DOI: 10.5061/dryad.g4f4qrfx9
title: Immunogenicity of autologous and allogeneic human primary cholangiocyte organoids

description: Primary human cells cultured in 3D organoid format have great promise as
 potential regenerative cellular therapies, but their immunogenicity has
 not yet been fully characterized. In this study, we use in vitro
 co-cultures and in vivo humanized mouse experimental models to examine the
 human immune response to autologous and allogeneic primary cholangiocyte
 organoids (PCOs). Our data demonstrate that PCOs upregulate the expression
 of HLA-I and HLA-II in inflammatory conditions. The immune response to
 allogeneic PCOs is driven by both HLA-I and HLA-II and is substantially
 ameliorated by donor-recipient HLA matching. Autologous PCOs induce a
 low-level immune infiltration into the graft site, while allogeneic cells
 display evolving stages of immune rejection in vivo. Our findings have
 important implications for the design and clinical translation of
 autologous and allogeneic organoid cellular therapies.
description: One representative mouse per group was selected, PCO and
 matrigel-only kidneys were sectioned (5mm) and slides sent to Nanostring
 for analysis. The slide preparation for WTA followed standard procedure.
 Briefly, for WTA assay, after baking for 3 hours in 65°C, slides were
 processed on Leica automation platform with a protocol includes three
 major steps: 1) slide baking and dewax, 2) Antigen Retrieval for 20 min at
 100°C, 3) 1µg/ml Proteinase K treatment for 20 min. Slides were then
 incubated with GeoMx WTA assay probe cocktail overnight. The following
 day, slides were washed and incubated with morphology markers before
 loading onto the GeoMx machine. The conjugated antibodies used were: KRT7
 (1:200, abcam, ab215855, clone [KRT7/760]), CD3 (1:100, Origene,
 UM000048BF, clone [UMAB54]), CD45 (1:200, CST, 13917BF, clone [D9M8I]), in
 addition to nuclear staining (Syto 83, ThermoFisher Scientific,
 S11364).  On the GeoMx machine, slides were scanned for
 fluorescence and Region of Interest (ROI) were selected. Barcodes were
 subsequently collected followed by barcoding reading done with Illumina
 NGS platform.
description: # Immunogenicity of autologous and allogeneic human primary cholangiocyte
 organoids #### ABSTRACT Primary human cells cultured in 3D organoid format
 have great promise as potential regenerative cellular therapies, but their
 immunogenicity has not yet been fully characterized. In this study, we use
 *in vitro* co-cultures and *in vivo* humanized mouse experimental models
 to examine the human immune response to autologous and allogeneic primary
 cholangiocyte organoids (PCOs). Our data demonstrate that PCOs upregulate
 the expression of HLA-I and HLA-II in inflammatory conditions. The immune
 response to allogeneic PCOs is driven by both HLA-I and HLA-II and is
 substantially ameliorated by donor-recipient HLA matching. Autologous PCOs
 induce a low-level immune infiltration into the graft site, while
 allogeneic cells display evolving stages of immune rejection *in vivo*.
 Our findings have important implications for the design and clinical
 translation of autologous and allogeneic organoid cellular therapies. ####
 GENERAL INFORMATION Title of Dataset: Immunogenicity of autologous and
 allogeneic human primary cholangiocyte organoids * Author Information A.
 Principal Investigator Contact Information Name: Kourosh Saeb-Parsy
 Institution: Department of Surgery, University of Cambridge Address: Box
 202. Level 9. Addenbrooke's Hospital · Hills Road *Cambridge* CB2 0QQ
 * Associate or Co-investigator Contact Information Name:
 Sandra Petrus-Reurer Institution: Department of Surgery, University of
 Cambridge Address: Box 202. Level 9. Addenbrooke's Hospital · Hills
 Road *Cambridge* CB2 0QQ Date of data collection (single date, range,
 approximate date): 2022-2023 Information about funding sources that
 supported the collection of the data: (MR/S020934/1, Medical Research
 Council (MRC) UK Regenerative Medicine Platform, G116517, MRC Confidence
 in Concept, and ERC: 741707, European Research Council Grant New-Chol ####
 SHARING/ACCESS INFORMATION Licenses/restrictions placed on the data: CC0
 1.0 Universal (CC0 1.0) Public Domain Links to publications that cite or
 use the data: * Petrus-Reurer, Sandra & Tysoe, Olivia & Lei,
 Winnie & Mairesse, Maelle & Tan, Thomas & Rehakova, Sylvia
 & Mahbubani, Krishnaa & Jones, Julia & Brodie, Cara & Han,
 Namshik & Betts, Catherine & Vallier, Ludovic & Saeb-Parsy,
 Kourosh. (2024). Immunogenicity of autologous and allogeneic human primary
 cholangiocyte organoids. 10.1101/2024.01.11.574744. Links to other
 publicly accessible locations of the data: None Links/relationships to
 ancillary data sets: None Was data derived from another source? No
 Recommended citation for this dataset: * Petrus-Reurer, Sandra &
 Tysoe, Olivia & Lei, Winnie & Mairesse, Maelle & Tan, Thomas
 & Rehakova, Sylvia & Mahbubani, Krishnaa & Jones, Julia &
 Brodie, Cara & Han, Namshik & Betts, Catherine & Vallier,
 Ludovic & Saeb-Parsy, Kourosh. (2024). Immunogenicity of autologous
 and allogeneic human primary cholangiocyte organoids.
 10.1101/2024.01.11.574744. #### DATA & FILE OVERVIEW This is a
 freeform section for you to describe how the data are structured and how a
 potential consumer might use them. Be as descriptive as necessary. Keep in
 mind that users of your data might be new to the field and unfamiliar with
 common terminology, metrics, etc. File List: * Annotation.csv * Matrix.csv
 * Metadata.csv Relationship between files, if important: * The samplename
 in Metadata.csv corresponds to the column in Matrix.csv * The in
 Metadata.csv corresponds to the in Annotation.csv Additional related data
 collected that was not included in the current data package: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: Annotation.csv Comma-delimited spreadsheet
 containing gene target properties information. Number of variables: 13
 Number of cases/rows: 18676 Variable List: * TargetName: Name given to
 target that may include the gene name; target level name may not be unique
 whereas probe level name is unique; in the case of multiple gene targets
 only one gene name is given * HUGOSymbol: Comma-delimited list of name of
 gene(s) with &gt;=95% identity over the length of probe; human gene names
 follow HUGO gene nomenclature; * TargetGroup: Target group as defined by
 the DSPDA software during analysis; multiple groups of targets can be
 user-defined; “All Targets” is a default group containing all targets in
 an assay * AnalyteType: AnalyteType Type of biological target, either RNA
 or Protein * CodeClass: Class of target used for analysis; value is either
 Control, Negative, or Endogenous * ProbePool: Integer value that
 identifies a single pool of probes; in the case multiple assays are used
 together, there would be more than one integer value in this list *
 MinCount: Minimum count observed for the target from all segments *
 MaxCount: Maximum count observed for the target from all segments *
 MedianCount: Median count measured for the target from all segments *
 GeomeanCount: Geometric mean of all counts for the target from all
 segments * NumberOfProbesIncluded: Number of probes included in the target
 in analysis after QC * NumberOfProbesTotal: Number of probes included in
 the target prior to QC * GeneID: The field identifies the one NCBI gene ID
 from the "SystematicName" column recommended for use in pathway
 analysis Missing data codes: NA (not applicable) Specialized formats or
 other abbreviations used: NA
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: Matrix.csv Comma-delimited spreadsheet
 containing Q3 (third quantile) normalized gene-counts matrix. Samples are
 specified in columns and genes (Variables) are in rows. Number of
 variables: 14766 Number of cases/rows: 91 Variable List: * ID_REF:
 TargetName Missing data codes: NA Specialized formats or other
 abbreviations used: NA
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: Metadata.csv Comma-delimited spreadsheet
 containing the meta data describing the FFPE samples from mouse model
 engrafted with human cholangiocyte organoids under kidney capsule by
 sample name. Number of variables: 22 Number of cases/rows: 91 Variable
 List: * Sample.name: sample name corresponding to the column names in
 matrix. * source.name: Type of cells analysed in the sample; value is
 either KRT+ cells or CD45+ cells * organism: The organism which the
 samples were derived * Tag: Type of humanisation and HLA class matching;
 value is either Non_Humanised, Full_Mismatch_Control, Full_Mismatch,
 Autologous, Partial_Mismatch, Partial_Mismatch_Control, or
 Autologous_Control * ROI.number: Numeric identifier for ROI; all segments
 within one ROI will have the same ROI number * AOISurfaceArea: Segment
 surface area, in pixels after conversion of scan in mm * AOINucleiCount:
 AOINucleiCount Segment nuclei count derived using the following algorithm
 in DSPDA v2.4 or earlier. * ROICoordinateX: x-coordinate of the ROI center
 on the Raw Channel Image exported from DSPDA, x starts on the left with 0
 and extends right, in pixels * ROICoordinateY: y-coordinate of the ROI
 center on the Raw Channel Image, y starts at the top with 0 and extends
 down, in pixels * RawReads: Number of read pairs initially inputted into
 the GeoMx NGS Pipeline * AlignedReads: Number of reads remaining after
 alignment to the barcode whitelist * DeduplicatedReads: Number of reads
 remaining after removal of PCR duplicates (i.e. unique UMIs) *
 TrimmedReads: Number of read pairs remaining after quality and adapter
 trimming * StitchedReads: Number of reads remaining after stitching
 overlapping sequences of Read 1 and Read 2 * SequencingSaturation:
 Sequencing saturation is related to sequencing depth and dependent on the
 given library complexity and reads sequenced; Proportion of UMIs in
 library sequenced more than once, sequencing saturation = (1 -
 deduplicated reads / aligned reads) * ScanWidth: Width of the largest
 rendered scan image, in pixels after conversion of scan width from mm;
 used to calculate the ROI center of an exported image using the following
 equation (result in pixels with origin (0,0) at the bottom left): Export X
 coordinate = (ROICoordinateX -ScanOffsetX) * (Export width/ScanWidth) *
 ScanHeight: Height of the largest rendered scan image, in pixels after
 conversion of scan height from mm; used to calculate the ROI center of an
 exported image using the following equation (result in pixels with origin
 (0,0) at the bottom left): Export Y coordinate = Export height -
 ((ROICoordinateY -ScanOffsetY) * (Export height/ScanHeight)) *
 ScanOffsetX: x offset of the start of the rendered scan image relative to
 the raw image, in pixels; used to calculate the ROI center of an exported
 image  using the following equation (result in pixels with origin (0,0) at
 the bottom left):  Export X coordinate = (ROICoordinateX -ScanOffsetX) *
 (Export width/ScanWidth) * ScanOffsetY: y offset of the start of the
 rendered scan image relative to the raw image, in pixels; used to
 calculate the ROI center of an exported image using the following equation
 (result in pixels with origin (0,0) at the bottom left):  Export Y
 coordinate = Export height - ((ROICoordinateY -ScanOffsetY) * (Export
 height/ScanHeight)) * LOQ: LOQ is equivalent to the geomean of negative
 probes in a given segment plus a user-defined number of standard
 deviations [geomean(neg probes)*geoSD(neg probes)^X, where X is a
 user-defined value (default = 2)]. The default value of 2 should be used
 if a slightly higher false positive rate (&lt;5%) is acceptable in
 exchange for higher sensitivity. Consider changing the value to 2.5 to
 minimize false positive calls (~1%), although this may decrease
 sensitivity slightly. The LOQ threshold can be used downstream to filter
 out targets that are not detected in at least a certain percentage of
 segments or when making binary detected/not detected calls. * description:
 Description of the sample. * platform: The platform accession number in
 GEO. Missing data codes: NA (not applicable) Specialized formats or other
 abbreviations used: NA #### CODE/SOFTWARE * BHOT_Analysis.Rmd: Analysis of
 immunogenicity associated pathways enrichment from differential expression
 results BHOT data
 ([https://onlinelibrary.wiley.com/doi/full/10.1111/ajt.16059).](https://onlinelibrary.wiley.com/doi/full/10.1111/ajt.16059\).) * CD45_DE_Analysis.html: Analysis of differentially expressed genes between humanised and non-humanised samples CD45+ cells. * CellDecon_Analysis.Rmd: Analysis to estimate cell abundance in CD45+ spatial gene expression data. * KRT_DE_Analysis.Rmd: Analysis of differentially expressed genes between humanised and non-humanised samples cholangiocytes.