No statistical methods were used to predetermine sample size. The experiments were not randomized. Investigators were not blinded to allocation during experiments and outcome assessment, unless otherwise mentioned.
Human ovarian carcinoma tissues were procured under protocols approved by the Committee for the Protection of Human Subjects at Dartmouth–Hitchcock Medical Center (no. 17702); and under a protocol approved by the H. Lee Moffitt Cancer Center (MCC no. 19767 and MCC no. 18974). Part of the tumour tissues were either snap-frozen for protein extraction, or were freshly dissociated and cryopreserved. Viable cells in thawed samples were gated for FACS analysis using viability dyes (BioLegend). We have analysed tissue microarrays (TMAs) procured from the Moffitt Cancer Center Tissues Core, which include 93 HGSOC and some control tissues (MCC cohort) (approval MCC no. 50264). Tumour tissue from 261 HGSOCs from 2 prospective cohort studies (the Nurses’ Health Study (NHS) and NHSII22) and from 180 HGSOCs from a population-based case–control study (New England Case–Control Study (NECC cohort)) were used. In brief, the NHS enrolled 121,700 female registered nurses, aged 30–55, residing in 1 of 11 states of the USA in 1976. Similarly, the NHSII enrolled 116,429 female registered nurses aged 25–42 residing in 1 of 14 states of the USA in 1989. At baseline, women completed a mailed baseline questionnaire on their lifestyle and medical history. Questionnaires to collect updated information have since been mailed biennially. Incident epithelial ovarian cancer cases were identified from questionnaires, reports from family or linkage to the National Death Index, and confirmed by medical record review or cancer registry linkage. The study protocol was approved by the institutional review boards of the Brigham and Women’s Hospital and Harvard T. H. Chan School of Public Health, and those of participating registries as required. Return of the questionnaires is considered to imply informed consent.
The NECC used state-wide cancer registries and hospital tumour boards to identify 2,040 cases of epithelial ovarian cancer from eastern Massachusetts and New Hampshire from 1992 to 200823. Women were ineligible if they were younger than age 18, did not have a telephone, did not speak English, moved, died, had a prior bilateral oophorectomy or their physician declined permission to contact. The institutional review boards at Brigham and Women’s Hospital and Dartmouth Medical School approved the study protocol, and each participant provided written informed consent.
The NHS and NHSII requested paraffin-embedded tissue blocks containing representative tumour samples from cases of epithelial ovarian cancer with a pathology report. The NECC accessed tumour blocks from patients, most of whom were diagnosed at Brigham and Women’s Hospital. In NECC, funding was available to obtain tissue blocks for only a subset of cases, oversampling high-grade serous tumours. Tissue blocks were reviewed to verify histology and grade and make TMAs. TMAs were arrayed at the Dana–Farber/Harvard Cancer Center Specialized Histopathology Core by taking at least three, and up to six, core biopsies with a 1.0 mm (NECC) or 0.6 mm (NECC, NHS and NHSII) diameter from ovarian cancer tissue blocks and re-embedding the cores into a single block. All specimens of HGSOC used and analysed in this study are described in Supplementary Table 10.
OVCAR3, A549, NIH-H23, HEK-293T, K562 and THP1 cells were purchased from ATCC; OVCAR4, OVCAR5 and OVACR8 cells were procured from National Cancer Institute; Kuramochi cell line was procured from JCRB Cell Bank. Human OSE cells were purchased from ScienCell Research Laboratories (no. 7310). We transduced OVCAR3 tumour cells with a luciferase-expressing vector (OVCAR3luci). To make OVCAR3 cells as target of NY-ESO-1-specific T cells, OVCAR3luci cells were transduced to express NY-ESO-1 (OVCAR3luci-NY-ESO-1). Tumour-sorted CD45−EpCAM+ primary HGSOC cells were cultured continuously in R10 medium (RPMI-1640, 10% FBS, penicillin (100 IU ml−1), streptomycin (100 μg ml−1), l-glutamine (2 mM), sodium pyruvate (0.5 mM)) (ThermoScientific) until they adhere and grow similar to a cell line. All cell lines, except OSE, were routinely cultured in R10 medium. OSE cells were routinely cultured in recommended complete medium, purchased from ScienCell Research Laboratories. Cell lines routinely tested for negative mycoplasma contamination.
CRISPR–Cas9-mediated ablation of PIGR in OVCAR3 cells
CRISPR RNA (crRNA) targeting PIGR 5′-CUUCACAACAGAGCGACGAUGUUUUAGAGCUAGAAA-3′ (IDT) was reconstituted to make 100 μM in nuclease-free duplex buffer (IDT), and then mixed at equimolar concentration with Alt-R CRISPR–Cas9 trans-acting crRNA (tracrRNA), Atto550 (IDT) in a sterile PCR tube. Guide RNA was prepared by annealing crRNA and tracrRNA duplexes by heating at 95 °C for 5 min in PCR thermocycler, followed by gradual slow cooling to room temperature. Nine μl of crRNA–tracrRNA duplexes was mixed with 6 μl (180 pmol) of TrueCut Cas9 Protein v.2 (Invitrogen), followed by incubation at room temperature for 10 min to form Cas9 ribonucleoproteins (RNPs). OVACR3 or primary human HGSOC cells were suspended into final concentration of 5 × 106 cells per ml. Fifteen microlitres of the Cas9 RNPs was added to 100 μl of the cell suspension and electroporated using Neon Tranfection System (ThermoFisher), performed at 1170 V, 30 ms for 2 pulses. Cells were then cultured in R10 medium. Electroporation was confirmed by analysing Atto500 (tracrRNA) signal by flow cytometry on the next day, and loss of pIgR from the OVACR3 cells or primary HGSOC cells was confirmed by western blot five days later.
Mice and tumours
Female, 4–6-week-old Rag1-deficient (Rag1−/−) mice and NSG mice of the same age group were procured from Charles River Laboratories and Jackson Laboratory, respectively; and maintained by the animal facility of H. Lee Moffitt Cancer Center, with a 12 light/12 dark cycle, at about 18–23 °C with 40–60% humidity. Mouse experiments were approved by the Institutional Animal Care and Use Committee at the University of South Florida.
Flank tumours were initiated by injecting 1 × 107 OVCAR3 or autologous primary human HGSOC cells (wild-type or PIGR-ablated) cells. Tumour volume was calculated as: 0.5 × (L × W2), in which L is length and W is width. Tumour tissues were dissected mechanically into single-cell suspensions for flow cytometry, or retained for RNA and protein isolation.
Intratumoural or peritumoural injections of antibodies were done on several days, starting from day 7 after the tumour challenge, at a dose of 100 μg per 20 g body weight.
Natural killer cells were depleted from RAG1-deficient mice by intraperitoneal injections of 200 μg of NK1.1-neutralizing antibodies (anti-NK1.1, BioXCell, PK136, BE0036) 3 days before tumour challenge, followed by 100-μg injections on every 3 or 4 days.
Tumour volumes in mice were measured using code names on the cages and ear tags, instead of specific information about the treatments that the mice received. Apart from this, no blinding method was used for mouse studies.
Ovarian-cancer-sorted B cell immortalization, antibody purification, proteome array and pepsin digestion
Cryopreserved single-cell suspensions of HGSOC were thawed in 37 °C water bath followed by clearing of the cryoprotectant medium by centrifugation. After performing annexin V+ dead cell removal, viable single-cell samples were bead-sorted with human CD19 microbeads (Miltenyi Biotec, 130-050-301), according to manufacturer’s recommendation. Isolated CD19+ B cells were counted and 2 × 105 B cells per ml R10 were expanded for 5 days using human B cell expansion reagents (R&D Systems, CDK005), according to manufacturer’s recommendation. Expanded B cells were then challenged with Epstein–Barr virus (ATCC-VR-1492) added at 1:10 ratio, and kept in R10 without expansion supplements. In 2 to 3 weeks, most of the Epstein–Barr-virus-infected B cells had died; very few sustained the challenge and become immortalized. Immortalized B cells were grown to confluency. The conditioned medium was collected and concentrated using filters (Millipore Amicon, UFC900325). Part of the concentrated supernatants was analysed for reactivity against human protein antigens, using a proteome array that includes about 81% of all human proteins spotted into glass slides (CDi Lab).
Specific antigen-reactive B cells were FACS-sorted using tetramers, prepared by biotinylated peptides (GenScript) and PE-labelled streptavidin (BioLegend, 405203), and grown. Peptides were added from a 5.0 mg ml−1 stock to B cells in suspension at a final concentration of 1.0 μg per 107 B cells in 200 μl medium and incubated for 30 min at 4 °C, followed by a wash with PBS and incubation with streptavidin–PE added at 1:20 dilution. We used the peptides HGIPPSCCMNETDCNP and TQSYVRALTMDSKKRI to sort TSPAN7- and BDNF-specific B cells, respectively, from the pool of immortalized B cells.
Sorted cells were diluted in fresh medium and distributed in round-bottom 96-well plates in a way such that each well received one cell. Additional 10,000 B cells, irradiated with 100 Gy X-ray and 30 min UV, were added to each well as feeder cells. Then individual clones of B cells were grown over a period of 1 month. Conditioned medium was collected, concentrated using Amicon filters, and from the concentrated medium, human IgA or IgG were purified using purification kits (LigaTrap, LT-146KIT and LT-095KIT) according to recommended protocols.
To prepare F(ab′)2 fragments, Fc regions of human IgA or IgG antibodies were pepsin-digested using a pepsin digestion kit (ThermoScientific, 44988) according to the manufacturer’s recommendation.
CD3 T cells were bead-sorted from peripheral blood lymphocytes and transduced with a retroviral construct of NY-ESO-1 TCR or human FSH–CER14. NY-ESO-1 TCR was made using a sequence corresponding to an HLA-A2-restricted TCR that recognizes SLLMWITQC, corresponding to residues 157 to 165 of NY-ESO-113 (publicly available http://www.google.com/patents/US8143376), was purchased from IDT and ligated into pBMN-I-GFP. In luciferase-compatible 96-well plates, 10,000 OVCAR3luci-NY-ESO-1 or OVCAR3luci cells were placed. After 4 h wells were washed, fresh medium was added and cells were treated with non-specific, native human IgA (Abcam, ab91025) or IgG (Abcam, ab98981) at 0.5 μg ml−1 final concentration. After 2 h incubation at 37 °C, we added the appropriate number of T cells per well in a final volume of 200 μl. Cells were incubated for 4 h or 16 h in a 37-°C incubator, and checked for cytotoxicity using the luciferase assay (Promega).
For the autologous cytotoxicity assay, total CD3+ T cells, CD19+ B cells and CD45−EpCAM+ tumour epithelial cells were sorted after removal of dead cells. The B cells were immortalized, and IgA or IgG antibodies from concentrated supernatants of growing B cell culture-conditioned medium were separated as described in ‘Ovarian-cancer-sorted B cell immortalization, antibody purification, proteome array and pepsin digestion’. The tumour cells were grown in R10 medium to make continuous cultures. Ten thousand tumour cells were placed in 96-well plates. After 4 h wells were washed, fresh medium was added and cells were treated with autologous B-cell-derived whole or pepsinized IgA, autologous IgG, or irrelevant whole or pepsinized human IgA at 0.5 μg ml−1 final concentration. After 2 h incubation at 37 °C, we added 10,000 T cells per well in a final volume of 200 μl. Cells were incubated for 16 h in a 37-°C incubator. Total apoptotic cells (annexin V+) and viable cells (annexin V−propidium iodide−) were analysed by flow cytometry.
Wild-type or PIGR-ablated OVACR3 cells were incubated with irrelevant IgA or IgG for 2 h, FSH–CER T cells were added (1:1) and after 16 h incubation in a 37-°C incubator, total apoptotic cells (annexin V+) and viable cells (annexin V−propidium iodide−) were analysed by flow cytometry.
Ten thousand OVCAR3luci cells were placed in 96-well plates and after 4 h wells were washed followed by incubation with or without anti-TSPAN7–IgA (0.5 μg ml−1) antibodies for 2 h at 37 °C, and then splenic myeloid cells from tumour-bearing mice, pre-incubated with isotype or anti-CD351 blocking antibody for 30 min in ice, were added to the OVACR3 cells at 1:1 ratio and incubated for 12 h, and checked for cytotoxicity using the luciferase assay (Promega).
Cytotoxicity was calculated as (maximum viability control – individual well)/(maximum viability control – maximum death control) × 100, as a percentage.
FFPE TMAs were immunostained using the PerkinElmer OPAL 7-Colour Automation IHC kit on the BOND RX autostainer (Leica Biosystems) and the following anti-human antibodies: CD3 (Dako, A0452, 1:100), CD4 (Cell Marque, EP204, 104R-25, 1:100), CD8 (Dako, C8/144B, M7103,1:800), CD19 (Dako, LE-CD19, M7296, 1:50), CD20 (Dako, L26, M0755, 1:800), CD138 (Dako, MI15, M7228, 1:500), pIgR (Abcam, ab96196, 1:100), IgA (Abcam, EPR5367-76, ab124716, 1:1000), IgG (Abcam, EPR4421, ab109489, 1:500), and pan-cytokeratin (PCK, Dako, AE1/AE3, M3515, 1:200). Nuclei were stained with DAPI. Precisely, tissues were baked at 65 °C for 2 h then transferred to the BOND RX (Leica Biosystems) followed by automated deparaffinization, and antigen retrieval using OPAL IHC procedure (PerkinElmer). Autofluorescence slides (negative control) were included, which use primary and secondary antibodies omitting the OPAL fluorophores. Slides were scanned and imaged with the PerkinElmer Vectra3 Automated Quantitative Pathology Imaging System. Multilayer TIFF images were exported from inForm v.2.4.8 (PerkinElmer) and loaded into HALO v.3.0.311.328 (Indica Labs) for quantitative image analysis. Each fluorescent fluorophore is assigned to a dye colour and positivity thresholds were determined per marker on the basis of published nuclear or cytoplasmic staining patterns. Quantifications in tumour islets and stroma were distinguished by PCK staining. Datasets were exported with cytoplasmic, nuclear and total cell counts for each fluorescent marker from the sample set. Cell segmentation files were generated through inForm and dot plots were generated and analysed by FCS Image v.7.0. Standardization of multiplex immunohistochemistry staining experiments with appropriate positive and negative control tissues, including isotype control antibodies, are summarized in Extended Data Figs. 7, 8. Precisely, human tonsil sections were used as a positive control for CD3, CD4, CD8, CD19, CD20, CD138, IgA, IgG and PCK, and as a negative control for pIgR. Healthy kidney tissue sections were used as a positive control for pIgR. Glioblastoma sections were used as a negative control for CD3, CD4, CD8, CD19, CD20, CD138, IgA, IgG and PCK. Respective positive-control tissue adjacent sections were stained with isotype control antibodies to rule out false-positive staining.
Whole or pepsinized, non-antigen-specific or specific, IgA or IgG antibodies were conjugated with APC-conjugation kit (Abcam). Fifty thousand OVACR3 cells (wild type or PIGR-ablated) were placed onto a coverslip within 6-well plates and after 12 h treated with the antibodies. After different hours of incubations, cells were washed, fixed and mounted using a DAPI-containing mounting reagent (CST). Images were captured in a confocal microscope (Leica SP8) using LAS X (v.22.214.171.12476) software. Quantitative acquisition was performed using Zeiss Imager Z2 upright microscope using ZEN 2.3 (blue edition) software. CZI image files were imported into Definiens Tissue Studio v.4.7 (Definiens) to quantify cellular CY5 intensity. The software was used to run nucleus and cell detection algorithms to segment each cell, nucleus and cytoplasm and calculate the mean CY5 intensity within in these compartments. Intensity and size thresholds were set to minimize false-positive detection caused by artefacts and background fluorescence.
Xenograft tumour FFPE sections were stained for TUNEL+ cells using TUNEL Alexa Fluor 647 Imaging assay kit (Thermo) according to the manufacturer’s recommendation. Tile images were captured with an ORCA-Flash 4.00 V3 CMOS camera (Hamamatsu Photonics K.K.) and Zen 2 Blue software (Carl Zeiss) and stored in CZI file format. The software was also used to stitch the image tiles into whole-slide images. Representative images were exported to 8-bit TIFF format. CZI image files were imported into Definiens Tissue Studio v.4.7 to quantify the number of TUNEL+ cells. The software was used to run a nucleus detection algorithm on the CY5 channel of the whole-slide image. Intensity and size thresholds were set to minimize false-positive detection caused artefacts and background fluorescence. An adjacent section for each tumour was stained with haematoxylin and eosin, scanned using automated slide scanner (Aperio-Leica Scanner Console (v.126.96.36.199)) and the images was examined for necrotic holes within the tissues. Each tissue section was manually annotated to determine the area of the entire tissue section and large necrotic holes. The number of TUNEL+ cells was normalized by total tissue area minus large necrotic hole areas that appear in many of the tumour sections.
Antibody uptake analysis in xenograft tumours
Xenograft-tumour FFPE sections were rehydrated, antigen was retrieved, blocked and incubated with anti-human IgA antibodies overnight, followed by incubation with Alexa Fluor 647-conjugated secondary antibodies (CST, 4414) and mounted. CZI image files were imported into Definiens Tissue Studio v.4.7 to quantify the number of positive foci in the tissue. The software was used to run a nucleus detection algorithm on the DAPI channel and a spot detection algorithm on the CY5 channel. Intensity and size thresholds were set to minimize false-positive detection caused by artefacts and background fluorescence. The number of positive foci per tissue was normalized by total number of nuclei.
Flow cytometry was performed by staining with Zombie NIR (BioLegend) or Zombie Yellow (BioLegend) or DAPI (ThermoScientific) viability dye, blocking with anti-CD16/32 (BioLegend), and staining for 30 min at 4 °C with the following anti-human antibodies: CD45 (BD Biosciences, HI30, 1:300), CD3 (BD Biosciences, SK7, 1:200), CD19 (BD Biosciences, HIB19, 1:200), CD20 (BioLegend, 2H7, 1:200), CD38 (BD Biosciences, HIT2, 1:200), CD138 (BioLegend, MI15, 1:200), CD27 (BD Biosciences, M-T271, 1:200), IgA (Tonbo Biosciences, 35-8016-M001, 1:20), IgG (BioLegend, M1310G05, 1:200), IgM (BioLegend, MHM-88, 1:200), IgD (BD Biosciences, IA6-2, 1:100), IgE (BD Biosciences, G7-26, 1:100), EpCAM (BD Biosciences, KS1/4, 1:200), pIgR (ThermoScientific, PA5-35340, 1:50) or tetramers against TSPAN7 or BDNF. For intracellular staining for immunoglobulin isotypes, cells were first incubated with surface staining antibodies (30 min in ice), followed by fixation (30 min at room temperature) (eBioscience) and finally incubation with the antibodies for intracellular markers in the permeabilization buffer (eBioscience) (45 min at room temperature).
Mouse xenograft-tumour single-cell suspensions or splenocytes were blocked with Fc blocker (BioLegend) and analysed by flow cytometry after incubation for 30 min at 4 °C with following anti-mouse antibodies: CD45 (BioLegend, 30-F11), CD11b (BioLegend, M1/70), CD351 (BioLegend, TX61) or with APC-conjugated human IgA. Splenocytes from RAG1-deficient mice were mechanically dissociated and red blood cells were removed, followed by neutralization of Fcα/μ receptor (Fcα/μR) by incubation with CD351-neutralizing antibodies (BioLegend, TX61, 137303) or with isotype controls (BioLegend, 400123) at a concentration of 2.0 μg per 106 cells in 100 μl volume for 30 min in ice. After washing, splenocytes were then incubated with APC-conjugated human IgA for another 30 min in ice and analysed by flow cytometry. Appropriate isotype controls and fluorescence minus one were run. Samples were subsequently run using BD FACS LSRII or sorted using BD FACS ARIA. Data were collected using BD FACS Diva v.8.0.1 and analysed using FlowJo v.10.7.1. Gating strategies used for flow cytometry analyses are summarized in Extended Data Figs. 9, 10.
OVCAR3 cells in culture in 2% FBS-containing RPMI medium were treated with or without 0.5 μg ml−1 of natural human IgA or IgG for 24 h. Total RNA was isolated from cultured cells using RNA isolation kit (Qiagen) and analysed for RINe. An Illumina NextSeq 550 instrument was used to generate 75-basepair paired-end RNA-sequencing (RNA-seq) reads. Base calls were converted to FASTQ files using bcf2fastq (v.2.20). Paired-end RNA-seq reads were aligned to the GRCh37 human reference genome using STAR24 (v.2.5.3a) following adaptor trimming by cutadapt25 (v.1.8.1). For OVCAR3 cells, uniquely mapped reads were counted by feautreCounts26 (v.1.5.3) using Gencode V30 transcript annotations for human. Differential expression analysis was performed using DESeq2 (v.1.30.0)27. Heat maps were generated using z-score-transformed log2(1 + normalized count).
For each differential expression analysis comparing antibody treated groups with the untreated group, genes were ranked based on −log10(P value) × (sign of log2(fold change)). The preranked gene list was used to perform preranked GSEA28 (v.4.0.2) to assess enrichment of hallmarks, curated gene sets and Gene Ontology29 terms in MSigDB28. The resulting normalized enrichment score and false-discovery-rate-controlled P values were used to assess the IgA-induced transcriptome changes.
IgA transcytosis and pIgR LC-MS/MS
One hundred thousand OVCAR3, OVCAR4, OVCAR5 or primary HGSOC-tumour cells were placed in 6-well plates and washed with PBS after 12 h before treating with or without brefeldin A (1 μg ml−1) or wortmannin (1 μM) in serum-free RPMI medium. After 4 h cells were treated with or without native human IgA (Abcam, ab91025) or IgG (Abcam, ab98981) at 0.5 μg ml−1 final concentration. After 12 h, conditioned medium was collected and filtered for contaminant debris removal. Proteins were extracted from the conditioned medium, reduced by DTT, digested by trypsin and subjected to LC–MS/MS analysis by the Moffitt Cancer Center Proteomics Facility. MaxQuant (v.188.8.131.52) was used to analyse the data, identify and quantify the proteins30.
Western blot and co-immunoprecipation
Cells and mechanically dissociated tumour samples were lysed in RIPA buffer (ThermoScientific) with protease–phosphatase inhibitor cocktail (SigmaAldrich) and cleared by centrifugation. Proteins were quantified by BCA assay (ThermoScientific). Membranes were blotted with anti-pIgR (Abcam, ab96196, 1:500), anti-IgA (Abcam, EPR5367-76, ab124716, 1:2,500), anti-phospho-ERK1/2 (CST, D1H6G, 5726, 1:1,000), anti-ERK1/2 (CST, L34F12, 4696, 1:2,000), anti-DUSP5 (Abcam, EPR19684, ab200708, 1:1,000) and anti-β-actin (CST, 13E5, 5125 or 4970, 1:5,000) antibodies. For TSPAN7 and BDNF, isolated IgA antibodies from concentrated conditioned medium from TSPAN7- and BDNF-reactive B cells were used. Immunoreactive bands were developed using horse radish peroxidase-conjugated secondary antibodies (CST, 7074, 1:5,000; CST, 7076, 1:5,000; Abcam, ab97215, 1:5,000) and enhanced chemiluminescence substrate (GE HealthCare).
HGSOC tumour tissue chunks were pulverized and lysed using nonreducing nondenaturing lysis reagent provided in the co-immunoprecipitation kit (Pierce) used. Pellets of OVCAR3 cells, CD45+ and CD45− fractions of human ovarian cancer ascites were also lysed with the same nondenaturing lysis reagent. Cell-free human ovarian cancer ascitic fluids were filtered, concentrated and diluted with the nondenaturing lysis reagent. Proteins were immunoprecipitated using anti-human IgA antibody (Abcam, EPR5367-76, ab124716, 1:20) and eluted following the manufacturer’s instructions. Elutes and inputs were immunoblotted for pIgR and IgA. Conditioned medium from transcytosis experiment in OVCAR3 (wild-type or PIGR-ablated) cells was concentrated and immunoprecipitated with anti-human IgA antibody and part of elutes were western-blotted for the secretory component of pIgR (Abcam, SC-05, ab3924, 1:500) or analysed by LC–MS/MS.
Analysis of TCGA data
Molecular and clinical data from TCGA for ovarian serous cystadenocarcinoma (designated OV) were downloaded from the cBio Cancer Genomics Portal (http://www.cbioportal.org/), Broad Firehose website (https://gdac.broadinstitute.org/) and Genomic Data Commons Data Portal (https://portal.gdc.cancer.gov/). A total of 428 patients with matched clinical information and tumour RNA-seq data was used in this study. Raw RNA-seq reads were aligned to the GRCh37 human transcriptome using STAR24 (v.2.5.3a). Uniquely aligned reads were counted against Gencode v.19 using htseq-count31 (v.0.6.1) and then normalized using DESeq227 taking into account batches and RNA composition bias. All statistical analysis and visualization was performed using log2-transformed normalized count. Kaplan–Meier survival analysis and the log-rank tests were performed to compare overall survival between groups.
10X Genomics single-cell V(D)J (BCR) sequencing and recombinant antibody production
Single-cell V(D)J (BCR) sequencing was performed using the 10XGenomics Chromium system. A single-cell suspension derived from HGSOC-sorted B cells was analysed for viability using the Nexcelom Cellometer K2 and then loaded onto the 10X Genomics Chromium Single Cell Controller at a concentration of 1,000 cells per μl to encapsulate around 5,000 cells per sample. In brief, the single cells, reagents and 10X Genomics gel beads were encapsulated into individual nanolitre-sized gel beads in emulsion and then reverse transcription of poly-adenylated mRNA was performed inside each droplet. The cDNA libraries were then completed in a single bulk reaction using the10X Genomics Chromium NextGEM Single Cell V(D)J Reagent Kit and enriched for B cell receptor sequences using the VDJ primers provide in the kit. Following PCR purification, fragment DNA libraries were prepared according to the 10X Genomics protocol. The V(D)J enriched libraries were sequenced to at least 5,000 reads per cell on the Illumina NextSeq 500 instrument. Demultiplexing, barcode processing, alignment and gene counting were performed using the 10X Genomics CellRanger V(D)J (v.3.1.0) software.
BCR reads sequenced by V(D)J assay were aligned to GRCh38 reference transcriptome using Cell Ranger VDJ (v.3.1.0, 10X Genomics). BCR heavy and light chains were assembled and annotated using cellranger vdj with –reference = refdata-cellranger-vdj-GRCh38-alts-ensembl-3.1.0 to determine clonotypes. Recombinant dimeric IgA antibodies were produced by Genscript. In brief, corresponding DNA sequences for immunoglobulin heavy chain, light chain and J chain were synthesized, and the complete sequence was subcloned into pcDNA3.4 vector and expressed in HD 293F cells. Dimeric IgA1 antibodies were eluted from cell culture supernatants. Molecular weight and purity were analysed by SDS–PAGE and high-performance liquid chromatography.
Statistics and reproducibility
Unless mentioned otherwise, all data are presented as mean with s.e.m. Two-tailed t-tests (unpaired and paired, as appropriate) were performed between two groups, and one-way ANOVA were performed for comparisons between more than two groups, unless indicated otherwise. Pearson correlation analysis was performed for correlation analysis. To compare IgA+ and IgG+ or IgM+ cell percentage in B cells, plasmablasts and plasma cells, two-way ANOVA followed by Dunnett’s ad hoc tests for multiple comparison was performed on arcsine-transformed percentage data (IgA versus IgG or IgM, P value = 1 × 10−10). Analyses were carried out in Graph Pad Prism (v.7.0) or R (v.3.6.1) software. A significance threshold 0.05 for P values was used. Box plots were generated using gglot2 in R (3.6.1) with following parameters: horizontal black lines with each box present median values; boxes extend from 25th to 75th percentile of values; whiskers extend to a maximum of 1.5 × interquartile range (75th percentile–25th percentile) beyond the boxes; the lowest dots are the minimum values and highest dots are the maximum values for each box. All experiments are represented by several biological replicates or independent experiments, unless otherwise mentioned. The number of replicates per experiment is indicated in the legends. Bar plots are mean ± s.e.m. with overlaid data points representing independent experiments or replicates. All western blot analyses were independently replicated at least two times in case of human-derived specimens and three times in case of in vitro experiments, with similar results. Co-immunoprecipitation experiments were independently replicated a minimum two times with similar results. For all multiplex immunohistochemistry of TMAs, individual tumours have replicated cores (2–6, from different areas of the tumour) on the TMAs, and averaged quantification values from replicated cores were used. The TMA slides were scanned twice, analysed, and used with <1% data variation. CRSIPR-mediated knockdown experiments were repeated three times independently. Additional information and test results of statistical analysis are provided in the figure legends and Supplementary Tables 1–12. No sample size calculations were performed before the study for human specimens. For most functional in vitro analyses, sample sizes were chosen on the basis of the availability of target cells. Mouse experiments used at least five mice per group per experiments. Because this study focuses on ovarian cancer, only female mice were included in the experimental design. The experiments were not randomized. HGSOC tumour and ascites specimens were obtained from de-identified patients and were not randomized. Peripheral blood mononuclear cells from de-identified donors without cancer were acquired and analysed. Mice were not intentionally randomized. Tumour volumes in mice were measured using code names on the cages and ear tags, instead of specific information about the treatments that the mice received. Apart from this, no blinding method was used for mouse studies. RNA-seq, BCR sequencing, multiplex immunohistochemistry quantifications, fluorescence microscopy quantifications or LC–MS/MS were performed with unidentifiable demarcation. In case of in vitro experiments, samples were often assigned code numbers to facilitate blinded flow cytometry, microscopy and luciferase assay. After all data were collected, the results were analysed and decoded. For analysis of human specimens, blinding is not applicable as no interventions were tested.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.