Childhood brain tumours instruct cranial haematopoiesis and immunotolerance [scRNAseqTCR postTx]

Recent research has revealed a remarkable role for immunosurveillance in healthy and diseased brains, dispelling the notion that this organ is a passive immune-privileged site. Better understanding of how this immunosurveillance operates could improve the treatment of neurological diseases. Here, using a novel genetically engineered mouse model of ZFTA-RELA ependymoma–a childhood brain tumour–we characterised an immune circuit between the tumour and antigen presenting, haematopoietic stem/progenitor cells (HSPCs) in the skull bone marrow. The presentation of antigens in the cerebrospinal fluid (CSF) by HSPCs to CD4+ T cells, biased HSPCs lineages toward myelopoiesis and polarised CD4+ T-cells to regulatory T cells (T-regs), culminating in tumour immunotolerance. Remarkably, a single infusion of antibodies directed against cytokines enriched in the CSF of mice bearing ZFTA-RELA ependymomas, choroid plexus carcinomas or Group-3 medulloblastoma–all aggressive childhood brain tumours–disrupted this process and caused profound tumour regression. These data unmask a mechanism by which skull bone marrow-derived HSPCs and CD4+ T cells cooperate to promote the immunotolerance of childhood brain tumours. Antibodies that disrupt this immunosurveillance could prove an effective therapy for these cancers that are less toxic than current treatments. Overall design: Tumour/brain samples from age-matched 6-8 week-old EPZFTA-RELA (NestinCreERT2;Fus1;RFPTdT) mice were mechanically dissociated using sterile surgical scalpels into ~1 mm3 cubes, and dissociated using the mouse tumour dissociation kit (Miltenyi Biotec) using the gentleMACS Octo Dissociator (Miltenyi Biotec). After dissociation, the samples were then filtered through 100 µm meshes and washed with 2% fetal bovine serum in RPMI, spun down 420 g for 5 minutes. Samples were resuspended in 40% percoll and centrifuged at 600 x g for 10 minutes. Supernatant was removed and washed with 2% fetal bovine serum in RPMI and resuspended on 0.05% BSA PBS solution. Samples were stained with DAPI (0.2 µg/ml). Samples were centrifuged, resuspended in FACS buffer with anti-CD16/32 (FC block; Biolegend) diluted 1:50 in FACS buffer and fluorescently conjugated antibodies (CD45-PE and anti-Ter 119 FITC) at 4°C, followed by for 20 minutes. Cells were sorted using the Influx Cell Sorter (BD Biosciences) or FACsAria II (BD Biosciences) into 1% BSA coated 1.5 mL Eppendorf tubes with 500 µL of DMEM