Ependymoma instructs cranial haematopoiesis and immunotolerance

ZFTA-RELA ependymoma presents a significant clinical challenge due to the lack of effective treatments, driving the need to explore novel immunotherapeutic strategies. Recent studies have identified the skull bone marrow as a source of meningeal immune cells critical for central nervous system (CNS) immunity, yet the role of these cells in childhood CNS tumor surveillance and pathology remains unclear. Here, we demonstrate that intracranial childhood ependymoma tumors harbour a unique myeloid cell population, including hematopoietic stem cells (HSCs) not from the blood, but supplied by adjacent skull bone marrow, ontogenetically distinct from their blood-derived counterparts. Our findings challenge the conventional view of HSCs as passive players in the CNS, revealing that antigen-specific interactions between skull bone marrow-derived HSCs and CD4+ T cells actively promote immunotolerance in childhood brain tumors by enhancing local myelopoiesis and inducing regulatory T cell polarization. This work uncovers a previously unrecognized cellular network linking the skull to brain tumors in children and underscores the therapeutic potential of modulating skull bone marrow haematopoiesis to shift the local immune environment. Targeting this skull-specific immune pathway offers a promising avenue for developing localized immunotherapies to improve treatment outcomes. Overall design: Age-matched 6-8 week-old EPZFTA-RELA and NestinCreERT2 mice were intravenously injected with CD45-PE 3 minutes prior to schedule 1. For skulls the dura was peeled and removed with fine forceps. Both the tibia and skull were then cut into small pieces using sterile scissors and mechanically dissociated in FACS buffer with a pestle, followed by a filtration step through a 70-µm cell strainer. Samples were centrifuged for 5 minutes at 420 x g. For peripheral bone marrow, both tibia were flushed with 0.05% BSA PBS with 0.05% EDTA, filtered through 7- µm meshes and washed with 2% fetal bovine serum in RPMI and resuspended on 0.05% BSA PBS solution. The whole intact deep cervical lymph nodes were mashed through a 70 µm cell strainer, using a sterile syringe plunger, and washed with 5 mL of FACS buffer. Deep cervical lymph node samples were then filtered through 70 µm meshes. Samples were centrifuged for 5 minutes at 420 x g.. Brains were macrodissected based on TdTomato fluorescent signal to harvest the tumor and region-matched brain in non-tumor bearing animals. Tumor/brain samples were mechanically dissociated using sterile surgical scalpels into ~1 mm3 cubes, and dissociated using the mouse tumor 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. 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 (anti-CD45 APC 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.