GoKawiilSex as a biological variable
In consideration of the clinical context, in which TNBC71, ANRE72,73 and their co-occurrence73,74 are predominantly observed in female patients, we have proactively chosen to model the underlying biology in female mice to maximize translational relevance. Furthermore, there are considerable differences in symptom frequency and severity between male and female patients with ANRE75,76. This sexual dimorphism may be partly explained by sex hormone activity, as ER signalling directly modulates NMDAR expression28,77 in the brain.
Ethical oversight
Human-participant oversight was provided by the Northwell Health Institutional Review Board (IRB 20-0150) for the prospective TNBC cohort and the South Central-Oxford A Research Ethics Committee (REC16/YH/0013) for the ANRE study. Vendor-procured specimens received IRB approvals from their respective institutions, as was verified by each vendor. The authors affirm that all human research was performed in accordance with the relevant guidelines and regulations, and that informed consent was obtained. Human samples included in this study are summarized in Supplementary Table 6.
Animals
All animal experiments were performed in strict accordance with the Cold Spring Harbor Laboratory (CSHL) Institutional Animal Care and Use Committee (IACUC) and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice were kept in specific-pathogen-free conditions under a 24 h 12 h–12 h light–dark cycle. Food and water were provided ad libitum, and the temperature and humidity were maintained at 21–23 °C and 40–60% relative humidity. Mice were allowed to acclimatize for at least 1 week after their arrival to the Cold Spring Harbor Laboratory animal facility. Adult female mice between 9 and 14 weeks of age were used. All mice were randomly assigned to experimental groups, and the sample size was predetermined according to the type of experiment, on the basis of common practice. For the majority of experiments, the investigators were not blinded to group allocation as they performed both the experiment and analysis, making blinding not possible. For tumour experiments, 8-week-old female wild-type BALB/c mice were obtained from Jackson Laboratory. As per our IACUC-approved protocol, a tumour size of greater than 20 mm was defined as a humane end point and this limit was never exceeded in any of the experiments. For antibody infusion experiments, 8-week-old female wild-type C57BL/6 mice were obtained from Jackson Laboratory. Outbred wild-type adult female X. laevis frogs (aged 2–4 years) were ordered from Xenopus 1.
Patient samples
Tumour and matched plasma specimens were obtained from three cohorts of individuals with stage I–III TNBC who received surgical resection as part of standard clinical management. All studies were approved by Institutional Review Boards, and written informed consent was obtained from all of the participants. All specimens were fully de-identified before analysis. The Northwell Health cohort (n = 48) was established through an academic partnership under IRB protocol 20-0150. Peripheral blood samples were collected either before or after surgery on the day of surgical resection. Tumour tissue was available for a subset of patients, depending on the availability of excess clinical material. Tumour specimens were viably preserved by mincing fresh tissue and storing in cryopreservation medium at −80 °C. The vendor-supplied cohort (n = 5) comprised paired plasma and tumour specimens obtained from Biochain Institute under IRB-approved protocols with the participants’ informed consent. Tumour sections were preserved as cold acetone-fixed frozen tissue and stored at –80 °C. A third cohort consisted of a single frozen tumour section obtained from Origene (patient ID: FR001D525), collected under IRB-approved protocols with the participant’s informed consent. This sample was used for Visium HD spatial transcriptomic profiling. TNBC status was confirmed in all cases by immunohistochemistry according to standard clinical criteria: ER and progesterone receptor (PR) expression each <1% of tumour nuclei, and HER2 scored as 0–1+ by immunohistochemistry or 2+ with negative fluorescence in situ hybridization. Plasma samples from all cohorts were processed using standardized protocols and stored at –80 °C until analysis. For the Northwell cohort, DFS data were abstracted by manual review of electronic health records by the Northwell biorepository team. DFS was defined as the interval from surgery to first recurrence or death, with censoring at the most recent clinical follow-up. To assess the normal range of anti-NMDAR antibody titres, a panel of plasma samples from healthy women 18–30 years of age (n = 23) was obtained from BioIVT under IRB-approved protocols with the participants’ informed consent. All of the patient samples in this study served as a discovery set, and no formal power calculation was performed.
B cell isolation and antibody cloning
The OX1 antibody was isolated from one patient with a confirmed diagnosis of anti-NMDAR encephalitis (ANRE), on the basis of cerebrospinal fluid antibody testing, with written informed consent as part of a research study approved by the University of Oxford with ethical approval REC16/YH/0013. Peripheral blood mononuclear cells (PBMCs) were isolated from patient blood samples and, after thawing, PBMCs were labelled using fluorophore-conjugated biotinylated GluN1-ATD baits along with B cell surface marker antibodies (CD19, CD20, IgG) for 30 min at 4 °C. GluN1-ATD-binding B cells were enriched by fluorescence-activated cell sorting and sorted into 96-well plates containing single-cell culture medium. After 10–14 days of culture, the cell culture supernatant was prescreened for NMDAR reactivity using GluN1-transfected HEK293 cells by IF microscopy. Positive wells were identified, and antibody sequences were determined using the RNA extraction using the RNeasy kit (Qiagen), followed by reverse transcription and amplification of heavy- and light-chain immunoglobulin variable regions using primers specific for human IgG, followed by Sanger sequencing.
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