Beriglobin

The following monoclonal antibodies were used to evaluate surface expression on PBMCs and allergen-specific TCC: PD-1-PE (EH12.2H7), BTLA-PE (MIH26), CD4-PE (OKT4) and appropriate isotype controls (MOPC-21) all purchased from Biolegend (San Diego, CA). LAG-3-PE (3DS223H) and CTLA-4-PE (14D3) were purchased from eBioscience (San Diego, CA). Cells were stained in FACS buffer (PBS, 1% BSA, 0.1% NaN₃) for 30 min. For immune checkpoint stainings, 10 mg/mL Beriglobin (CSL Behring) were added to prevent unspecific binding to F_c-receptors. Exclusion of dead cells from analysis was done using 7-AAD (Biolegend) where appropriate. Intracellular CTLA-4 expression was determined using the Cytofix/Cytoperm kit from BD Biosciences according to the manufacturer’s instructions.
To assess the effect of coinhibitory pathways, the following monoclonal blocking antibodies were added at a final concentration of 8 μg/ml: functional grade PD-1 (EH12.2H7; LEAF), PD-L1 (29E.2A3; LEAF) and PD-L2 (MIH18; LEAF) from Biolegend, CTLA-4 (Ipilimumab, Yervoy), BTLA, LAG-3 and a mouse IgG1 κ isotype control antibody (MOPC-21; LEAF, Biolegend). Blocking antibodies to BTLA and LAG-3 was described previously^{35 (link)}. Flow cytometry analysis was performed using FACSCalibur and LSRFortessa flow cytometers (BD Bioscience). FlowJo software (version 10.0.6., Tree Star, Ashland, OR) was used for data analysis.

Thymus tissue was obtained from children undergoing cardiac surgery. Parents gave informed written consent. The study was approved by the Regional Ethical Board at the University of Gothenburg. The tissue was embedded in Optimal cutting temperature compound (OCT) (Histolab Products AB) in isopentane pre-cooled with liquid nitrogen, and 7 μm sections were cut and fixed in cold acetone. After rehydration in PBS and blocking with Protein Block (Dako), tissue sections were stained with biotinylated monoclonal antibodies (5 μg/ml) and anti-cytokeratin 5 (BioLegend), and detected with anti-rabbit-AlexaFluor488, streptavidin-AlexaFluor555, and Hoechst, and mounted with ProLong Gold Antifade mountant (all Life Technologies). For flow cytometry, thymocyte single cell suspensions were prepared by enzymatic (DNase I and Liberase TH, Roche) and mechanical (gentleMACS^TM Dissociator, Miltenyi Biotec) treatment. Cells were blocked with Beriglobin (CSL Behring) and stained with biotinylated monoclonal antibodies (10 μg/ml) and Fixable Viability Dye 506 (Life Technologies), followed by Streptavidin APC (BD Bioscience). For intracellular staining, cells were fixed with the Transcription Factor Staining Buffer Set (Life Technologies).

Stimulations were stopped by incubation in 2mM EDTA for 5 min. Surface staining was performed for 15 min in the presence of 1 mg/ml of Beriglobin (CSL Behring) with the following fluorochrome-conjugated antibodies titrated to their optimal concentrations as specified in Supplementary Table 2: anti-CD3-FITC (Miltenyi), anti-CD4-VioGreen (Miltenyi), anti-CD8-VioBlue (Miltenyi), anti-CD38-APC (Miltenyi), and anti-HLA-DR-PerCpVio700 (Miltenyi). During the last 10 min of incubation, Zombie Yellow fixable viability staining (Biolegend) was added. Fixation and permeabilization were performed with eBioscience™ FoxP3 fixation and PermBuffer (Invitrogen) according to the manufacturer’s protocol. Intracellular staining was carried out for 30 min in the dark at room temperature with anti-4-1BB-PE (Miltenyi), anti-CD40L-PEVio770 (Miltenyi) and anti-CD40L-PECy7 (Biolegend), anti-IFN-γ-A700 (Biolegend) and anti-TNF-α-BV605 (Biolegend). All samples were measured on a MACSQuant^®Analyzer 16 (Miltenyi). Instrument performance was monitored prior to every measurement with Rainbow Calibration Particles (BD Biosciences).

Dulbecco's Modified Eagle Medium (DMÈM) was obtained from BioWhittaker and supplemented with 100 IU ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin and LPS-free FCS (BioWhittaker). IFN-β/α (PBL), and ProLong Gold, DAPI, TRIzol, Poly I:C (all from Invitrogen) Lipofectamine 2000 (Invitrogen) were used in the experiments described below. HSV-1 (strains KOS and McKrae) was grown in Vero cells. The Vero cells used were from the lab stock. The titres of the stocks used were 8–14 × 10⁹ PFU ml⁻¹. Titres were determined by plaque assay on Vero cells. Beriglobin was used to neutralize extracellular HSV-1 (CSL Behring) and fully neutralized the virus in the dilutions used to calculate titres. Both strains were used for infection of mice, while only KOS strain was used for in vitro stimulation. GFP-HSV-1 (HSV-1-expressing eGFP driven by the CMV promoter) and HSV-1 McKrae were kind gifts from David Leib, while HSV-1 (KOS) was a gift from Peter O'Hare.

Isolated PBMCs were stained first with Aqua live/dead (Life Technologies) and incubated at room temperature in the dark for 30 min. After washing, they were suspended in 50 μl of filtered Flow Cytometry Buffer FCB (1X PBS (Gibco), 0.5% BSA (Sigma-Aldrich) 2 mM EDTA (Thermo Fischer Scientific)) and 50 μl of Staining Buffer (FCB 2% Beriglobin (CSL Behring)) and stained with anti CD3-PercP Cy5.5 (eBioscience), anti CD4-FITC, (BD Biosciences) followed by an incubation at 4 °C in the dark for 30 min. Anti-IL-10-PE, anti-IL-13-BV711, anti-IL-2-BV785, anti-IFNγ-BV421, anti-TNFα-BV605 and anti-IL4-PE CF594 (all Biolegend) intracellular staining was done according to manufacturer’s recommendations (BD Biosciences). Cell acquisition was performed using a Spectral Cell Analyzer cytometer SP6800 (Sony Biotechnology) 15-color cytometer, with 100,000 cells per tube as the total number of cells acquired. Polyfunctional CD4⁺ T cells were defined as CD4⁺ T cells expressing any combination of IFN-γ, IL-2 or TNF. IL-10, IL-4 or IL-13 producing CD4+ T cells are defined as CD3+CD4+IL-10+, CD3+CD4+IL-10−IL-4+ and CD3+CD4+IL-10−IL-13+, respectively.