of a global network, are being met by working in collaboration with bodies such as the International Stem Cell Banking Initiative (ISCBI) and the Global Alliance for iPSC Therapies (GAiT). Ethics approval was granted by an Institutional Human Research Ethics Committee, and government approval has been obtained to use banked cord blood for this purpose. New issues of whole-genome sequencing and the relevant donor safeguards and protections were considered with input from clinical genetics services, including the rights and information flow to donors, and commercialization aspects. The success of these processes has confirmed feasibility and utility of using banked cord blood to produce clinical-grade iPSC lines for potential cellular therapies.

A naturally selected dimorphism within the HLA-B44 supertype alters class I structure, peptide repertoire, and T cell recognition.

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the alpha2 helix (B*4402 Asp156-->B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this "minimal" mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.

Natural HLA class I polymorphism controls the pathway of antigen presentation and susceptibility to viral evasion.

HLA class I polymorphism creates diversity in epitope specificity and T cell repertoire. We show that HLA polymorphism also controls the choice of Ag presentation pathway. A single amino acid polymorphism that distinguishes HLA-B*4402 (Asp116) from B*4405 (Tyr116) permits B*4405 to constitutively acquire peptides without any detectable incorporation into the transporter associated with Ag presentation (TAP)-associated peptide loading complex even under conditions of extreme peptide starvation. This mode of peptide capture is less susceptible to viral interference than the conventional loading pathway used by HLA-B*4402 that involves assembly of class I molecules within the peptide loading complex. Thus, B*4402 and B*4405 are at opposite extremes of a natural spectrum in HLA class I dependence on the PLC for Ag presentation. These findings unveil a new layer of MHC polymorphism that affects the generic pathway of Ag loading, revealing an unsuspected evolutionary trade-off in selection for optimal HLA class I loading versus effective pathogen evasion.

TCD8 response in diverse outcomes of recurrent exposure to hepatitis C virus.

To analyse the immune correlates in a setting of recurrent exposure to hepatitis C virus (HCV), we studied T(CD8) responses in injecting drug users (IDUs) with different disease outcomes. Ex vivo HCV-specific T(CD8) responses assessed by interferon-gamma (IFNgamma) enzyme-linked immunospot (ELISPOT) were comparable in human lymphocyte antigen (HLA)-matched IDUs with spontaneous HCV clearance or persistent infection. A detailed characterization of these T(CD8) cells in age and HLA-matched IDUs demonstrated that HCV clearance and protection from reinfection correlated with HCV-specific T(CD8) cells that could proliferate in vitro, possessed cytotoxic potential and produced IFNgamma and tumour-necrosis factor-alpha, rather than with the circulating frequency of responding T(CD8) cells determined ex vivo. While validating the importance of multifunctional T(CD8) in mediating protection in IDUs with recurrent exposure to HCV our findings highlight that the magnitude and/or breadth of HCV-specific T(CD8) determined in ex vivo ELISPOT may not be the sole determinant of protection especially in a setting of recurrent exposure.

Review article: Luminex technology for HLA antibody detection in organ transplantation.

Since its inception in the early 1960s, the serologically based complement-dependent cytotoxicity (CDC) assay has been the cornerstone technique for the detection of human leucocyte antigen (HLA) antibodies, not only in pre-transplant renal patients, but also in other forms of organ transplantation. Recently, solid phase assays have been developed and introduced for this purpose, and in particular the Flow-based bead assays such as the Luminex system. This latter assay has proved to be far more sensitive than the CDC assay and has revealed pre-sensitization in potential transplant recipients not detected by other methods of HLA antibody detection. However, the clinical implications of this increased sensitivity have not been convincingly demonstrated until recently. This technology for HLA antibody detection permits the evaluation of the clinical importance of antibodies directed at, for example, HLA-DPB1 and HLA-DQA1, which has not been possible to date. There are Luminex issues, however, requiring resolution such as the ability to distinguish between complement fixing and non-complement fixing antibodies and determination of their relative clinical significance. Luminex technology will permit a re-evaluation of the role of HLA antibodies in both early and late antibody-mediated rejection.

Risk Indices in Deceased-donor Organ Allocation for Transplantation: Review From an Australian Perspective.

Over the last decade, organ donation and transplantation rates have increased in Australia and worldwide. Donor and recipient characteristics for most organ types have generally broadened, resulting in the need to consider more complex data in transplant decision-making. As a result of some of these pressures, the Australian software used for donor and recipient data management is currently being updated. Because of the in-built capacity for improved data management, organ allocation processes will have the opportunity to be significantly reviewed, in particular the possible use of risk indices (RIs) to guide organ allocation and transplantation decisions. We aimed to review RIs used in organ allocation policies worldwide and to compare their use to current Australian protocols. Significant donor, recipient, and transplant variables in the indices were summarized. We conclude that Australia has the opportunity to incorporate greater use of RIs in its allocation policies and in transplant decision-making processes. However, while RIs can assist with organ allocation and help guide prognosis, they often have significant limitations which need to be properly appreciated when deciding how to best use them to guide clinical decisions.

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.