Clone l243

Macrophages were harvested with a cell scraper and fixed with 4% paraformaldehyde for 20 min, followed by cell staining with antibodies specific for human HLA-ABC, dilution 1:100 (clone W6/32, BioLegend, UK, Cat. No. 311418) or HLA-DR dilution 1:25 (clone L243, BioLegend UK, Cat. No. 307606), for 30 min. Determination of the rate of infection was performed based on the percentage of GFP positive cells, on fixed cells 24 h post-infection, after washing several times with PBS to remove extracellular bacteria. Samples were run on an LSR II flow cytometer and the data analysed using FlowJo (TreeStar Inc, Ashland, USA).
HeLa cells infected with ChAdOx1 and MVA cloned with each selected antigen were used detect antigen expression by western blot, using the iBind Western System (Invitrogen, UK). Antigens were detected with a primary Histidine Tag antibody, diluted 1:1000 BioRad, Cat. No. MCA1396, clone AD1.1.10, followed by a secondary polyclonal antibody, Goat Anti-Mouse IgG Fc (Alkaline Phosphatase), dilution 1:1000 (Abcam, UK, Cat. No. ab98710).

After harvesting, iMoDCs were immediately transferred to 24-well plates in RPMI medium and co-cultured for 24 h in a 20:1 ratio with A2058 cells (control) or A2058 cells previously treated with 22 µM lepadin A (1) or with 2 µM doxorubicin (2). Flow cytometry analysis was performed by staining with CD86 antibody (PE-Vio770, Clone REA968, Invitrogen, Thermo Fisher Scientific), CD83 (APC, Clone REA714, Invitrogen, Thermo Fisher Scientific), CD91 (PE, Clone A2MR-a2, Invitrogen, Thermo Fisher Scientific), HLA-DR (FITC, Clone L243, Biolegend) and Fixable Viability Stain 780 (APC-Vio770, BD Horizon). The viable HLA DR⁺ cell population was selected for surface expression markers analysis according to the gating strategy reported in the Supporting Information (Additional file 2: Fig. S5). All data were acquired by a MACSQuant® Analyzer 16 (Miltenyi Biotec, California, USA) and analysed by FlowJo 9 Software (Tree Star, Inc., Ashland, OR USA).

PBMCs were isolated from buffy coats as described earlier and pan-DCs (including myeloid and plasmacytoid DCs) were further enriched by negative selection using EasySep^TM human pan-DC pre-enrichment kit (STEMCELL Technologies) according to the manufacturer´s instructions. Cell purity of enriched live (fixable viability stain 780 ^– from BD Biosciences), lineage negative cells (CD14^– (clone M5E2, BD Biosciences), CD16^– (clone 3G8, BD Biosciences), CD3^– (clone UCHT1 BD Biosciences), CD20^– (clone 2H7, Biolegend) or CD19^– (clone HIB19, Biolegend), CD56^– (clone MEM-188, Biolegend), but HLA DR⁺ (clone L243, Biolegend)) was confirmed by flow cytometry. Isolated DCs were seeded at 1 × 10⁶ cells/ml in 96-well plates with 200 μl/well complete culture medium. Following 1–2 h rest, DCs were primed with 10% L. reuteri-CFS or were kept in RPMI as the control. Following 24 h incubation, cells were collected and washed twice with warm PBS and fresh medium with RA (1,15 μg/ml) or without RA was added. On day 4, the medium was replaced and on day 6 cells were re-activated with 10 μg/ml of Pam3SCK4 for 24 h. Supernatants were collected following 24 h priming with the first stimuli on day 1 and following 24 h stimulation with the second stimulus on day 7. Supernatants were stored at −20°C until further analysis.