Cd8α clone 53 6

Cell surface staining was performed at 4°C for 30 min in FACS buffer (10% FBS, 2.5mM EDTA in PBS). ILC were identified among CD8α⁻ (clone 5.3–6.7, Biolegend), CD3i⁻ (clone 17A2, Biolegend), IL‐7Rα⁺ (clone A7R34, Biolegend), B220⁻ (clone RA3‐6B2, eBioscience), CD11b⁻ (clone M1/70, eBioscience), CD11c⁻ (clone N418, eBioscience), CD3⁻ (clone 145‐2C11, eBioscience), and CD5⁻ (clone 53–7.3, eBioscience). Thymic iNKT cells were identified as mCD1d/PBS57⁺ and TCRβ⁺ (clone H57‐597, eBioscience). Intracellular cell staining was performed at room temperature for 1 h in permeabilization buffer (eBioscience). ILC2 and ILC3 subsets were identified using Abs against GATA3⁺ (clone TWAJ, eBioscience) and RORγt⁺ (clone AFKJS‐9, eBioscience). Staining for RANKL expression was performed in two steps using biotinylated RANKL (clone: IK22/5, eBioscience) and streptavidin‐PECy7 (Molecular Probes) at 4°C for 30 min in FACS buffer. Samples included Spherotech Accucount blank particles to enable calculation of cell frequency and were acquired using a Fortessa (BD). Samples were analyzed using FlowJo (FlowJo, LLC). Full gating strategy for all flow cytometry data identifying thymic ILC populations is shown in Supporting Information Fig. 1A.

Spleens and brains were harvested at indicated days p.i. Brains were minced and digested with collagenase (100 U/ml in DMEM with 2% FBS, 200 U/ml penicillin, 200 g/ml streptomycin, 2 mM L-glutamine, 5 μM HEPES, 1 μM MgCl₂, 1 μM CaCl₂) for 30 min at 37°C, passed through a 70 μm nylon cell strainer (BD Biosciences). Cells were isolated by centrifugation on a 44%:66% Percoll gradient. Spleens were passed through a 70 μm nylon cell strainer, then treated with ACK buffer (0.15 M NH₄Cl, 1mM KHCO₃, 1mM Na₂EDTA, pH 7.0) to lyse RBCs. Cells were surface-stained in FACS Buffer (PBS, pH 7.2 with 1% BSA, 0.1% sodium azide) for 30 min at 4°C with mAbs to CD8α (clone 53–6.7; Biolegend), CD44 (clone IM7; eBioscience), CD45.1 (clone A20; Biolegend), CD62L (clone MEL-14; BD Biosciences), CD69 (clone H1.2F3; BD Biosciences), PD-1 (clone RMP1-30; Biolegend), CD11a (clone 2D7; BD Biosciences), CD49d (clone MFR4.B; BioLegend), KLRG1 (clone 2F1; BD Biosciences), and CD127 (clone AFR34; Biolegend). Samples were collected on a BD LSR Fortessa or FACSCanto10 flow cytometer. Fluorescence-minus-one (FMO) samples were used to set positive gates for each surface molecule examined.

For samples not sorted, cells were washed with PBS and stained in FACS buffer (2% FBS, 2 mM EDTA, and 0.001% NaN₃). All gating strategies are represented in Supplementary Fig. 9. Cells were first stained for H-2K^b/SIY-pentamer (PE; ProImmune) for 10 min at room temperature at a 1:20 dilution, followed by staining with remaining antibodies for 20 min on ice. Antibodies against the following molecules were used: CD3ε (clone 17A2, BioLegend, 100216), Thy1.2 (clone 30-H12, BioLegend, 105320), CD45.2 (clone 104, BioLegend, 109806), CD8α (clone 53-6.7, BioLegend, 100747), CD4 (clone RM4-5, BioLegend, 100547), PD-L1 (clone 10 F.9G2, BioLegend, 124312), CD19 (clone 6D5, BioLegend, 115545), I-A/I-E (clone M5/114.15.2, BioLegend, 107630), and H-2K^b (clone AF6-88.5, BioLegend, 742862). All antibodies were used at a 1:100 dilution. Fixable Viability Dye eFluor 506 or 780 (eBioscience) was used for live/dead discrimination and was used at a 1:200 dilution. All flow cytometric analysis was conducted on either an LSRFortessa or X20 (BD) and analyzed using FlowJo software (Tree Star).

ReCl₃·6H₂O (Er, Yb, Y > 99%), oleic acid (OA), oleylamine (OM), 1-octadecene (ODE), yttrium trifluoroacetate ((CF₃COO)₃Y), sodium trifluoroacetate (CF₃COONa), sodium hydroxide (NaOH), ammonium fluoride (NH₄F), N-hydroxy-succinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), fluorescein isothiocyanate (FITC), methanol, ethanol, acetone, cyclohexane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), and polyacrylic acid (PAA) were purchased from Sigma-Aldrich. All chemicals were used without further purification. DAPI and anti-CD86-FITC (clone GL1) were purchased from Thermo Fisher. Anti-CD69-PE (clone H1.2F3), anti-CD62L-APC (clone MEL-14), and anti-CD3 (clone 145-2C11) were purchased from BioLegend. Alpha-Amino-omega-carboxy poly(ethylene glycol) hydrochloride (H₂N-PEG-COOH*HCL, MW 3.000 Dalton) was purchased from Iris Biotech. Cell Titer 96 AQueous MTS Reagent Powder was purchased from Promega. Anti-CD45.1 (clone A20), CD8α + (clone 53–6,7), and anti-CD43 (clone 1B11) were purchased from BioLegend.