Mice

Mice were housed in the University of Pennsylvania Department of Pathobiology vivarium according to institutional guidelines with 12 h light/dark cycles, ambient temperature ranging from 20 °C to 25 °C and ambient humidity ranging from 35% to 55%. C57BL/6J (stock number 000664, RRID:IMSR_JAX:000664), CD45.1 (B6.SJL-Ptprc^a Pepc^b/BoyJ, stock number 002014, RRID:IMSR_JAX:000664), Nur77^GFP (stock number 016617, RRID:IMSR_JAX:016617), OT-I (stock number 003831, RRID:IMSR_JAX:003831) and BALB/c (stock number 000651, RRID:IMSR_JAX:000651) mice were purchased from Jackson Laboratories and bred at the University of Pennsylvania. Snap25^Cre mice were generously provided by H. Zeng at the Allen Institute for Brain Science, and Stat1^flox/flox mice were generously provided by L. Hennighausen at the National Institutes of Health⁹². Snap25^Cre mice were bred with Stat1^flox/flox mice at the University of Pennsylvania to generate Snap25^Cre–Stat1^flox/flox (Stat1^ΔNEU) progeny. Cohorts of male mice were used for all studies, and all mice are on a C57BL/6 background unless otherwise noted. Ethical oversight of all animal studies was approved by the University of Pennsylvania Institutional Care and Use Committee (protocol 805045.)

Infections and T-cell transfers

T. gondii parasites were maintained in culture in human foreskin fibroblasts (HFFs). HFFs were purchased from American Type Culture Collection and were periodically tested for mycoplasma contamination using the MycoScope PCR Mycoplasma Detection Kit (Genlantis, catalogue number MY01050.) To isolate tachyzoites for infection, T. gondii-infected HFFs were gently scraped from flasks, passed 5 times through a 26G syringe and washed with PBS. Mice were infected at 8 weeks of age by intraperitoneal injection with 5,000 tachyzoite parasites grown in vitro.

For OT-I T-cell transfers, whole splenocytes from Naive Nur77^GFP CD45.1⁺CD45.2⁺ OT-I mice were processed as described below. The fraction of OT-I cells in the splenocytes was determined by flow cytometry, and 5,000 OT-I cells were transferred intravenously into mice at the indicated time point before or after infection.

IFNγ blockade and sulfadiazine treatment

In vivo blockade of IFNγ signalling was performed by intraperitoneal injection of 200 μg per dose of rat IgG1 anti-IFNγ (clone XMG1.2, BioXcell) or IgG1 anti-horseradish peroxidase for control mice (HRPN, BioXcell). Injections were administered 2 times per week for 4 weeks before collection and analyses for parasite burden.

Where indicated, mice were treated with the anti-parasitic drug sulfadiazine (Sigma-Aldrich, S8626-25G) via drinking water. Sulfadiazine was reconstituted in dimethylsulfoxide to 50 mg ml⁻¹ and added to drinking water at a final concentration of 0.25 mg ml⁻¹. Sulfadiazine-treated water was administered starting at 21 d.p.i. and refreshed every 3 days for 4 weeks.

Tissue processing and cell counting

To generate single-cell suspensions for flow cytometry, spleens were passed through a 40 μm filter, and red blood cells were lysed for 3 min at room temperature in ammonium-chloride-potassium (ACK) lysis buffer. Brains were diced into 1 mm pieces and digested at 37 °C and 5% CO₂ for 1.5 h with 250 μg ml⁻¹ collagenase/dispase and 10 μg ml⁻¹ DNase and then passed through a 70 μm filter. Leukocytes were then isolated through a 30% and 60% percoll gradient and density centrifugation at 900 g for 25 min. Whole blood was collected through submandibular bleed into 0.05 mM EDTA in PBS. Cells were pelleted, and red blood cells were lysed for 3 min at room temperature in ACK lysis buffer.

For quantification of cellularity and live leukocytes isolated from tissues, a fraction of processed cell suspensions was stained with Guava ViaCount Reagent (catalogue number 11-25209, 240 ml) and analysed on a Guava easyCyte flow cytometer according to manufacturer’s protocol.

Generation of transgenic parasites

To generate Δbfd1 parasites, Pru-tub1-OVA-tdTomato parasites⁶⁸ were mechanically lysed from host cells by scraping and syringe releasing through a 27G needle. Parasites were pelleted for 10 min at 1,000 g, resuspended in Cytomix (10 mM KPO₄, 120 mM KCl, 150 mM CaCl₂, 5 mM MgCl₂, 25 mM HEPES, 2 mM EDTA) and combined with a DNA transfection mixture to a final volume of 400 μl. The final transfection mixture was supplemented with 2 mM ATP and 5 mM glutathione. Two guide RNAs contained on the Cas9-expressing pU6-Universal⁹³ (Addgene 52694) plasmid (Supplementary Data 1) were transfected to target the regions immediately upstream and downstream of the BFD1 locus as previously described¹². A pTUB-mNeonGreen repair template with homology arms matching the 40 bp regions flanking the cut sites was transfected to allow for sorting of ∆bfd1 parasites by fluorescence-activated cell sorting (FACS). After sorting, parasites were plated at limiting dilutions to allow for screening of clonal parasites. A ∆bfd1 clone was confirmed by PCR and sanger sequencing (Supplementary Data 1). However, despite these parasites being mNeonGreen positive, the mNeonGreen repair template was not found within the BFD1 locus, indicating that this construct had randomly integrated into the parasite genome.

Generation of bag1-OVA parasites was performed by first generating the pbag1OVA/sagCAT plasmid (Supplementary Data 1) which contains (1) the BAG1 promoter upstream of (2) the last 31 amino acids of the T. gondii major glycosyl-phosphatidylinositol-anchored surface antigen, P30, containing the signal sequence for targeting this protein to the parasitophorous vacuole, followed by (3) amino acids 140–386 of ovalbumin (OVA) and (4) a 3′ untranslated region of the dihydrofolate reductase-thymidylate synthase gene (3′dhfr). The OVA-3′dhfr fragment was subcloned in place of the red fluorescent protein (RFP)-3′dhfr fragment of the pbag1RFP/sagCAT plasmid⁹⁴ via BglII/NotI restriction digests. Transfections of type II Pruginaud (Pru) strain parasites were performed using electroporation, and stably expressing parasites were selected with chloramphenicol. Clonal parasite lines were generated by limiting dilution.

Immunohistochemistry

Brains were dissected by a sagittal cut along the midline, collected in 10% formalin, embedded in paraffin and sectioned. For identification of T. gondii parasites, slides were hydrated, and antigen was retrieved with 0.01 M sodium citrate buffer at pH 6.0, endogenous peroxidase was blocked with 0.3% H₂O₂, and sections were blocked with 2% goat serum. Parasites were detected with rabbit anti-T. gondii polyclonal antibody (gift from F. Araujo, Palo Alto Medical Foundation, 1:1,000) followed by biotinylated goat anti-rabbit IgG antibody (Vector Laboratories). Avidin-biotin complex (ABC) reagent and 3,3′-diaminobenzidine (DAB) substrate (Vector Laboratories) were used according to manufacturer’s protocol to visualize parasite staining, and haematoxylin staining was applied to visualize nuclei. Images were acquired with a Leica DM6000 Widefield Fluorescence Microscope using LAS X version 3.7.4.23463 software.

Pathological assessment

Brains were collected and processed for sectioning as described above. Haematoxylin and eosin (H&E)-stained sagittal sections of the brain, including the cerebral cortex and basal nuclei (Fig. 5 and Extended Data Fig. 5) or cerebral cortex and basal nuclei, hippocampus, thalamus, midbrain, cerebellum, pons and medulla (all other assessments) were assessed by a board-certified veterinary pathologist. The type of inflammatory cells and semiquantitative scores for the severity of parameters of interest (inflammation, haemorrhage, gliosis, necrosis and presence of parasites) were recorded for individual animals.

Flow cytometry

Single-cell suspensions were plated at up to 2 × 10⁶ cells. Cells were Fc receptor blocked for 20 min with 0.5 μg ml⁻¹ αCD16/32 (Clone 2.4G2) and 0.25% normal rat serum in FACs buffer (2% BSA and 0.02 mM EDTA in PBS) at 4 °C. If staining for tetramer, cells were then washed and incubated with 1:200 to 1:400 dilution of tetramer in FACs buffer at room temperature for 30 min. Following Fc blocking or tetramer stain, surface stain was applied, and cells were incubated for 30 min at 4 °C. Cells were then washed and resuspended in 0.1% paraformaldehyde in FACs buffer. If staining for intracellular antigens, cells were fixed and permeabilized with eBioscience Foxp3/Transcription Factor Staining Buffer Set according to manufacturer’s protocol. Cells were then stained for intracellular antigens in permeabilization buffer for 30 min at 4 °C. Cells were washed and resuspended in FACs buffer. Stained cells were acquired on a BD LSR Fortessa, BD FACSymphony A5 or BD FACSymphony A3 using BD FACS DIVA v9.0 software. Analysis was performed with FlowJo v10.7.2. Gate placement was determined based on samples stained with all fluorescent antibodies used in the flow panel except the marker of interest. Gating strategies used to identify the populations analysed in these studies can be found in Extended Data Fig. 1d (OT-I T cells) and Supplementary Fig. 6 (CNS immune populations).

The following antibodies and reagents were used for staining: B220: BUV496, BD Biosciences: 612950, clone: RA3-6B2, RRID:AB_2870227, dilution 1:300; CD3: APC-ef780, Invitrogen: 47-0032-82, clone: 17A2, RRID:AB_1272181, dilution 1:300; CD3: BUV737, BD Biosciences: 612803, clone: 17A2, RRID:AB_2738781, dilution 1:300; CD3e: PE-cf594, BD Biosciences: 562286, clone: 145-2C11, RRID:AB_11153307, dilution 1:300; CD4: BUV496, BD Biosciences: 612952, clone: GK1.5, RRID:AB_2813886, dilution 1:200; CD4: BV650, Biolegend: 100555, clone: RM4-5, RRID:AB_2562529, dilution 1:400; CD4: FITC, eBioscience: 11-0041-85, clone: GK1.5, RRID:AB_464892, dilution 1:200; CD4: APC-ef780, Invitrogen: 47-0041-82, clone: GK1.5, RRID:AB_11218896, dilution 1:300; CD8a: BUV563, BD Biosciences: 748535, clone: 53-6.7, RRID:AB_2872946, dilution 1:200; CD8a: BUV615, BD Biosciences: 613004, clone: 53-6.7, RRID:AB_2870272, dilution 1:200; CD8a: BV650, Biolegend: 100742, clone: 53-6.7, RRID:AB_2563056, dilution 1:200; CD8b: APC-ef780, Invitrogen: 47-0083-82, clone: eBioH35-17.2, RRID:AB_2573943, dilution 1:200; CD11a: BUV805, BD Biosciences: 741919, clone: 2D7, RRID:AB_2871232, dilution 1:300; CD11b: BV650, Biolegend: 101259, clone: M1/70, RRID:AB_2566568, dilution 1:500; CD19: BUV395, BD Biosciences: 563557, clone: 1D3, RRID:AB_2722495, dilution 1:300; CD45: AF647, Biolegend: 103124, clone: 30-F11, RRID:AB_493533, dilution 1:200; CD45.1: ef450, Invitrogen: 48-0453-82, clone: A20, RRID:AB_1272189, dilution 1:200; CD45.1: BV711, Biolegend: 110739, clone: A20, RRID:AB_2562605, dilution 1:200; CD45.1: PE-Cy7, Biolegend: 110730, clone: A20, RRID:AB_1134168, dilution 1:200; CD45.2: APC, Biolegend: 109814, clone: 104, RRID:AB_389211, dilution 1:200; CD45.2: BV711, Biolegend: 109847, clone: 104, RRID:AB_2616859, dilution 1:200; CD69: BUV737, BD Biosciences: 612793, clone: H1.2F3, dilution 1:200; CD69: PerCP-Cy5.5, eBioscience: 45-0691-82, clone: H1.2F3, RRID:AB_1210703, dilution 1:400; CD103: PE, eBioscience: 12-1031-81, clone: 2E7, RRID:AB_11150242, dilution 1:200; CD103: BV605, Biolegend: 121433, clone: 2E7, RRID:AB_2629724, dilution 1:200; CD107a: PE-Cy7, Biolegend: 121620, clone: 1D4B, RRID:AB_2562146, dilution 1:300; CD127: BV421, Biolegend: 135027, clone: A7R34, RRID:AB_2563103, dilution 1:200; CTLA-4: APC-R700, BD Biosciences: 565778, clone: UC10-4F10-11, RRID:AB_2739350, dilution 1:200; CX3CR1: BV785, Biolegend: 149029, clone: SA011F11, RRID:AB_2565938, dilution 1:400; CX3CR1: PerCP-Cy5.5, Biolegend: 149009, clone: SA011F11, RRID:AB_2564493, dilution 1:400; F4/80: APC-ef780, eBiosciences: 47-4801-82, clone: BM8, RRID:AB_2735036, dilution 1:200; GFP: AF488, Biolegend: 338008, clone: FM264G, RRID:AB_2563288, dilution 1:300; H-2Kb: AF647, Biolegend: 116512, clone: AF6-88.5, RRID:AB_492917, dilution 1:200; I-A/I-E: AF700, Biolegend: 107622, clone: M5/144.15.2, RRID:AB_493727, dilution 1:300; I-A/I-E: BV711, Biolegend: 107643, clone: M5/144.15.2, RRID:AB_2565976, dilution 1:1200; IFNγ: BUV737, BD Biosciences: 612769, clone: XMG1.2, dilution 1:200; Ki-67: BV470, BD Biosciences: 566109, clone: B56, RRID:AB_2739511, dilution 1:200; KLRG1: BUV395, BD Biosciences: 740279, clone: 2F1, RRID:AB_2740018, dilution 1:200; Ly-6C: BV785, Biolegend: 128041, clone: HK1.4, RRID:AB_2565852, dilution 1:600; Ly-6G: BUV563, BD Biosciences: 612921, clone: 1A8, RRID:AB_2870206, dilution 1:400; NK1.1: BUV395, BD Biosciences: 564144, clone: PK136, RRID:AB_2738618, dilution 1:300; PD-1: BV421, Biolegend: 135221, clone: 29 F.1A12, RRID:AB_2562568, dilution 1:200; PD-1: BV605, Biolegend: 135220, clone: 29 F.1A12, RRID:AB_2562616, dilution 1:200; PD-1: BV785, Biolegend: 135225, clone: 29 F.1A12, RRID:AB_2563680, dilution 1:200; T-bet: AF647, Biolegend: 644804, clone: 4B10, RRID:AB_1595466, dilution 1:200; TCRβ: PerCP-Cy5.5, Biolegend: 109228, clone: H57-597, RRID:AB_1575173, dilution 1:500; TCRβ: ef450, Invitrogen: 48-5961-82, clone: H57-597, RRID:AB_11039532, dilution 1:200; Tetramer MHCI (OVA): PE, NIH Tetramer Core, peptide: SIINFEKL, dilution 1:300; Tetramer MHCI (Tgd057): APC, NIH Tetramer Core, peptide: SVLAFRRL, dilution 1:300; Tetramer MHCII (AS15): APC, NIH Tetramer Core, peptide: AVEIHRPVPGTAPPS, dilution 1:400; Tim3: BV605, Biolegend: 119721, clone: RMT3-23, RRID:AB_2616907, dilution 1:200; TNF: APC, Invitrogen: 17-7321-82, clone: MP6-XT22, RRID:AB_469508, dilution 1:200; Vα2 TCR: BUV615, BD Biosciences: 751416, clone: B20.1, RRID:AB_2875415, dilution 1:400; Vα2 TCR: PE, Biolegend: 127808, clone: B20.1, RRID:AB_1134183, dilution 1:300; Viability: GhostDye Violet 510, TONBO Biosciences: 13-0870-T100, dilution 1:300; Viability: GhostDye Red 780, TONBO Biosciences: 13-0865-T100, dilution 1:300.

T-cell peptide restimulation

Whole splenocytes or brain leukocytes were plated at a constant cell concentration. Cells were incubated with 1 μM SIINFEKL (OVA peptide), SVLAFRRL (Tgd057 peptide) or AVEIHRPVPGTAPPS (AS15 peptide) and fluorescently labelled αLAMP1 antibody for 2 h, followed by a further 2 h with Protein Transport Inhibitor Cocktail (Invitrogen: 00-4980-03). Cells were analysed for degranulation and cytokine production by flow cytometry.

Fluorescent imaging

Brains were collected from mice after cardiac perfusion with heparin (10 U ml⁻¹) in 0.9% saline followed by 4% paraformaldehyde, fixation in 4% paraformaldehyde for 24 h and cryoprotection at 4 °C in 30% sucrose for at least 24 h. Sagittal sections (40 μm) were cut on a freezing microtome and stored at 4 °C in cryoprotectant (0.05 M sodium phosphate buffer with 30% glycerol and 30% ethylene glycol). Staining and imaging for neurons, astrocytes, dolichos biflorus lectin and OVA in infected brains was performed using the following reagents and antibodies: anti-OVA, Abcam: ab181688, dilution 1:200; DBA, Vector laboratories: B1035, dilution 1:500; anti-GFAP, DAKO: Z0334, dilution 1:200; anti-MAP2, Abcam: ab5392, dilution 1:2,000; anti-NeuN, Millipore: MAB3778, dilution 1:200; anti-Neurofilament, Abcam: ab4680, dilution 1:20,000. Anti-SAG1 (DG52), dilution 1:500 and SRS9, dilution 1:2,000 antibodies were gifted by J. Boothroyd^95,96. Stained sections were mounted on slides using ProLong Diamond Anti-Fade Mountant with DAPI (Invitrogen P36962) according to manufacturer’s protocol. Images were acquired on a Zeiss LSM 880 inverted confocal microscope (University of Arizona, Imaging Core) and Nikon E600 upright widefield microscope (University of Pennsylvania, Penn Vet Imaging Core); images were analysed using Zen 2.6 blue edition software and counted using ImageJ version 1.53 v software.

For quantification of cyst size in WT and Stat1^ΔNEU mice, perfused brains were frozen in Optimal Cutting Temperature Compound (Fisher Scientific, catalogue number 23-730-571), and 10 μm sections were cut on a vibratome. Sections were mounted with ProLong Diamond Anti-Fade Mountant with DAPI. Sections were imaged on a Leica SP5-II at ×60 magnification. Cysts were defined as vacuoles with >32 parasites present. Cyst area was quantified using Imaris (v9.7.2) microscopy imaging software, and images were generated with LAS AF (v2.7.3.9723) software. Five to 20 vacuoles per brain were quantified.

Imaging of bag1-OVA parasites in vitro was performed using HFFs cultured on eight-well chamber slides. Cells were infected at multiplicity of infection (MOI) = 3 for 3 h. Stress-induced cyst formation was performed by culturing infected cells under cyst-inducing conditions for 4 days: cells were incubated without supplemental CO₂ in RPMI media (pH 8.0) without sodium bicarbonate, supplemented with 1% FBS, 10 mg ml⁻¹ HEPES, 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin. Media was replaced every 48 h (ref. ⁶⁴). Cells were then stained with anti-OVA (Abcam ab181688) and DAPI nuclear stain. Slides were imaged on a Nikon E600 upright widefield microscope (University of Pennsylvania Imaging Core).

Quantification of serum IFNγ

Blood was collected by submandibular bleed and clotted at room temperature for 1 h. Serum was separated through centrifugation for 10 min at 14,000 g. Serum was diluted between 1:5 and 1:20, and IFNγ levels were assayed by BD Mouse IFNγ Flex Set Cytometric Bead Array according to manufacturer’s protocol. Beads were analysed by flow cytometry on a BD Canto.

Quantification of parasite burden by quantitative PCR

Sagittal sections of brain tissue were snap frozen and stored at −20 °C. DNA was isolated using QIAGEN DNeasy Blood and Tissue Kit according to manufacturer’s protocol. DNA quality and concentration was assessed on a Nanodrop ND-1000 UV–Vis Spectrophotometer. About 200 ng of DNA was used to quantify parasite burden by quantitative PCR (qPCR) with Power SYBR Green Master Mix and primers specific for the T. gondii B1 gene (Supplementary Data 1). qPCR was performed on Applied Biosystems ViiA7 with the following conditions: hold phase for 2 min at 50 °C and 10 min at 95 °C; PCR phase (occurs 50 times) for 15 s at 95 °C and 1 min at 60 °C.

Statistical information

Statistical tests were run in Prism software version 10.2.3 (Graphpad). Data were analysed by two-sided Student’s t-tests, two-sided Student’s t-tests with Bonferroni–Dunn correction for multiple tests, two-way analysis of variance (ANOVA) or two-sided Mann–Whitney tests where indicated. Not significant, P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Precise P values for significant trends are included in the corresponding figure legends.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.