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

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Wai H. Lim1,2* , Brigitte Adams3, Stephen Alexander4,5, Antonia H. M. Bouts6, Frans Claas7, Michael Collins8,9, Elisabeth Cornelissen10, Heather Dunckley11, Huib de Jong12, Lloyd D’Orsogna13,14,15, Anna Francis16,17, Sebastiaan Heidt7, Jean Herman18, Rhonda Holdsworth19, Joshua Kausman20,21,22, Rabia Khalid23,24,

Jon Jin Kim25,26,27, Siah Kim4, Noël Knops18,28, Vasilis Kosmoliaptsis29,30,31, Cynthia Kramer7, Dirk Kuypers32,33, Nicholas Larkins2,24,34, Suetonia C. Palmer35, Chanel Prestidge36, Agnieszka Prytula37, Ankit Sharma24,38, Meena Shingde39, Anne Taverniti24, Armando Teixeira‑Pinto23, Peter Trnka16,17, Francis Willis2,34,40,

Daniel Wong2,41 and Germaine Wong23,24,38

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Abstract

Background: Parental donor kidney transplantation is the most common treatment option for children and adoles‑ cents 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 diferences in immunological compatibility between maternal and paternal donor kidneys and ascertain how this afects kidney allograft outcomes in children and adolescents with kidney failure.

Methods: This longitudinal observational study will recruit kidney transplant recipients aged ≤18years 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 compat‑ ibility. 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 stratifcation for the primary and secondary outcome measures among young people accepting a parental donor kidney for transplantation. This study design will allow us to specifcally 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 fndings will explore potentially diferential 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).

Keywords: Kidney transplant, Children, Adolescents, Parental donor, Immunological profle, Human leukocyte antigen, Antibody, Rejection, Allograft loss

Background

Kidney transplantation is the treatment of choice for peo- ple with kidney failure, conferring a signifcant survival advantage compared to people treated with long-term dialysis [1]. Live donor kidney transplantation is pre- ferred because survival rates of patients and allografts are substantially longer in comparison with deceased donor transplants [2–4]. In addition, living donation facilitates pre-emptive transplantation, avoiding the deleterious efects on health, psychosocial and educational outcomes of dialysis treatment on children during critical periods of growth and development [3].

Most pediatric and adolescent kidney transplant recipients require subsequent transplants and following allograft failure are often highly sensitized (with development of multiple anti-human leukocyte antigen [HLA] antibodies that may hinder future transplant potential) and at higher risk of death [5]. Selecting the donor kidney associated with the best possible long-term allograft outcome is vital. Parental donors account for over 60% of live donor kidneys for pediatric and adolescent patients with kidney failure in the United Kingdom, Australia and New Zealand [6, 7]. However, it is unclear whether maternal versus paternal donation is associated with diferential allograft outcome. A recent population cohort study showed that kidney transplants from mater- nal donor kidneys were associated with an up to a 60% greater risk of acute rejection and allograft loss compared to paternal donor kidneys [8]. Tis fnding is contradic- tory to the previously held paradigm that exposure to non-inherited maternal antigen (NIMA) may incite a lesser immunological response compared to exposure to non-inherited paternal antigen (NIPA). Previous cohort studies and experimental models have suggested that exposure of a child to the NIMA during pregnancy may lead to NIMA-specific tolerance. In one study, sibling transplants expressing NIMA were associated with lower risk of rejection and superior allograft outcomes com- pared to transplants expressing the NIPA [9–11]. Given that parental donors are the predominant source of live donor kidneys for pediatric and adolescent patients with kidney failure, this study will help inform whether HLA matching at the epitope, amino acid and physiochemi- cal properties level may enhance the understanding of the differential antigenicity and immunogenicity of NIMA and NIPA on allograft outcomes following paren- tal donor kidney transplantation. Te fndings from this study may potentially have important clinical implications in the selection of the appropriate parental donor kidney for transplantation.

The over-arching objective of this study is to address the current knowledge gap of the “NIMA paradox”by identifying potential diferences in immunological com- patibility between maternal and paternal donor kidneys and how this compatibility afects allograft outcomes. Te study aims include:

(i) Detailed immunological profling for pediatric and adolescent recipients of parental donor kidneys with and without adverse allograft outcomes of the development of de novo donor-specifc anti-HLA antibody (DSA), any acute rejection or allograft loss;

(ii) Determine the association between different immunological risk profles and adverse allograft outcomes; and

(iii) Determine whether the associations between the immunological risk profles and adverse allograft outcomes are modifed by parental status and other clinical factors.

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Methods/design

Study design and setting

The INCEPTION study is a longitudinal observational study that will recruit pediatric and adolescent transplant recipients aged ≤18years who have received parental donor kidneys between January 1990 and December 2020 (Fig. 1). Corresponding parental donors will also be

recruited for inclusion in the study. Adult kidney trans- plant recipients of parental donor kidneys aged > 18 years at time of transplantation or recipients of deceased donor kidney transplants will be excluded. Funding for the INCEPTION study is provided by the National Health and Medical Research Council (NHMRC) Ideas Grant (Application ID: APP1184595, funding duration 2020- 2023), the Department of Health Western Australia and the Telethon-Perth Children’s Hospital Research Fund (funding duration 2017-2020) and Starship Foundation Clinical Research Grant (Auckland, New Zealand). Te reporting and conduct of the study will adhere to Te Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines [12].

Tis study will be conducted across 4 countries includ- ing Australia (5 transplant centres), New Zealand (1 transplant centre), United Kingdom (UK, 13 transplant centres) and the Netherlands (3 transplant centres). Te INCEPTION study has been registered with the Aus- tralian New Zealand Clinical Trials Registry (ANZCTR) prior to recruitment, with the trial registration number of ACTRN12620000911998 (registration approved 14th September 2020).

Study recruitment and data capture In Australia and New Zealand, parental donor-recip- ient pairs that fulfl the inclusion criteria will be identi- fed through the Australia and New Zealand Dialysis and Transplant (ANZDATA) registry and forwarded to local site investigators. Only donor-recipient pairs recorded in the registry as living and residing in Australia and New Zealand will be approached for participation. Each donor-recipient pair will provide written or verbal informed consent or age-appropriate assent by the site study investigators or delegates before participation. Te Sir Charles Gairdner Osborne Park Health Care Group Human Research Ethics Committee (Perth, Western Australia, Australia) approved the research application in December 2018, with reciprocal ethics approvals cover- ing all participating sites in Australia and New Zealand. Consent for New Zealand participation was obtained from the New Zealand Health and Disability Ethics Com- mittee (Ministry of Health, Wellington, New Zealand). Verbal consent will be utilized during the current pan- demic of COVID-19 infection, with the HREC of each participating site granting the approval to this amend- ment if appropriate. In the United Kingdom (approval from the United Kingdom National Health Service [NHS] in England and Wales) and the Netherlands (approval from the Medical Ethics Review Committee of the Amsterdam Academic Medical Center), project-specifc waiver of consents for serum or deoxyribonucleic acid (DNA) biobanking and conduct of research studies relat- ing to kidney transplant outcomes have been granted, and the conduct of this study have received updated eth- ics approvals.

Study procedure

In Australia and New Zealand, all consented donor-recipient pairs will attend a single study visit to have 20 mL of blood drawn for separation into serum/plasma and deoxyribonucleic acid (DNA) isolation. Tis blood sampling will be taken from all donor-recipient pairs regardless of the availability of prior stored serum/plasma and DNA for testing. Te timing of this additional blood test for recipients will coincide with their routine clinical blood withdrawal where possible. Donor and recipients will also consent to allow access of their health records from ANZDATA registry (or equivalent healthcare regis- tries in other countries), Organ Matching (Australia-spe- cifc) and other country-specifc Blood Service systems (a computer data system that stores information relating to organ allocation and donor kidney details, including information on donor and recipient HLA typing and pre- transplant and de novo DSA) and local health records from their respective hospitals. For donor-recipient pairs in the UK and the Netherlands, the ethics approval will allow for the utilization of the stored serum/plasma and DNA for testing (where required), with no additional blood sampling to be requested from these pairs.

Defning detailed donor‑recipient immunological risk profles

High resolution HLA typing

Over the last decade, HLA-typing has evolved from sero- logical typing to high resolution molecular typing cover- ing HLA-A, −B, −C, −DRB1/3/4/5, −DPA1, −DPB1, −DQA1 and -DQB1 loci, providing a more comprehen- sive and accurate assessment of tissue compatibility in transplantation. High-resolution molecular HLA typ- ing for the 11 HLA-loci will be performed using a Next Generation Sequencing (NGS) or an alternative high- resolution typing technique by the respective tissue typ- ing laboratories in each country. Given NGS HLA-typing across all HLA alleles is considered standard practice for living donor transplantation in many transplanting cent- ers, re-typing of donors/recipients will not be undertaken unless typing across all HLA alleles at the high-resolution level is not available.

Quantifying HLA‑eplet mismatches

Epidemiological studies have consistently shown an association between an increasing number of epitope or eplet mismatches and an increased risk of adverse allograft outcomes, including the development of de novo DSA, antibody mediated rejection (AMR), transplant glomerulopathy (TG) and/or premature allograft loss [13, 14]. High-resolution molecular typing provides the oppor- tunity for a more comprehensive and accurate assess- ment of HLA compatibility in transplantation. However,compatibility based on the many thousands of HLA alleles is fundamentally impossible, but the variation in HLA alleles can be explained by diferences in a relatively low number of epitopes, which are part of the HLA pro- tein, made up of polymorphic amino acid residues. Eplets are short discontinuous sequences of amino acid residues within each HLA epitope that can theoretically elicit a B-cell driven immune response in the recipients (immu- nogenicity) [15, 16]., Terefore, compatibility assessment focusing on eplet mismatches, calculated using a computer program known as HLAMatchmaker [16, 17], is likely to have the potential to improve prediction of adverse allograft outcomes [18]. Te identifcation and quantifcation of the respective location and number of eplet mismatches at HLA class I (HLA-A, −B, −C) and class II (HLA-DRB1/3/4/5, −DPA1, −DPB1, −DQA1 and -DQB1) loci will be determined by entering the 2-feld molecular HLA typing for each donor and recipi- ent pair into the HLAMatchmaker to enable a comparison of the HLA eplet-profles of each donor/recipient pair (HLA-ABC v2.0 and HLA-DRDQDP v2.1; http:// www.eplets.net).

Donor‑recipient amino acid polymorphisms and physicochemical HLA incompatibility

Eplets are theoretically defned (combinations of) amino acids that have been subject to change, making clinical integration of this approach difcult [19–21]. Assess- ment of the physicochemical disparity between donor and recipient HLA, in addition to comparing the inter- locus and intralocus amino acid polymorphisms in antibody-accessible regions of the HLA molecules may improve risk stratifcation and predict adverse outcomes post-transplant [22–24]. Te specifcity and stability of antigen-antibody binding is determined by the struc- tural compatibility and the electrostatic and hydropho- bic interactions between the two molecules [25]. Donor and recipient HLA class I and class II mismatches will be compared at the sequence level to derive the number and physicochemical disparity of amino acid polymor- phisms on donor HLA molecules using the Cambridge HLA immunogenicity algorithm [22–24]. Te solvent accessibility of these amino acid polymorphisms will also be assessed using the HLA Epitope Mismatch Algorithm (EMMA; https://hla-emma.com) and Cambridge HLA immunogenicity algorithm [24, 26]. Te electrostatic dis- similarity between donor and recipient HLA molecules at the structural level will be assessed using the recently described electrostatic mismatch score (EMS-3D) [27]. Tese scores may provide additional predictive value for class I and II allo-antibody responses but the clini- cal applicability of one or more of these scores remains unclear [24, 27].

Quantifying the role of T‑cell help in DSA development

Until recently, the contribution of T cells to DSA devel- opment was not quantifable. T-cell help is essential in the development of immunoglobulin (Ig)-G DSA by pro- moting the diferentiation and proliferation of antigen- specifc naïve B cells into memory B-cells and plasma cells. Peptides derived from donor HLA molecules are presented by HLA class II molecules of recipient B cells to cognate helper T cells, which then provide signals and cytokines to these B cells to undergo afnity maturation, class switching and diferentiation into plasma cells [28]. We will utilize the NetMHCIIpan software (www.cbs.dtu. dk/services/NetMHCII-2.3 and www.cbs.dtu.dk/servi ces/NetMHCIIpan-3.2) [29] to determine the pan-spe- cifc binding of peptides to MHC class II alleles of known sequence using pre-specifed afnity thresholds and pep- tide ranking and other approaches in development by the study team [22, 23, 30]. In addition, we will also uti- lize the predicted indirectly recognizable HLA epitopes (PIRCHE) computational algorithm, which is designed to predict indirectly recognizable HLA-derived donor peptides that may induce T-cell allorecognition and lead to the production of donor-specifc anti-HLA IgG anti- bodies (https://www.pirche.com/pirche/#/features/bioin formatics) to quantify the“amount”of T- cell help [31]. A high number of PIRCHE, likely to represent a higher number of“theoretical”T-cell epitopes that can be pre- sented by recipient HLA class II molecules, may correlate with clinical alloreactivity but the clinical signifcance of PIRCHE on long-term allograft outcomes is yet to be determined.

Outcome measures

Te primary outcome is the composite of de novo DSA (defned as mean fuorescence intensity [MFI] of over 500), or any biopsy-proven acute rejection or allograft loss (defned as returning to dialysis or death with func- tioning allograft) after parental donor kidney trans- plantation up to 60-months post-transplant. Secondary outcomes are individual components of the primary composite outcome (development of de novo DSA, any biopsy-proven acute rejection, allograft loss at 60-months post-transplant but also extended follow-up to 120-months post-transplant), acute or chronic AMR (including the presence of TG) or Chronic Allograft Damage Index (CADI) score of > 1 on allograft biopsy post-transplant, allograft function (creatinine-derived estimated glomerular fltration rate [eGFR] using the Chronic Kidney Disease-Epidemiology Collaboration [CKD-EPI] equation for recipients aged ≥16years [32] or bedside Schwartz equation for recipients aged < 16 years [33–35]), and urine proteinuria.

De novo DSA

Post-transplant sera of all recipients are tested pre-transplant, when clinically indicated or routinely at prespecified time-points post-transplant (in some centres) for de novo DSA directed against either the maternal or paternal donor HLA across HLA-A, −B, −C, −DRB1, − DRB3/4/5, −DPA1, −DPB1, −DQA1 and -DQB1 alleles. In brief, an aliquot of a single antigen bead (SAB) mixture will be incubated with a small volume of patients’ sera (approximately 50 μl) containing the anti-HLA anti- body as per manufacturer’s instructions. Te analysis will be undertaken on a Luminex (or equivalent) platform and the reactivity will be determined with the manufacturer’s software and expressed as mean fluorescence intensity (MFI) for each mismatched HLA. Antibod- ies to these HLA alleles with MFI of varying thresholds (from MFI ≥ 500) will be considered a“positive result” because we and others have shown a consistent association between the presence of pre-transplant DSA with MFI ≥500 and a heightened risk of rejection after kidney transplantation [36, 37]. The MFI threshold value of ≥500 is defined using the Luminex platform. Te time- points for the monitoring and testing for pre-transplant and de novo DSA will be undertaken according to the standard policies of each participating site; typically, pre-transplant, annually post-transplant and/or when clinically indicated (during episodes of allograft dysfunction or acute rejection).

Assessment for acute rejection and CADI

Episodes of biopsy-proven acute and chronic rejection (diagnosed on protocol or clinical indication biopsies) post-transplant will be assessed and reported according to the validated Banf classification [38]. Consistent with this, each rejection episode will be categorized as acute, chronic or acute on chronic; and further stratifed to cellular, vascular, antibody-mediated or mixed pattern rejections. The most severe type of rejection in those with a mixed pattern will be considered the dominant lesion (i.e. vascular or antibody-mediated > cellular rejections). TG (Banf cg 1-3) will be coded according to the Banf classifcation and is considered a morphological manifestation of chronic AMR [39].

Te CADI score quantifies the amount of chronic damage to the allograft, with the score calculated from a total of six parameters of: (a) difuse or focal inflammation and (b) fbrosis in the interstitium, (c) mesangial matrix increase and (d) sclerosis in glomeruli, (e) inti- mal proliferation of vessels, and (f) tubular atrophy; with each individual parameter being scored between 0 and 3 as described in other studies [40]. Te CADI score is a globally standardized scoring system based on pre-defned criteria and is considered part of standard reporting in all pathology laboratories. Previous studies have consist- ently shown that an elevated CADI score correlates with acute rejection as well as chronic rejection and allograft loss up to 4 years post-transplant, indicating that CADI score has important prognostic signifcance [41–45].

Renal allograft biopsies (protocol or clinical indication biopsies) are frequently undertaken after kidney trans- plantation and are typically prepared into formalin-fxed parafn-embedded tissue blocks or stored as frozen tis- sues. Representative images or whole slide imaging (using digital microscopy or site-specifc systems such as the Sysmex system according to site availability) for histological scoring to capture the abnormal fndings will be requested from each histopathology department, which will comprise of at least one Periodic acid–Schif (PAS) and one Trichrome-stained images (where pos- sible). If multiple biopsy specimens were available in the frst 12 months post-transplant, only tissues from two of these biopsies (one between 0 and 6 months and the other between > 6-12 months if available) will be imaged. A nominated blinded pathologist will re-score the stored digital images (where available) in accordance with the established CADI parameters and Banf criteria for rejec- tion and chronicity [41, 46].

Clinical and laboratory parameters

In addition to the immunological profling and outcome measures, a number of donor and recipient character- istics will be extracted from registries, databases and local hospital healthcare records where appropriate and available. Data to be extracted include donor factors of age, sex, body mass index, donor relationship to the recipient, comorbid conditions and race; recipient factors of age, sex, body mass index, race, socio-economic status (SES; derived from post-code in Australia or alterna- tive SES parameters in other countries), prior episodes of non-adherence to immunosuppressive agents or missed appointments (as recorded in healthcare records), comorbid conditions (including complex syndrome, uro- logical, liver or cardiac diseases) and dialysis duration; and transplant-related factors of transplant era, ischaemic time, sensitization status of percentage class I and II panel reactive antibody (%PRA), types and intra-patient variability of the calcineurin-inhibitor levels; all of which are known to be associated with the clinical outcomes of interest. Information relating to measures of potential sensitization to maternal antigens, including complications in maternal pregnancy including infections, breast-feeding and prior pregnancies will be sought from respective donors (where available).

Specifc outcome measures include acute rejection (and Banf classifcation) at any time point post-transplant (types, severity and response to treatment), allograft function (creatinine and eGFR at 3 and 6 months, then annually post-transplant), allograft failure (including causes of allograft failure) and death (including causes of death) will be extracted from ANZDATA registry or relevant sources in other countries; and kidney biopsy reports, rejection-related treatment and response to treatment, intensity of immunosuppression (therapeu- tic drug levels for calcineurin inhibitor [CNI] at 3, 6, 12, 24, 36, 48, and 60 months post-transplant), proteinuria (urine protein/creatinine ratios at 3, 6, 12, 24, 36, 48, and 60 months post-transplant if available) and number/dura- tion of all-cause and cause-specifc hospitalisations will be extracted from local hospital and laboratory data. Te data will be entered into a password-protected database (unique to each site) at least up to 5-years post-transplant (or until allograft loss up to 10-years post-transplantation using linkage data to ANZDATA and other country-spe- cifc registries).

Sample size calculation

Te sample size calculation is based on the primary com- posite outcome of de novo DSA, any episodes of acute rejection and allograft loss. In our ANZDATA registry study comparing the allograft outcomes of maternal and paternal donor kidney transplants, a greater propor- tion of kidney transplant recipients who had received maternal donor kidneys experienced any rejection epi- sode compared with recipients of kidneys from paternal donors (37 and 27%, respectively; p < 0.001) [4]. Data relating to the development of de novo DSA are not avail- able from ANZDATA registry. Assuming that up to 40% of recipients may develop de novo DSA, acute rejection or lose their allografts within 60 months post-transplant, a sample size of 479 donor/recipient pairs (1:1 allocation to maternal and paternal door kidneys) will be required to achieve a power of 80% with a two-sided signifcance of 5% for detecting a diference of 0.10 between mar- ginal proportions after applying continuity correction (and correlations score of 0.5 of at least 4 observations [for the composite outcome measures], and accounting for potential missingness of data of up to 10%) (Fig. 2). Te expected recruitment of 520 parental donor/recipi- ent pairs in the retrospective study should have adequate sample size and sufcient power to address the study question. Sample size calculation was determined using the expected time-averaged diference (TAD) between two means from continuous, correlated data using the GEE method in PASS Sample Size Software.

Between 2015 and 2018, there were 182 pediatric and adolescent patients aged ≤18years with kidney fail- ure in Australia who received a kidney transplants, with parental donor kidney transplants contributing 40% of total transplants. Tis compared with 30 patients in New

Zealand (67% parental donor kidney transplants) and 30 patients in the Netherlands (30-40% parental donor kid- ney transplants) in the same time-period. In the United Kingdom, there were 760 pediatric and adolescent patients who received kidney transplants between 2010 and 2015, with approximately 340 (45%) parental donor kidney transplants. Tese recent fgures suggest that the target sample size (to recruit 520 donor/recipient pairs between 1990 and 2020) is achievable.

Statistical analysis

Data will be presented as mean ± standard deviation (SD) or number (proportion) for continuous and categori- cal variables, respectively, with means and proportion compared using t-test and the chi-squared test where appropriate. We will develop models with diferent num- bers of archetypes and choose which to use as the fnal model according to the residual sum of squares using the“elbow”method [47]. Te archetypal models will assign scores based on a combination of pre-transplant immunological and clinical (donor, recipient and transplant) parameters to each recipient using the time from transplant to the composite primary outcomes of de novo DSA, any episodes of acute rejection and allograft loss; with the scores totalling 1. Each parameter will be assigned to a single archetype cluster on the basis of the highest archetype score (corresponding to “high immu- nogenic risk”profle, comprising of immunological risk factors). Additional models accounting for pre- and post- transplant factors (such as donor age, non-adherence) will also be constructed. A principal component analy- sis will be constructed to visualize the data matrix used as the input for the archetypal analysis. Te principal component analyses will produce two main results: (i) a correlation circle, and (ii) a projection of the individu- als. Te correlation circle allows for a graphical exami- nation of the relationships among the pre-transplant immunological and clinical parameters and the graphical parameter contribution of the axes (positive or negative contribution: vector direction; strength of the contribu- tion: vector length when projected on the axis). We will identify distinct groups (i.e. archetypes), each compris- ing of a well-defned set of immunological and clinical parameters that may improve the risk stratifcation for “adverse immunological outcomes”for those accepting a parental donor kidney for transplantation, and separately for those who have received maternal or paternal donor kidneys. Te Australian and New Zealand cohorts will be the derivation cohort for these archetypes, which will be validated in the cohorts from the United Kingdom and the Netherlands. We will next seek to build a predictive model (combined cohorts) to examine the associations between the archetype clusters and other pre-specifed covariates and the primary outcome using univariate Cox proportional hazards regression models. A multivariable Cox model will be constructed by selecting covariates by Group Lasso and Doubly Robust Estimation (GLiDeR), a method of variable selection to identify confounders using an adaptive group lasso approach that simultane- ously performs coefcient selection, regularisation, and estimation across the treatment and outcome models. Bootstrap resampling with replacement or subsampling without replacement will be used to investigate and quan- tify model stability. Separate models will be constructed for each of the secondary outcomes. Te results of the model and covariates will be reviewed to ensure clinical relevance. Deviance and score residuals will be plotted to evaluate for poorly predicted individuals or individu- als that may have had large infuences on model parame- ters. Te performance of the integrated prognostic model will be assessed by computing a non–time-specifc area under the curve (AUC) using the measure of concord- ance from Somer’s Dxy statistic. For assessment of model calibration, patients will be risk-stratifed on the basis of their predictive index, which is the linear combination of the product of the multivariate model β coefcients and their individual covariate values. Kaplan-Meier survival curves will then be plotted for patients who are strati- fed into quintiles of their predictive indices. Te valida- tion cohort’s AUC will be determined and predicted and observed mortalities will be compared. Te association between archetypes and adverse outcomes will be exam- ined in the prospective cohort, and to examine how the addition of other newly developed novel immunological assays will improve the test performance of the models.

Discussion

Te INCEPTION study including parental donor kid- ney transplant recipients across 4 countries refects a study cohort of diverse ethnic distributions, which will allow for the generalizability of fndings. Te presence of local site study investigators and the implementation of site-specifc study processes and procedures will ensure that we achieve the target recruitment. Te investiga- tors/authors (and delegates) of the protocol manuscript and appointed consumer and registry representative(s) of each country will form the steering and data moni- toring committee, which will convene bi-annually and address issues relating to the conduct of the research and adherence to study protocol and processes. Any change in protocol or data capture will need to be approved by the steering and data monitoring committee prior to implementation.

Storage of genetic materials and serum for related future research projects

With the current funding restrictions for this study, not all HLA and non-HLA genes or identifcation of anti- HLA and non-HLA antibodies will be possible. Genetic materials and sera obtained from patients (and corre- sponding donors) will be stored for a period of 15 years after completion of the initial study until future funding for additional projects become available. Te steering and data monitoring committee of this study will provide oversight of potential research proposals regarding the use of these specimens and de-identifed health informa- tion for future related projects.

Confdentiality, data storage and record retention

All data will be managed in a confdential manner. Data will be stored on a secure server and only authorized investigators in the study team or their delegates will have access to the data. Health-related data will be retained for at least 15 years after completion of the project and relevant publications. Te additional DNA and sera that are extracted/isolated and stored will be kept in a local or central facility, which will be pre-identifed prior to com- mencement of study. If participants withdraw consent during the research project, the study doctor and rel- evant study staf will not collect additional information, although personal and other relevant information already collected will be retained to ensure that the results of the research project can be measured properly and to com- ply with law. Genetic materials and sera obtained for the purpose of this study will be kept locally or centrally for 15 years following completion of the initial study, after which the specimens will be destroyed and discarded appropriately.

Dissemination of outcomes of project

It is anticipated that the results of this research project will be published and/or presented in a variety of forums, including national and international medical conferences. In any publications and/or presentations, only aggregate information will be provided in such a way that no par- ticipants can be identifed. Te results of these fndings will also be disseminated through a newsletter summa- rising the salient fndings to all study investigators, site investigators and other relevant people directly involved in the care of kidney transplant recipients. A summary of the study fndings in lay person’s language will be dis- seminated to the participants.

Te INCEPTION study fndings will provide evi- dence to support or refute the apparent contradic- tory paradigm of diferential outcomes associated with maternal versus paternal transplants, which can inform healthcare providers, clinicians, patients and their families of potential risks and expected long-term allo- graft outcome after accepting maternal compared to paternal donor kidneys. As children and adolescents have a higher need for maximizing transplant survival, the INCEPTION study is critical to supporting the evidence base to improve survival particularly for chil- dren and adolescents with kidney failure. In addition, kidney transplantation is associated with improved cognitive functioning [48, 49], health related quality of life [50], educational attainment and life participa- tion [51]. Hence maximizing frst allograft survival has ensuing benefts through increased social integra- tion and labour productivity at the societal level. Te INCEPTION study is important to improve the under- standing of the immunological diferences between accepting a kidney from the mother or father, which has not been possible until the recent development and availability of cutting-edge technology. Tis proposed program of work will enable an improved selection of the best available parental donor kidneys for children and adolescents with kidney failure, with subsequent improvements in the health outcomes for this at-risk population. Tere is considerable uncertainty regarding the utility, clinical application and signifcance of some of these methods and this study will systematically evaluate and validate the predictive power of combin- ing the existing and novel pre-transplant B- and T cell molecular mismatch approaches to establish the infu- ence of genetic compatibility in determining adverse allograft outcomes.

Additionally, this resource will further stimulate research interest in this and related areas leading to further improvements in kidney transplants and patient out-comes for this and other populations. Specifcally, these include:

1) Te predictive value of novel assessment of donor/ recipient gene compatibility for adverse allograft and patient outcomes such as acute rejection, allograft loss, recurrence of primary kidney disease and other complications occurring after kidney transplantation.

2) Te potential for individualizing immunological risk assessment to reduce adverse allograft outcomes in kidney transplant recipients.

3) Establishment of an important resource that will comprise the largest cohort of pediatric and adolescent kidney transplant recipients worldwide, with the availability of high-resolution HLA typing (using the most advanced typing technique in NGS sequencing) and complete allograft and patient data. The improvement of outcome in this cohort of patients is critical because of their projected lifespan, burden of chronic disease and the likelihood that these recipi- ents will require repeat transplantation and continuing long-term utilization of healthcare resources in the treatment of their disease burden.

Abbreviations

AMR: Antibody-mediated rejection; AUC: Area under the cruve; ANZDATA: Australia and New Zealand Dialysis and Transplant; ANZCTR: Australian New Zealand Clinical Trials Registry; CADI: Chronic Allograft Damage Index; CKD‑ EPI: Chronic Kidney Disease‑ Epidemiology Collaboration; DSA: De novo donor‑ specifc anti‑ HLA antibody; DNA: Deoxyribonucleic acid; EMS‑3D: Electrostatic mismatch score; eGFR: Estimated glomerular filtration rate; GLiDeR: Group Lasso and Doubly Robust Estimation; HLA: Human leukocyte antigen; Ig: Immunoglobulin; MFI: Mean fuorescence intensity; NHMRC: National Health and Medical Research Council; NGS: Next-generation sequencing; NIMA: Non‑inherited maternal antigen; NIPA: non-inherited paternal antigen; PRA: Panel reactive antibody; PAS: Periodic acid–Schif; PIRCHE: Predicted indirectly recognizable HLA epitopes; SES: Socioeconomic status; STROBE: Strengthen‑ ing the Reporting of Observational Studies in Epidemiology; TG: Transplant glomerulopathy; UK: United Kingdom.

Acknowledgments

We would like to acknowledge Dr. Sean Kennedy, Dr. Hugh McCarthy and Dr. Fiona Mackie from the Sydney Children’s Hospital, Sydney, New South Wales for participating in this study.

Authors’ contributions

WL and GW conceptualized and designed the study and were responsible for the initial draft. FC, AF, SH, VK, CK, JK, PT, NL, AT, AT‑ P assisted in the design of the study. BA, SA, AB, FC, MC, EC, HD, HdJ, LD, AF, SH, JH, RH, JK, RK, JJK, SK, NK, VK, CK, DK, NL, SP, CP, AP, AS, MS, AT, AT‑ P, PT, FW, DW critically appraised and helped to prepare the draft. WL and GW were responsible for the final draft. The author(s) read and approved the final manuscript.

Funding

Funding for the INCEPTION study is provided by the National Health and Medical Research Council (NHMRC) Ideas Grant (Application ID: APP1184595, funding duration 2020‑2023), the Department of Health Western Australia and the Telethon‑ Perth Children’s Hospital Research Fund (funding duration 2017‑2020) and Starship Foundation Clinical Research Grant (Auckland, New Zealand). All funding was subjected to vigorous full external peer-review processes.

Availability of data and materials

Data sharing not applicable to protocol paper. Future study data will be presented within the manuscript and additional supporting files (where appropriate), but the availability of these data (in public repositories) will be according to each country‑specifc governance process.

Declarations

Ethics approval and consent to participate

Ethics approval was granted by Sir Charles Gairdner Osborne Park Health Care Group Human Research Ethics Committee (PRN: RGS930), with reciprocal approvals for all sites in Australia. Written or verbal consents will be obtained from all participants in Australia, with the process of verbal consents approved by the ethics committee during the COVID‑ 19 pandemic.

Consent for publication

Consents will be sought with participant recruitment.

Competing interests

The authors declare that they have no competing interests.

Author Details

1 Department of Renal Medicine, Sir Charles Gairdner Hospital, Perth, Western Australia 6009, Australia. 2 Medical School, University of Western Australia, Perth, Australia. 3 Department of Pediatric Nephrology, Hôpital Universitaire des Enfants‑ Reine Fabiola, Brussels, Belgium. 4 Department of Nephrology, Westmead Children’s Hospital, Sydney, Australia. 5 Faculty of Medicine and Health, University of Sydney, Sydney, Australia. 6 Department of Pediat‑ ric Nephrology, Emma Children’s Hospital, Amsterdam University Medical Center, Amsterdam, The Netherlands. 7 Department of Immunology, Leiden University Medical Centre, Leiden, The Netherlands. 8 Department of Renal Medicine, Auckland District Health Board, Auckland City Hospital, Auckland,