Functional Role of Skull Lymphoid Structures in Central Nervous System Immunosurveillance

Animals, mouse strains and housing conditions

Specific pathogen-free (SPF) C57BL/6J mice (JAX, 000664) were obtained from The Jackson Laboratory. The following genetically modified and transgenic mouse 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 knockout (B6.129S7-Ifng^tm1Ts/J, JAX, 002287)⁴⁵; Lta knockout (B6.129S2-Lta^tm1Dch/J, JAX, 002258)⁴⁶; Aqp4 knockout (B6(Cg)-Aqp4, RIKEN, RBRC10053)⁴⁷; and muMT (B6.129S2-Ighm^tm1Cgn/J, JAX, 002288) mice⁴⁸. All mice were maintained in temperature-controlled conditions at 22 °C and humidity-controlled conditions of 33–39%, under a 12 h–12 h light–dark cycle. Food and water were available ad libitum.

Gnotobiotic mice were maintained in plastic flexible-film isolators within a dedicated gnotobiotic facility and housed under the same 12 h–12 h light–dark cycle. Mice were weaned onto autoclaved food and had unrestricted access to food and water. Germ-free (GF) mice were fed γ-irradiated AAD (Teklad custom diet, TD.130916) for 4 weeks to establish the AAD-fed GF cohort. Unless otherwise indicated, experiments used 8-week-old male mice. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Washington University in St Louis (23-0145).

Cell lines, engineered tumour cells and AAV vectors

The murine brain cancer cell line GL261 was provided by G. Dunn⁴⁹. The mouse glioma cell line CT2A was purchased from Sigma-Aldrich (SCC194). To generate CT2A cell lines expressing HEL-OVA, full-length OVA DNA was obtained from Addgene (64599). DNA encoding HEL-linker-OVA-IRES-eGFP was cloned into pLX208 vectors, and CT2A cells were transduced with the resulting lentiviral vectors. Transduced cells were selected using 100 µg ml⁻¹ hygromycin (Invitrogen) and maintained in medium supplemented with 50 µg ml⁻¹ hygromycin.

Cell lines were passaged with trypsin/EDTA (Gibco, 25300) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning, 10-013-CM) containing 10% FBS (Gibco, 26140) and 1% penicillin–streptomycin (Gibco, 15140). All cell lines were tested and confirmed to be free from mycoplasma contamination. Cell-line identity was verified by short-tandem-repeat analysis.

The following adeno-associated virus (AAV) vectors were used: 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).

Intracranial, intrahindbrain and peripheral tumour and AAV injections

For intracranial and intrahindbrain procedures, mice were anaesthetized and secured in a stereotaxic instrument (Kopf). Anaesthesia was induced with 5% isoflurane and maintained at 1–2%, or was induced with an intraperitoneal (i.p.) ketamine–xylazine cocktail containing ketamine at 100 mg per kg and xylazine at 10 mg per kg. Ophthalmic ointment was applied to prevent eye dehydration.

For intracranial AAV delivery, a small craniotomy was performed and 0.5 μl of AAV suspension, containing 1 × 10¹² viral genomes per ml and diluted in Dulbecco’s phosphate-buffered saline (DPBS; Gibco, 14190), was injected into the striatum. Injections were made at the following coordinates: anteroposterior (AP), 1.5 mm; mediolateral (ML), 1.5 mm; and dorsoventral (DV), −2.5 mm. A Nanoliter 2020 injector (World Precision Instruments) fitted with a fine glass capillary was used at a rate of 100 nl min⁻¹.

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

Antibody, cytokine, tamoxifen and OVA injections

To induce Cre recombination, recipient mice received daily i.p. injections of tamoxifen (0.1 mg per g; Sigma-Aldrich, T5648) dissolved in corn oil for five consecutive days. For T-cell depletion, mice were treated with 200 μg of anti-CD4 antibody (GK1.5, BioXcell, BE0003-1), anti-CD8a antibody (2.43, BioXcell, BE0061) or rat IgG2b isotype-control antibody (LTF-2, BioXcell, BE0090).

To enhance germinal-centre (GC) responses, mice received 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), with or without 10 μg i.p. or 2 μg s.c. recombinant IFNγ (BioLegend, 575308) and 1 μg i.p. or 0.2 μg s.c. recombinant IL-21 (BioLegend, 574506). Treatments were administered either i.p. or transcranially. To block GC formation, mice received 200 μg i.p. or 20 μg s.c. anti-CD40L antibody (MR-1, BioXcell, BE0017-1) or polyclonal Armenian hamster IgG control antibody (BioXcell, BE0091) by i.p. or transcranial administration. For transcranial delivery, reagents were mixed with 10 mg ml⁻¹ carboxymethylcellulose hydrogel (Sigma-Aldrich) to a final volume of 300 μl and injected s.c. beneath the scalp. For peripheral administration, anti-CD40L antibody was mixed with hydrogel and injected s.c. into the flank.

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

Splenectomy procedure

Lta-knockout 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 disinfected with iodine solution, and a 0.5 cm incision was made using a sterile scalpel. The spleen was exposed with smooth forceps, and its vessels and attachments were ligated using absorbable 5-0 sutures before the spleen was removed. The midline fascial defect was closed with 4-0 absorbable sutures, and the skin was closed with interrupted 4-0 non-absorbable sutures.

Deep cervical lymph-node ligation and removal

Deep cervical lymph-node (dCLN) ligation was performed as previously described^29,52. Briefly, mice were anaesthetized with a ketamine–xylazine cocktail, and an incision was made 5 mm above the clavicle. The sternocleidomastoid muscles were retracted, and afferent lymphatic vessels were ligated with nylon suture. Sham-operated mice underwent the incision and muscle retraction procedures but did not receive lymphatic ligation. For lymphadenectomy, the dCLNs were removed during the same procedure. Incisions were sutured, and mice recovered on a heating pad until responsive. Postoperative analgesics and prophylactic antibiotics were administered.

Adoptive transfer of T cells and B cells

Spleens were collected from donor mice, and primary naive CD4⁺ T cells, total CD4⁺ T cells or 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 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 administered intravenously through the retro-orbital route.

Single-cell preparation and flow cytometry analysis

For in vivo intravenous immune-cell labelling, anaesthetized mice received 7.5 μg of CD45-BV750 antibody (30-F11, BD Biosciences) through the retro-orbital route. After 3–5 min, mice were euthanized. Euthanasia was performed in a CO₂ chamber, followed by transcardial perfusion with DPBS. Single-cell suspensions were prepared from each organ. Bones were collected and cleaned of attached soft tissue, and the dura was removed from the skull. Bone-marrow single-cell suspensions were prepared as previously described^4,5. Briefly, bones were mechanically dissociated by chopping with scissors in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, 11875) containing 2% FBS.

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

Cells were incubated with anti-CD16/32 antibody (93, BioLegend) to block Fc receptors and stained with antibodies against 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 Vα2 (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 flow-cytometry staining, cells were fixed and permeabilized with the FoxP3 staining kit (BioLegend) according to the manufacturer’s instructions. Antibodies against 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. Viable cells were identified with Zombie NIR fixable viability dye (BioLegend). For intracellular cytokine detection, cells were stimulated for 4 h at 37 °C 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). Antibodies were diluted 1:200. Samples were acquired using an Aurora spectral flow cytometer (Cytek), and data were analysed with FlowJo v.10.8.1 (TreeStar). Gating strategies and fluorescence-minus-one controls are provided in Supplementary Figs. 5 and 6.

Reverse-transcription quantitative PCR

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

eGFP transcripts were detected using the following primers: forward, 5′-AAGGGCATCGACTTCAAGG-3′; reverse, 5′-TGCTTGTCGGCCATGATATAG-3′. Transcript abundance was normalized to Actb, and relative expression was calculated using the comparative ΔΔC_t method.

Immunofluorescence staining and imaging

Mice were perfused intracardially with DPBS followed by 4% paraformaldehyde (PFA), and samples were subsequently fixed in 4% PFA. Bone samples were decalcified in 5% EDTA at 4 °C for 3 days, with the EDTA solution replaced daily. For cryosectioning, samples were dehydrated in 30% sucrose at 4 °C for 3 days, embedded in Fisher Healthcare Tissue-Plus OCT compound (Fisher Healthcare, 23-730-571), frozen and sectioned at 20–50 μm using a Leica Biosystems cryostat.

For immunofluorescence staining, tissues were blocked in 0.2% Triton X-100 in DPBS containing 5% goat or donkey serum and incubated with primary antibodies against 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), eFluor 660-conjugated rat anti-mouse B220 (RA3-6B2, Thermo Fisher Scientific) and polyclonal anti-GFP (Thermo Fisher Scientific, A10262). Secondary antibodies were 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) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific, A-21206).

For in vivo labelling, mice received intravenous injections 40 min before perfusion containing 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), eFluor 660-conjugated anti-mouse B220 (RA3-6B2, Thermo Fisher Scientific), Alexa Fluor 594-conjugated anti-mouse CD4 (GK1.5, BioLegend) or Alexa Fluor 647-conjugated anti-mouse CD4 (GK1.5, BioLegend). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, D9542).

The ADAPT-3D protocol was used for whole-mount tissue clearing⁵³. Whole-mount dura staining was performed as previously described²⁷. Images were acquired using wide-field microscopy (Olympus SLIDEVIEW VS200) or a Stellaris confocal microscope (Leica) with ×10, ×20 or ×40 objectives. Leica Application Suite v.4.2.1.23810 and Nikon Elements v.5.2.0 were used for image acquisition, and Fiji v.2.14.0/1.54j was used for image analysis. The Fiji colocalization plugin was used to identify colocalized cells. Cell counts and density measurements were performed with QuPath v.0.6.0⁵⁴. Fluorescent-protein intensity and coverage were quantified using Fiji. Background fluorescence was determined from negative controls, including non-fluorescent samples or regions, and values were not normalized. Representative single-plane images of whole-mounted skulls are shown in Supplementary Fig. 7.

Single-cell RNA sequencing and B-cell receptor analysis

Publicly available datasets were downloaded from the Gene Expression Omnibus (GEO) under accession numbers GSE184766 (ref. ⁴) and GSE233304 (ref. ⁸). For T-cell and B-cell single-cell RNA-sequencing (scRNA-seq) analyses, single-cell suspensions were prepared from the skull and sternum of healthy 8-week-old male C57BL/6J mice as described above. Cells were treated with anti-CD16/32 antibody (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 anti-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 antibody-secreting-cell (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 sorted using a FACSAria II system (BD Biosciences).

For GC B-cell scRNA-seq, single-cell suspensions were prepared from skull and tumour tissue 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⁻ viable cells were sorted using a FACSAria III system (BD Biosciences).

Cells were loaded onto a 10x Genomics Chromium platform to generate gel-bead-in-emulsion partitions. cDNA libraries for gene expression and B-cell receptor (BCR) profiling were prepared using the Single Cell 5′ Library & Gel Bead Kit and Mouse B Cell Chromium V(D)J Reagents Kits. Libraries were sequenced on an Illumina NovaSeq 6000 system and aligned to the mm10 genome using the CellRanger pipeline (10x Genomics; v.1.1.0). Matrices were imported into Seurat v.4 or v.5 for analysis⁵⁵, and R v.4.4.0 was used for statistical analyses.

Cells were excluded during quality control if they contained fewer than 200 or more than 6,000 unique features for T-cell data, fewer than 200 or more than 7,500 unique features for B-cell data, or fewer than 200 or more than 8,000 unique features for GC B-cell data. Cells with mitochondrial gene expression exceeding 20% for T- and B-cell datasets, or exceeding 5% in skull and 10% in tumour tissue for GC B-cell datasets, were also excluded. Doublets were removed. Filtered and normalized datasets were analysed using the Seurat pipeline. Objects were merged, and principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) were performed using an elbow-plot-based approach. Shared nearest-neighbour (SNN) clustering was optimized with the Louvain algorithm in the FindClusters function.

V(D)J receptor annotations were generated using the CellRanger vdj command. Filtered contig CSV files were imported into R, and productive barcodes were retained. The scRepertoire pipeline was used for scBCR/RNA-seq analysis⁵⁶. Cluster markers were identified with the FindMarkers function, and cluster identities were assigned manually. The Nebulosa package was used to visualize gene-expression density⁵⁷. Somatic hypermutation frequencies were calculated from BCR sequences processed with the Immcantation pipeline (https://immcantation.readthedocs.io/en/stable/)^58,59, using the IgBLAST and IMGT germline sequence databases. The Change-O package was used to process V(D)J annotations, clonal family sizes were calculated with Alakazam, and IGHV BCR mutation frequencies were calculated with SHazaM.

OVA protein measurement by ELISA

Brains infected with AAV-eGFP or AAV-OVA were chopped and homogenized through a 70 μm cell strainer. Following centrifugation, supernatants were collected and stored at −80 °C. Cerebrospinal fluid (CSF) from AAV-eGFP- or AAV-HEL-OVA-infected mice was collected as described previously⁵². OVA concentrations were measured using a sandwich OVA enzyme-linked immunosorbent assay (ELISA) kit (LS Bio, LS-F9540-1) according to the manufacturer’s instructions. Data were processed using BioTek Gen5 v.3.11 (Agilent).

Statistical analysis and data presentation

Data are presented as the mean ± s.e.m. Differences between two experimental groups were evaluated using two-tailed paired or unpaired Student’s t-tests or Mann–Whitney U-tests, as appropriate. Comparisons among more than two groups were performed using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. Dunnett’s correction was applied to paired samples. Two-way ANOVA with Sidak’s correction was also used, with paired comparisons applied when appropriate. Survival data were analysed using the log-rank test; the Holm–Sidak method was used to correct for multiple comparisons. All results are representative of at least two independent experiments. Single-cell BCR/RNA-seq data were analysed and visualized in R v.4.4.0 with RStudio. All other data were analysed and plotted using GraphPad Prism v.10.3.1.

Reporting summary

Additional information about the research design, experimental procedures and reporting standards is available in the Nature Portfolio Reporting Summary linked to this article.