Functional role of skull lymphoid structures in CNS immunosurveillance

Industry Updates

August 19, 2026

Functional role of skull lymphoid structures in CNS immunosurveillance

Animals

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-Il21^tm1.1Hm/DcrJ, JAX, 030295)³⁵, Il21r-knockout (B6.129-Il21r^tm1Kopf/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)-Jchain^{em1(icre/ERT2)Deep}/J, JAX, 035764)³⁹, ROSA-DTA (B6.129P2-Gt(ROSA)26Sor^tm1(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-Aicda^tm1(cre)Mnz/J, JAX, 007770)⁴², Ai14 (B6.Cg-Gt(ROSA)26Sor^{tm14(CAG-tdTomato)Hze}/J, JAX, 007914)⁴¹, UBC-GFP (C57BL/6-Tg(UBC-GFP)30Scha/J, JAX, 004353)^43,44, Ifng KO (B6.129S7-Ifng^tm1Ts/J, JAX, 002287)⁴⁵, Lta KO (B6.129S2-Lta^tm1Dch/J, JAX, 002258)⁴⁶, Aqp4 KO (B6(Cg)-Aqp4, RIKEN, RBRC10053)⁴⁷ and muMT (B6.129S2-Ighm^tm1Cgn/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.

For the intracranial tumour injection model, 1 × 10⁵ cancer cells in 2 μl of DPBS were injected into the brain (coordinates: AP, 2 mm; ML, 2 mm; DV, −3 mm) using a 26 G syringe (Hamilton, 80300) and an injector (KD Scientific)^50,51. After intracranial injection, the burr hole was sealed with bone wax (Surgical Specialities Corporation, 901). For intrahindbrain injection, the posterior scalp and neck of anaesthetized mice were shaved. The head was secured in a stereotaxic frame with the neck tilted. A midline incision was made, and the posterior nuchal musculature was divided to expose the occipital bone and the dura overlying the cisterna magna. In total, 1 × 10⁵ cancer cells suspended in 2 μl of DPBS were injected into the hindbrain with a 26 G syringe through the cisterna magna. For peripheral tumour injection, anaesthetized mice were shaved, and 5 × 10⁵ cancer cells in 100 μl of DPBS were injected s.c. into the flank.

Injections

For tamoxifen-induced Cre recombination, recipient mice were injected i.p. with 0.1 mg per g of tamoxifen (Sigma-Aldrich, T5648) dissolved in corn oil per day for five consecutive days. For T cell depletion, mice were treated with 200 μg of anti-CD4 (GK1.5, BioXcell, BE0003-1), anti-CD8a (2.43, BioXcell, BE0061) or rat IgG2b isotype control antibody (LTF-2, BioXcell, BE0090). For GC enhancement, 100 μg (i.p.) or 20 μg (s.c.) of anti-CD40 antibody (FGK4.5, BioXcell, BE0016) or rat IgG2a isotype control antibody (2A3, BioXcell, BE0089) was administered, with or without 10 μg (i.p.) or 2 μg (s.c.) of recombinant IFNγ (BioLegend, 575308) and 1 μg (i.p.) or 0.2 μg (s.c.) of recombinant IL-21 (BioLegend, 574506), either i.p. or transcranially. For GC blockade, 200 μg (i.p.) or 20 μg (s.c.) of anti-CD40L antibody (MR-1, BioXcell, BE0017-1) or polyclonal Armenian hamster IgG control antibody (BioXcell, BE0091) was injected i.p. or transcranially. For transcranial injections, materials were mixed with 10 mg ml⁻¹ of carboxymethylcellulose hydrogel (Sigma-Aldrich) in a total volume of 300 μl and injected s.c. under the scalp. For peripheral injections, anti-CD40L antibody was mixed with hydrogel and injected s.c. into the flank.

For intracranial OVA injections, 0.5 μl of 1 mg ml⁻¹ OVA-647 (Invitrogen) in DPBS was injected into the striatum in 1 min (coordinates: AP, 1.5 mm; ML, 1.5 mm; DV, −2.5 mm). For intracisternal OVA injections, 5 μl of 1 mg ml⁻¹ OVA-647 in DPBS was injected into the cisterna magna as described previously²⁷.

Splenectomy

Lta-KO mice were anaesthetized by i.p. injection of ketamine (40–80 mg per kg) and xylazine (5–10 mg per kg). The skin over the left abdomen was sterilized with iodine solution, and a 0.5 cm incision was made with sterile scalpel. The spleen was exposed using smooth forceps, and vessels and attachments were ligated with absorbable 5-0 sutures before excision of the spleen. The midline fascial defect was closed with 4-0 absorbable sutures, and the skin was closed with interrupted 4-0 non-absorbable sutures.

dCLN ligation and removal

dCLN ligation was performed as previously described^29,52. In brief, mice were anaesthetized with ketamine–xylazine cocktail. An incision was made 5 mm above the clavicle. The sternocleidomastoid muscles were retracted, and afferent lymphatic vessels were ligated with a nylon suture. Sham-surgery group received the incision and muscle retraction but were not ligated. For lymphadenectomy, dCLNs were removed during this process. Mice were sutured and recovered on a heating pad until responsive. After surgery, mice were given analgesic and prophylactic antibiotics.

Adoptive transfer

Spleens from donor mice were collected, and primary naive or whole CD4⁺ T cells and B cells were isolated using the EasySep Mouse Naive CD4⁺ T Cell Isolation Kit (StemCell Technologies, 19765), mouse CD4⁺ T cell isolation kit (StemCell Technologies, 19852) and the Mouse B Cell Isolation Kit (StemCell Technologies, 19854), respectively. A total of 2–5 × 10⁵ naive CD4⁺ T cells and 0.5–1 × 10⁶ B cells were resuspended in DPBS and intravenously injected through the retro-orbital route.

Single-cell isolation and flow cytometry

For i.v. immune labelling, mice were anaesthetized and received 7.5 μg of CD45-BV750 (30-F11, BD Biosciences) intravenously through the retro-orbital route. Then, 3–5 min later, mice were euthanized. Mice were euthanized using a CO₂ chamber and transcardially perfused with DPBS. Single-cell suspensions were prepared from each organ. Bones were collected and cleaned to remove attached soft tissues, and the dura was peeled from the skull. Single-cell suspensions from BM were obtained as previously described^4,5. In brief, bones were mechanically dissociated by chopping with scissors in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 11875) containing 2% FBS.

Dura, chopped LNs, brain and brain tumour tissues were digested with a solution containing 1 mg ml⁻¹ collagenase VIII (Sigma-Aldrich, C2139) and 0.5 mg ml⁻¹ DNase I (Thermo Fisher Scientific, EN0521) at 37 °C for 30 min. Whole spleens or single-cell suspensions from various organs were passed through 70-μm strainers. Cell suspensions from brain and tumour tissues were centrifuged with 30% Percoll (Sigma-Aldrich, 17-0891-01) to remove myelin debris. Red blood cells were removed using ammonium–chloride–potassium (ACK) lysis buffer (Gibco, A1049201).

Single-cell suspensions were treated with anti-CD16/32 antibody (93, BioLegend) to block Fc receptors, then stained with antibodies targeting the following molecules: CD45 (30-F11, BioLegend), CD3 (17A2, BioLegend), CD4 (GK1.5, BD Biosciences), CD8a (53-6.7, BioLegend), CD11b (M1/70, BD Biosciences), CD11c (N418, BioLegend), I-A/I-E (M5/114.15.2, BioLegend), NK1.1 (PK136, Thermo Fisher Scientific), CD19 (1D3, BD Biosciences), CD45R/B220 (RA3-6B2, BD Biosciences), IgD (11-26c.2a, BioLegend), IgM (II/41, Thermo Fisher Scientific), IgA (mA-6E1, Thermo Fisher Scientific), IgG (Poly4053, BioLegend), CD138 (281-2, BD Biosciences), TACI (8F10, BD Biosciences), CD38 (90, BioLegend), FAS (SA367H8, BioLegend), CXCR5 (L138D7, BioLegend), CXCR3 (CXCR3-173, BioLegend), CD154 (MR1, BD Biosciences), CXCR4 (L276F12, BioLegend), ICOS (7E.17G9, Thermo Fisher Scientific), PD-1 (RMP1-30, BD Biosciences), CD44 (IM7, BD Biosciences), CD69 (H1.2F3, BD Biosciences), CD62L (MEL-14, BioLegend), IL-21R (4A9, BioLegend), Thy1.2 (53-2.1, BioLegend), IgMa (DS-1, BD Biosciences), IgMb (AF6-78, BD Biosciences), TCR Va2 (B20.1, BioLegend), TCR Vβ5.1/5.2 (MR9-4, BD Biosciences), CD31 (390, BioLegend), PDPN (8.1.1, BioLegend), CR1/2 (7E9, BioLegend), CD16/32 (93, BioLegend), CD25 (PC61, BioLegend), Ly6C (AL-21, BD Biosciences), Ly6G (1A8, BD Biosciences) and CD103 (2E7, BioLegend).

For intracellular staining, cells were fixed and permeabilized using the FoxP3 staining kit (BioLegend) according to the manufacturer’s instructions, and antibodies targeting T-bet (4B10, BioLegend), BCL6 (K112-91, BD Biosciences), IFNγ (XMG1.2, BioLegend), TNF (MP6-XT22, BioLegend), GZMB (NGZB, Thermo Fisher Scientific), IL-10 (JES5-16E3, BD Biosciences) and FOXP3 (FJK-16s, Thermo Fisher Scientific) were used. Live cells were gated using Zombie NIR fixable viability dye (BioLegend). For intracellular cytokine staining, single-cell suspensions were stimulated with PMA/ionomycin (Cell Stimulation Cocktail; 00-4970-93, Thermo Fisher Scientific) in the presence of Protein Transport Inhibitor Cocktail (00-4980-93, Thermo Fisher Scientific) for 4 h at 37 °C. Antibodies were diluted 1:200 for staining. All samples were acquired on an Aurora spectral flow cytometer (Cytek), and data were analysed using FlowJo software v.10.8.1 (TreeStar). The gating strategies and FMO control are shown in Supplementary Figs. 5 and 6.

qPCR with reverse transcription

Tissues were isolated as described above. Total RNA was extracted from the brain and skull using TRIzol (Sigma-Aldrich, T9424), while RNA from dura was isolated using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, AM1931) according to the manufacturer’s instructions. Purified RNA was reverse transcribed into cDNA using the iScript Reverse Transcription Supermix for quantitative PCR (qPCR) (Bio-Rad, 1708841). qPCR was performed on the QuantStudio 6 Flex system (Applied Biosystems).

The following primers were used to detect eGFP. Forward: 5′-AAGGGCATCGACTTCAAGG-3′; reverse: 5′-TGCTTGTCGGCCATGATATAG-3′. Transcript levels were normalized to Actb, and relative gene expression was calculated using the comparative ΔΔC_t method.

Immunofluorescence

Mice were perfused with Dulbecco’s phosphate-buffered saline (DPBS) followed by 4% paraformaldehyde (PFA) through intracardiac injection. The samples were then fixed in 4% PFA. Bone samples were decalcified in 5% EDTA solution at 4 °C for 3 days, with daily EDTA changes. For cryosectioning, samples were dehydrated in 30% sucrose solution at 4 °C for 3 days, then embedded in Fisher Healthcare Tissue-Plus OCT compound (Fisher Healthcare, 23-730-571). The blocks were frozen and sectioned at 20–50 μm thickness using a cryostat (Leica Biosystems).

For immunostaining, tissues were blocked in 0.2% Triton X-100 in DPBS with 5% goat or donkey serum, then stained with primary antibodies, including: Armenian hamster anti-mouse CD3e (145, Thermo Fisher Scientific), rabbit anti-mouse CD20 (SP32, Thermo Fisher Scientific), APC-conjugated mouse-anti-mouse IgMa (MA-69, BioLegend), rat anti-mouse BLIMP1 (6D3, Thermo Fisher Scientific), rabbit polyclonal anti-mouse S1PR2 (Proteintech), Alexa Fluor 647-conjugated rat anti-mouse PD-1 (29 F.1A12, BioLegend), Alexa Fluor 594-conjugated rat anti-CD4 (GK1.5, BioLegend), Alexa Fluor 488-conjugated rat anti-mouse CD20 (SA275A11, BioLegend), polyclonal goat anti-mouse CXCL13 (R&D Systems), polyclonal goat anti-mouse BAFF (R&D Systems), CD35 chimeric recombinant rabbit antibody (8C12, Thermo Fisher Scientific), rat anti-mouse FDC (FDC-M1, BD Biosciences), rabbit polyclonal anti-mouse BCL6 (Thermo Fisher Scientific) and eFluor 660-conjugated rat anti-mouse B220 (RA3-6B2, Thermo Fisher Scientific), polyclonal anti-GFP (Thermo Fisher Scientific, A10262). Secondary antibodies included: Alexa Fluor 488-conjugated goat anti-Armenian hamster IgG (Jackson Laboratory, 127-545-099), Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, A32754), Alexa Fluor 647-conjugated donkey anti-goat IgG (Thermo Fisher Scientific, A32849), Alexa Fluor 647-conjugated donkey anti-rat IgG (Thermo Fisher Scientific, A78947), Alexa 488-conjugated donkey anti-rat IgG (Thermo Fisher Scientific, A48269), Alexa Fluor 647-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, A32795TR), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, A-21206).

For in vivo labelling, the following antibodies were injected intravenously 40 min before perfusion: 30–100 μg of Alexa Fluor 488-conjugated anti-mouse CD20 (SA275A11, BioLegend), Alexa Fluor 594-conjugated anti-mouse I-A/I-E (M5/114.15.2, BioLegend), coralite594-conjugated anti-mouse Thy1.2 (30-H12, Thermo Fisher Scientific), Alexa Fluor 594-conjugated anti-mouse CD31 (390, BioLegend) and eFluor 660-conjugated anti-mouse B220 (RA3-6B2, Thermo Fisher Scientific), Alexa Fluor 594-conjugated anti-mouse CD4 (GK1.5, BioLegend) and Alexa Fluor 647-conjugated anti-mouse CD4 (GK1.5, BioLegend). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9542).

For whole-mount staining, the ADAPT-3D protocol was used for tissue clearing⁵³. Whole-mount staining of the dura was performed as previously described²⁷. Images were acquired using the wide-field microscopy (Olympus SLIDEVIEW VS200) or Stellaris confocal microscope (Leica) with ×10, ×20 or ×40 objectives with Leica Application Suite v.4.2.1.23810 and Nikon Elements v.5.2.0 and analysed with Fiji software v.2.14.0/1.54j. The colocalization plugin in Fiji was used to identify co-localized cells. Cell counting and density calculation were performed with QuPath-0.6.0 software⁵⁴. The intensity and coverage of fluorescent proteins were analysed using Fiji software. The background fluorescence was determined using negative controls, such as non-fluorescent samples or regions. Values were not normalized. Representative single-plane images of whole mounted skulls are provided in Supplementary Fig. 7.

Single-cell transcriptomic analysis

Public data were downloaded from the Gene Expression Omnibus (GEO) under accession numbers GSE184766 (ref. ⁴) and GSE233304 (ref. ⁸). For T and B cell scRNA-seq analysis, single-cell suspensions from the skull and sternum of healthy 8-week-old C57BL/6J male mice were prepared as described above. Single-cell suspensions were treated with anti-CD16/32 (2.4G2) to block Fc receptors. For T cell isolation, cells were stained with PerCP/Cy5.5-conjugated anti-CD45 (30-F11, BioLegend), PE/Cy7-conjugated anti-CD11b (M1/70, BD Biosciences), PE-conjugated anti-Thy1.2 (30-H12, Thermo Fisher Scientific), eFluor 660-conjugated anti-B220 (RA3-6B2, Thermo Fisher Scientific), FITC-conjugated anti-CD4 (RM4-4, BD Biosciences) and Alexa Fluor 488-conjugated CD8a (4SM16, Thermo Fisher Scientific). Dead cells were labelled with DAPI. Live CD45⁺B220⁻CD11b⁻Thy1.2⁺CD4/CD8⁺ cells were sorted. For B cell and ASC isolation, cells were stained with PerCP/Cy5.5-conjugated anti-CD45 (30-F11, BioLegend), FITC-conjugated anti-CD3 (17A2, Thermo Fisher Scientific), Alexa Fluor 700-conjugated anti-CD19 (6D5, BioLegend), APC-conjugated anti-CD138 (281-2, Thermo Fisher Scientific), PE-conjugated anti-TACI (8F10, BD Biosciences) and Pacific Blue-conjugated anti-IgD (11-26 c.2a, BioLegend). Dead cells were labelled with Zombie-NIR viability dye (BioLegend). Live CD45⁺CD3⁻CD138⁻CD19⁺ B cells and live CD45⁺IgD⁻CD3⁻CD138⁺TACI⁺ ASCs were isolated using the FACSAria II (BD Biosciences) system. For GC B cell scRNA-seq, single-cell suspensions from skull and tumour tissues were prepared as described above, 14 days after tumour injection. GC B cells were isolated using the GC B cell (PNA) microbead kit (Miltenyi Biotec). Dead cells were labelled with DAPI, and DAPI⁻ live cells were sorted using the FACSAria III (BD Biosciences) system.

Cells were loaded onto a 10x Genomics Chromium platform for gel beads-in-emulsions. Single Cell 5′ Library & Gel Bead Kit and Mouse B Cell Chromium V(D)J Reagents Kits were used to generate cDNA libraries targeting BCRs and gene expression. Libraries were sequenced using the Illumina NovaSeq 6000 system. Data were aligned to the mm10 genome using the CellRanger software pipeline (10x Genomics; v.1.1.0). Matrices were loaded into Seurat v.4 or v.5 for analysis⁵⁵, and R v.4.4.0 was used for statistical analyses. Cells with unique feature counts <200 or >6,000 (for T cell data), <200 or >7,500 (for B cell data), and <200 or >8,000 (for GC B cell data) with >20% (for T and B cell data) and >5% (skull) or >10% (tumour tissue) (for GC B cell data) mitochondrial gene expression were excluded for quality control. Doublets were also removed. Filtered and normalized data were analysed with the Seurat pipeline. Objects were merged and principal component analysis (PCA) and UMAP analysis were conducted using an elbow plot. Shared nearest neighbour (SNN) clustering was optimized with the Louvain algorithm in the FindClusters function. V(D)J receptor annotation data were generated with the CellRanger vdj command. Filtered contig CSV files were imported into R, and productive barcodes were filtered. The scRepertoire pipeline was used for scBCR/RNA-seq data analysis⁵⁶. Cluster markers were identified using the FindMarkers function, and cluster identities were determined manually. The Nebulosa package was used to visualize gene expression density⁵⁷. Somatic hypermutation frequencies were calculated using BCR sequence data processed with the Immcantation pipeline (https://immcantation.readthedocs.io/en/stable/)^58,59 and the IgBLAST and IMGT germline sequence databases. The Change-O package was used to process V(D)J annotations in BCR data files, and clonal family sizes were calculated using the Alakazam package. BCR mutation frequencies of IGHV were calculated using the SHazaM package.

OVA measurement

AAV-eGFP-, AAV-OVA-infected brains were chopped, homogenized with 70 μm strainer. After centrifugation, supernatants were collected and stored at −80 °C. CSF from AAV-eGFP- or AAV-HEL-OVA-infected mice was collected as described before⁵². OVA was measured by enzyme-linked immunosorbent assay (ELISA) using the sandwich OVA ELISA kit (LS Bio, LS-F9540-1) according to the manufacturer’s instructions. BioTek Gen5 software v.3.11 (Agilent) was used.

Statistical analysis

All data are presented as the mean ± s.e.m. Differences between two experimental groups were analysed using a two-tailed paired or unpaired Student’s t-test and Mann–Whitney U-test. For comparisons involving more than two groups, one-way ANOVA with Tukey’s multiple comparison test was used. For paired samples, Dunnett’s correction was applied. Two-way ANOVA with Sidak’s correction was also used, with paired comparisons used when samples were paired. Survival data were analysed using the log-rank test, and if multiple comparisons were required, the Holm–Sidak method was used for correction. All data are representative of results from at least two independent experiments. scBCR/RNA-seq data were analysed and visualized using R software (v.4.4.0) with RStudio, and all other data were analysed and plotted with GraphPad Prism (v.10.3.1).