Nature 2020 01 30 Part.02

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nature research | reporting summary

October 2018

Methodology

Sample preparation Isolation of mononuclear cells and flow cytometry. Tissue was harvested and incubated in a digestion cocktail containing 1 mg ml

−1 collagenase D (Roche), 1 mg ml−1 collagenase A (Roche) and 30 μg ml−1 DNase I (Sigma-Aldrich) in complete RPMI (10% FBS)

at 37 °C for 30 min. Tissue was then filtered through a 70 μm filter. For brain tissues, cells were mixed in 4 mL of 25% Percoll

(Sigma-Aldrich) solution and centrifuged at 530g for 15 minutes without a brake. The Percoll layer was removed and cells were

diluted in 5 mL of 1% BSA. Cells were treated with ACK buffer, and resuspended in 1% BSA. At this point cells were counted using

an automated cell counter (Thermo fisher).

For tetramer experiments, staining was performed with antibodies (1:200) and tetramer (1:50) for 60 minutes at room

temperature. Cells were washed to remove excess antibodies and resuspended in 1% BSA with 10 μL of CountBright absolute

counting beads (Life technologies, OR) for multiparameter analyses on the LSR II flow cytometer (Becton Dickinson), and

subsequently analyzed using FlowJo software (10.5.3, Tree Star). For calculation of tetramer positive T cells in each organ this

calculation was used:

number of tetramer positive T cells * (# of input beads / # of counted beads) * (# of cells from automated counter / # of total

events in flow cytometry).

For cytokine stimulation, surface markers were first stained on ice for 30 minutes. After washing, cells were stimulated in

complete RPMI with 200μL of 1x Cell stimulation cocktail without protein transporter inhibitor (eBioscience Cell Stimulation

Cocktail, ThermoFisher) for 1 hour at 37C. 50 μL of 5x Cell stimulation cocktail with protein transporter (eBioscience Cell

Stimulation Cocktail, ThermoFisher) was added and incubated for an additional 4 hours. Cells were then fixed with 100μL 2%

formaldehyde on ice for 45 minutes. Cells were washed with 1x Perm/Wash Buffer (BD Cytofix/Cytoperm, BD Biosciences), and

then permeabilized with 1x Perm/Wash Buffer (BD Cytofix/Cytoperm, BD Biosciences) for 10 minutes on ice. Intracellular

antigens were stained on ice for 30 minutes.

For transcription factor staining, surface markers were first stained on ice for 30 minutes. Cells were then fixed with 100μL 2%

formaldehyde on ice for 45 minutes. Cells were washed with 1x Perm/Wash Buffer (eBioscience Foxp3/Transcription Factor

Staining Buffer Set, ThermoFisher), and then permeabilized with 1x FOXP3Perm/Wash Buffer for 10 minutes on ice. Intracellular

antigens were stained on ice for 30 minutes.

For AKT phosphorylation staining, surface markers were first stained on ice for 30 minutes. Cells were then fixed and washed

following BD Phosflow kit directions.

Instrument LSRII

Attune NxT Flow Cytometer

Software FlowJo 10.6.0

Cell population abundance n/a

Gating strategy All gates are shown in the supplementary information.

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.