GoKawiilAnimals
SPF WT C57BL/6J (JAX, 000664) mice were obtained from The Jackson Laboratory. The following strains were also purchased from The Jackson Laboratory: Il21-VFP (B6.Cg-Il21tm1.1Hm/DcrJ, JAX, 030295)35, Il21r-knockout (B6.129-Il21rtm1Kopf/J, JAX, 019115)36, OT-II (B6.Cg-Tg(TcraTcrb)425Cbn/J, JAX, 004194)37, MD4 (C57BL/6-Tg(IghelMD4)4Ccg/J, JAX:002595)38, Jchain-creERT2 (B6(129)-Jchainem1(icre/ERT2)Deep/J, JAX, 035764)39, ROSA-DTA (B6.129P2-Gt(ROSA)26Sortm1(DTA)Lky/J, JAX, 009669)40, Ai9 (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J, JAX, 007909)41, Thy1.1 (B6.PL-Thy1a/CyJ, JAX, 000406), AID-cre (B6.129P2-Aicdatm1(cre)Mnz/J, JAX, 007770)42, Ai14 (B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, JAX, 007914)41, UBC-GFP (C57BL/6-Tg(UBC-GFP)30Scha/J, JAX, 004353)43,44, Ifng KO (B6.129S7-Ifngtm1Ts/J, JAX, 002287)45, Lta KO (B6.129S2-Ltatm1Dch/J, JAX, 002258)46, Aqp4 KO (B6(Cg)-Aqp4<tm1.1Tsna>, RIKEN, RBRC10053)47 and muMT (B6.129S2-Ighmtm1Cgn/J, JAX, 002288)48 mice. All animals were housed in a temperature-controlled (22 °C) and humidity-controlled (33–39%) environment under a 12 h–12 h light–dark cycle and had ad libitum access to food and water.
Gnotobiotic mice were housed in plastic flexible film isolators in the gnotobiotic facility, also under a 12 h–12 h light–dark cycle. These mice were weaned onto autoclaved food, with ad libitum access to food and water. GF mice were fed γ-irradiated AAD (Teklad custom diet, TD.130916) for 4 weeks to establish AAD-fed GF cohort. Unless otherwise specified, 8-week-old male mice were used for experiments. All procedures were approved by the Institutional Animal Care and Use Committee of Washington University in St Louis (23-0145).
Cell lines and AAVs
The mouse brain cancer cell line GL261 was provided by G. Dunn49. The mouse glioma cell line CT2A was purchased from Sigma-Aldrich (SCC194). To generate HEL-OVA-expressing CT2A cell lines, full-length OVA DNA was obtained from Addgene (64599). The DNA encoding HEL-linker-OVA-IRES-eGFP was inserted into pLX208 vectors, and the cell lines were transduced with lentiviruses. Cells were selected with 100 µg ml−1 hygromycin (Invitrogen) and maintained with 50 µg ml−1 hygromycin supplementation.
Cell lines were passaged using trypsin/EDTA (Gibco, 25300) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Corning, 10-013-CM) supplemented with 10% FBS (Gibco, 26140) and 1% penicillin–streptomycin (Gibco, 15140). All cell lines were confirmed to be free of mycoplasma contamination. Cell lines were authenticated by short-tandem-repeat analysis.
pENN-AAV-hSyn-cre-WPRE-hGH (Addgene, 105553-AAV9), rAAV2/2-hSyn-DIO-eGFP-WPRE-hGH, rAAV2/2-hSyn-DIO-OVA-Flag-2A-eGFP-PA, rAAV2/9-hSyn-DIO-OVA-HEL-2A-eGFP-PA (Biohippo), AAV2-hSyn-OVA-P2A-eGFP-WPRE3-SV40polyA, and AAV2-hSyn-OVA-mCherry-P2A-eGFP-WPRE3-SV40polyA (AAVnerGene) were used.
Tumour and AAV injections
For intracranial and intrahindbrain injection, mice were anaesthetized and secured in a stereotaxic instrument (Kopf) using isoflurane (5% for induction, 1–2% for maintenance) or an intraperitoneal (i.p.) injection of a ketamine (100 mg per kg) and xylazine (10 mg per kg) cocktail. Ophthalmic solution was applied to the eyes to prevent dehydration.
For intracranial AAV injection, after a small craniotomy, 0.5 μl of 1 × 1012 viral genomes per ml of AAVs diluted in Dulbecco’s phosphate-buffered saline (DPBS; Gibco, 14190) was injected into the striatum (coordinates: anteroposterior (AP), 1.5 mm; mediolateral (ML), 1.5 mm; dorsoventral (DV), −2.5 mm) using a Nanoliter 2020 injector (World Precision Instruments) with a fine glass capillary at a rate of 100 nl min−1.
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