Defining the structural basis for human alloantibody binding to human leukocyte antigen allele HLA-A*11:01

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope−epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

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Clearly defined error bars
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Software and code
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Data collection
No software was used for data collection. Data analysis MIXCR (v2.0.2) was used for analyzing the next-generation sequencing results of B cell; briefly, to define complementarity-determining regions (CDRs) and framework regions, and germline genes and cluster reads likely to originate from the same antibody clone. The structural data set was processed with XDS and XscALE (version Jan 26 2018). The structure of the complex protein was determined by molecular replacement using PHASER-MR within the PHENIX software package (version 1.13-2998-000), and using the PDB code: 1W72 as a search probe. A model for the 3D structure of the complex HLA-A*11:01 with 2E3-Fab was built iteratively at the computer graphics using COOT (0.8.9.1), and refined using BUSTER (version 2.10.3). Structure visualization and modeling was performed using USCF Chimera (version 1.12) and Pymol (Open-source pymol 1.8.6.0).
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April 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The datasets generated during X-ray crystallography are available in the worldwide Protein Data Bank (wwPDB) repository, under accession code 6ID4. The data that support the findings of next-generation sequencing of patient B cells are deposited to figshare under DOI 10.6084/m9.figshare.7618520. All other data generated or analysed during this study are included in this published article (and its supplementary information files). Source data for Fig. 1a, Fig. 1b, Fig. 4b, Fig. 4c, Fig. 4d, Fig. 4e, Fig. 5a, Fig. 5b, Fig. 5c, Fig. 5d, Supplementary Fig. 1b, Supplementary Fig. 1c

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No sample size-based calculation was performed. Experiments were repeated at least three times where applicable. The exact number of repeats are provided in the figure legends. Antibody binding was tested against 20 different Epstein-Barr virus-transformed B lymphoblastoid cell lines (EBV-BCLs) that express common Asian HLA Class I types.
Data exclusions No data has been excluded.

Replication
Please note that the Clinical prognostic test on a patient sample was an official clinical test performed by the Health Sciences Authority (Singapore) laboratory that is internationally accredited to perform this test. This experiment was therefore not replicated.
The clinical prognostic test on monoclonal antibody 2E3 was also performed by the Health Sciences Authority (Singapore) laboratory. It was replicated and the reactive HLA Class I alleles identified were the same. Differences in the exact mean fluorescence intensity (MFI) values were expected because this is not a quantitative assay. ELISA, kinetic study, flow cytometry, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays were successfully repeated 3 times. Complement-dependent cytotoxicity (CDC) assays were successfully repeated 4 times. Sorting of human participant peripheral blood mononuclear cells (PBMCs) and subsequent next-generation sequencing was not repeated due to sample availability and the nature of the experiment.

Recruitment
The study was approved by the National Healthcare Group Domain Specific Review Board (NHG DSRB) which is accredited by the Association for the Accreditation of Human Research Protection Program (AAHRPP). The subject recruited in this study was recruited as part of a larger study to evaluate Anti HLA antibody specificity and antibody characteristics among prevalent renal transplant recipients on follow-up at the National University Centre for Organ Transplantation (NUCOT). Eligible subjects were approached for consent to participate in this study when they were seen in the NUCOT clinic for their regular clinic visit. The main components of the study protocol were: 1. Collection of 45 ml of blood; 2. Identification of Anti HLA antibody specificity from testing of serum samples from enrolled patients; 3. Collection of relevant clinical, laboratory and immunological information on enrolled patients with and without Anti HLA antibodies; 4. Analysis of Anti HLA specificity of antibodies by the Single Antigen Bead Luminex assay performed at an American Society for Histocompatibility and Immunogenetics (ASHI) certified laboratory in Singapore (HSA); 5. Identification of donor specificity of Anti HLA antibodies by examination of Donor and Recipient HLA typing.
Potential subjects were given the option of declining participation in this study with the assurance that their care would not be nature research | reporting summary

April 2018
affected. Blood collection will be performed at the next clinic visit so as to give additional time for the patients to withdraw from the study if they wished to do so. Subjects had the information given in a language they could understand with appropriate translation as required. All consents were witnessed by a third party and study procedures performed only after written and informed consent was obtained.
Inclusion criteria: 1. Adult kidney transplant recipients ie. between the ages 21 and 90 years old 2. Asian race/ethnicity 3. Have received a documented deceased or live donor kidney transplant and are currently on follow-up at the National University Centre for Organ Transplantation (NUCOT) in NUH, and who agree to provide an additional 45ml of blood for research purposes.
Exclusion criteria: 1. Intercurrent Illness 2. Pregnancy (clinically confirmed) 3. Unable to give informed consent 4. Kidney transplant from an Undocumented overseas donor The following possible sources of bias were considered in this study 1. The study design only included prevalent transplant patients with a functioning allograft. Hence those with a particularly pathogenic anti HLA antibody that led to graft loss would have been excluded. 2. All potential subjects were given information in a language they could understand and were given the opportunity to decline participation. All potential subjects were also given time to consider the study proposal and had their blood taken for the purpose of the study at a subsequent visit and were allowed to withdraw consent at any time. Assurance of care by the clinicians at NUCOT was given regardless of provision of consent. All consents were obtained in the presence of a third party. With this study design, it is possible that patients who declined participation may have done so due to unique socio-cultural characteristics that prevented them from understanding the nature of the study and with complying to clinical treatments. While these could potentially lead to non-adherence and the development of Anti HLA antibodies, these inter-relationships were deemed to be minimal and expected to have minimal impact on this study. 3. Exclusion of kidney transplants from undocumented donors could have introduced some bias as NUCOT is a tertiary referral institution and a proportion of the prevalent patients would have been necessarily excluded. However this was necessary so as to include only those cases whose donor and recipient HLA typing was available so that the specificity of the Anti HLA antibody could be identified and so as to clearly identify Donor Specificity.
Hence, bias if present was deemed to be largely minimized by ensuring a rigorous consent process.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
Cell surface binding assay: in-house EBV-BCLs were washed, stained with relevant antibodies at 4°C for 1 h, protected from light. Cells were then washed in staining buffer (PBS supplemented with 1% fetal bovine serum)and acquired. CDC assay: Target cells were incubated with antibody of interest for 1h, followed by a 3 h incubation with 10% baby rabbit complement serum (Cedarlane, #CL3441). Cells were washed in 1x PBS, stained with sytoxgreen and resazurin (ThermoFisher Scientific, #L34951) for 15 min, protected from light. Cells were kept in a cell incubator for all incubation steps. ADCC assay: PBMCS from healthy donors were isolated and Natural Killer cells were enriched for via magnetic beads-based negative selection (STEMCELL #19055). NK cells were stimulated with 1500 units/mL IFN-alpha (ThermoFisher Scientific, #PHC4814) and cultured overnight at 37°C. Target cells were labelled with 1000x diluted CFSE (ThermoFisher Scientific, #C34564) and incubated with antibody of interest for 1 h. NK cells were then added (at a ratio of NK cells : target cells = 4:1) to the target cells for 4 h incubation. Cells were kept in a cell incubator for all incubation steps. Cells were then washed with staining buffer and labeled with 7AAD (ThermoFisher Scientific, #A1310) at a final concentration of 20 ug/mL. EBV-BCLs were washed and co-incubated with the relevant antibodies at 37°C. Cells were then stained with markers for viability for 15 minutes, washed and acquired. FACS sorting: PBMCs from a kidney transplant recipient were resuspended in staining buffer and stained with mouse-anti-human CD27-AF647, mouse-anti-human CD19-PB, and goat-anti-human IgM-AF488 for 1 hour at 4°C, protected from light. Cells were washed, resuspended, and stained with propidium iodide (Sigma-Aldrich #P4864-10ML) at a final concentration of 10 ug/mL.

Software
FlowJo VX