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Immunoblots and immunoprecipitation were performed using standardized protocols with the antibodies listed in Supplementary Table 2^{58 (link)}. For flow cytometry, cells were washed with- and resuspended in 100 μL FACs buffer (phosphate-buffered saline, 2% fetal bovine serum). The cells were Fc-blocked (TruStain, Biolegend), stained (1 h) with the appropriate antibodies (Supplementary Table 2), washed and resuspended in 250 μL FACs buffer to be analyzed on a BD LSRFortessaX20. Compensation and data were analyzed using FlowJo. H2Kb-SIINFEKL pentamer (Proimmune) staining was performed with blood seven days post boost-immunization according to manufacturer’s protocol. The HLA-A2-RMSAPSTGGV tetramer was synthesized by the NIH Tetramer Core Facility. hCD155_AAD-tg mice were immunized with vector or peptide and bled one week after boost. Blood was lysed with ACK Lysing buffer (Gibco, A1049201), washed (FACs buffer), stained with tetramer (30 min, 20 °C), washed, Fc-blocked (see above), stained with CD8, CD19 and CD11b antibodies and analyzed as described above. For IHC, lymph nodes were harvested from euthanized mice, fixed in 4% parafomaldehyde, dehydrated in 70% ethanol, paraffin-embedded and sectioned (7 μM). Mounted sections were stained using the Ventana Discovery Ultra platform (Research Immunohistology Lab, Duke Dept. of Pathology).

After the mice were euthanized, lungs were perfused with 5 mL of PBS via the left ventricle. Lungs were mechanically homogenized and passed through a 70 μm cell strainer. Remaining RBCs were lysed using BD Pharm Lyse (BD Biosciences). Cells were treated with an Fc blocker (TruStain, BioLegend) prior to staining. Surface staining to identify leukocyte populations was then performed. For FoxP3 intracellular staining, cells were permeabilized with 0.1% saponin and stained for intracellular FoxP3. The cells were washed, resuspended in FACS buffer, and analyzed by using an Attune flow cytometer.

To cells in 300μl to 700μl PBS, TruStain (BioLegend) was added at 1μg/100μl for 10 mins on ice. Cell aliquots (10⁵ to 10⁶) were made up to 200μl with PBS and incubated with fluorescent marker conjugated antibodies directed against: CD45-FITC or CD45-APC (BioLegend) at 37°C, 25 mins. For ROS detection, 2μl of DCFH-DA was added to each sample at the same time as anti-CD45 and samples were incubated at 37°C, 15 mins. Samples were washed with 1ml PBS (centrifugation 400g, 5 mins) and either washed again and resuspended in 500μl PBS, 1% FCS for FACS or fixed in 100μl 2% buffered paraformaldehyde, 15 mins dark. Fixed cells were centrifuged and resuspended in 100μl PBS and stored in the dark, overnight, 4°C. Before analysis, fixed cells were pelleted and resuspended in 500μl PBS, 1% FCS. Samples were sorted using FACSCalibur or FACSAria (BD Biosciences) and data acquired using FlowJo and analysed using Kaluza 1.2 (Beckman-Coulter). Statistical analyses were carried out using GraphPad Prism 5, using paired T-tests.

T‐cell polyfunctionality was measured as previously described (Kim et al., 2017). Briefly, splenocytes were stimulated with 5 μg/mL cEDIII for 4 h in the presence of 5 μg/mL brefeldin A. Cells were then washed with PBS and stained with a viability dye (eFluor 780; 1:1000 dilution; Thermo Fisher, Hemel Hempstead, UK) alongside an Fc receptor blockade (TruStain, 1 μg/mL; Biolegend London, UK) for 20 min at 4 °C. Following this, cells were washed in flow cytometry buffer and then fixed in 100 μL BD Cytofix (Becton Dickinson, Oxford, UK) for 30 min at 4 °C. Cells were washed and then stained with the following antibodies at optimized concentrations for 45 min at 4 °C: CD3‐FITC, CD4‐PerCP‐Cy5.5, CD8‐Alexa Fluor 700, IFN‐γ‐PE Dazzle, IL‐2‐PE, IL‐17A‐PE‐Cy7 and TNF‐α‐APC (all from Biolegend). Fluorescence minus one and PMA/ionomycin‐stimulated cells were used to determine gating boundaries and serve as positive controls. Cells were then washed twice with permeabilization buffer and flow cytometry buffer and then acquired on a LSR II (Beckton Dickinson, Oxford, UK) instrument.

For staining cell surface markers, 1 × 10⁶ cells were washed in 1 × PBS buffer and resuspended in 100 μl FACS buffer. To block the Fc receptor, 5 μl of TruStain (Biolegend) was added and incubated on ice for 15 min. The cells were then washed and resuspended in 100 μl FACS buffer. FITC-conjugated anti-CD10 or SLAMF1 was added (2.5–5 μl) and incubated at room temperature for 30 min. For staining IRF4 and BCL6, we followed the methods described by Albu et al. [46 (link)]. Briefly, the cells were washed and incubated with an Fc blocker. The cells were then fixed with fixation buffer (3% formaldehyde, 0.1% saponin in PBS) and permeabilized with permeabilization buffer (1% FBS, 0.5% saponin, 10 μg/ml RNase A in PBS) before incubation with PE-conjugated anti-IRF4 (646403) or APC-conjugated anti-BCL6 (358505). Isotype control APC mouse IgG2b (400319) and PE mouse IgG2a (400213) served as controls. All antibodies were purchased from Biolegend. Flow cytometry was performed on a FACS Calibur, and data were analyzed using CellQuest software (BD Biosciences).