Assay diluent

The levels of IL-1β and IL-17 in the serum were determined using an Enzyme Linked Immuno-sorbent Assay employing Human cytokine ELISA set (R&D Systems, USA). ELISA was performed following the manufacturer’s protocol. The sensitivity of the kits ranges from 3.91 to 200pg/ml for IL-1β and 31.20 to 2000pg/ml for IL-17. Briefly, 96-well ELISA plate was coated overnight with either anti-human IL-1β, or IL-17 capture antibodies at 4°C. After washing, wells were blocked with 200 μl of assay diluents (BD Biosciences) at 37°C for 2hr. The plates were finally incubated with the standards and samples at 37°C for 2hr. Detection antibodies were added after washing and incubated at 37°C for 1hr. Further, after washing the plates, streptavidin-HRP conjugate was added to each well and incubated for 1hr at 37°C. Finally, plates were washed and coloured complex was developed using 3,3’,5,5’-tetramethylbenzidine substrate and reaction was stopped after 15min using 2N sulphuric acid. The absorbance was read at 450nm with a microtiter plate reader (Genetix GMB-580). Cytokine levels were determined with the help of standard curve and concentration was expressed as pg/mL.

IL-1β and IL-8 were measured from cell culture medium samples using BD OptEIA^TM assays and following the manufacturer’s protocol (San Diego, CA, USA). Where needed, samples were diluted in Assay Diluent (BD OptEIA^TM, San Diego, CA, USA). A commercial TMB substrate solution (BD OptEIA^TM, San Diego, CA, USA) was used for the IL-1β and IL-8 measurements. OD values were measured at a wavelength 450 nm with a reference wavelength of 655 nm using a spectrophotometer (Bio-Rad Model 550 with the Microplate Manager 5.2 programme; Bio-Rad Laboratories, Inc., Hercules, CA, USA)

Mouse splenocytes and lung cells were stimulated with antigens and assayed for production of IFN-γ as we published previously (6 (link)). Briefly, a single cell suspension of 1.0 × 10⁵ splenocytes or lung cells per well was seeded in U-bottom 96-well plates and incubated with T cell medium (Complete Advanced RPMI 1640 [Invitrogen] supplemented with 1 mM HEPES [Cellgro, Mediatech], 2 mM l-alanyl-l-glutamine [GlutaMAX Supplement, Gibco], 50 μM 2-mercaptoethanol [Sigma-Aldrich], 100 U/mL penicillin/100 μg/mL streptomycin, and 2% fetal bovine serum) alone or T cell medium supplemented with 2 μg/mL of N protein, 2 μg/mL of N peptide pool, or 2 μg/mL of M protein of SARS-CoV-2 or 5 × 10⁶ HI-LVS for 3 or 6 days. Afterwards, the culture supernatant fluid was collected, cell debris removed by centrifugation, and the supernatant fluid diluted 5-fold and stored in assay diluent (BD Biosciences) at −80°C until use. The production of mouse IFN-γ in the culture supernatant fluid was assayed using a mouse cytokine EIA kit (BD Biosciences) per the manufacturer’s instructions.

Human sCD155 concentrations in the culture supernatants of HeLa (2.0 × 10⁴ cells/100 µl/well) 24 h after the start of culture were measured by ELISA, as described (Iguchi-Manaka et al., 2016 (link)). For measurement of mouse sCD155, we established a cytokine bead array (CBA) assay. Anti-CD155 mAb (TX56) was conjugated to BD CBA Functional Beads (BD Bioscience) in accordance with the manufacturer’s protocol. 2 μl of the TX56 beads was incubated with a 50-µl sample of sera, culture supernatants, or tumor extraction at room temperature or on ice for 2 h, washed with the wash buffer (BD Bioscience), and then incubated with 0.5 µg/ml of rabbit anti-mouse CD155 polyclonal antibody generated in our laboratory in 50 µl of the Assay Diluent (BD Bioscience) at room temperature for 1 h. The TX56 beads were then washed with the wash buffer, incubated with 1 µl of PE-conjugated donkey anti-rabbit polyclonal IgG (BioLegend) in 50 µl of the Assay Diluent at room temperature for 1 h, washed again with the wash buffer, and analyzed by flow cytometry. The culture supernatants of sCD155/BL6 and mock/BL6 (2.0 × 10⁴ cells/100 µl medium/well) obtained 24 h after the start of culture, sera of peripheral blood of mice injected with sCD155/BL6 or mock/BL6, and sera from the pulmonary vein of tumor-colonized lungs after clamping were subjected to the CBA assay.

A single-cell suspension of 1.0 × 10⁵ splenocytes per well was seeded in U-bottom 96-well plates and incubated with T-cell medium alone, or T-cell medium supplemented with 2 µg/mL of recombinant SARS-CoV-2 protein or peptide antigens for 3 days. Afterward, the culture supernatant fluid was collected, cell debris removed by centrifugation, and the supernatant fluid stored in assay diluent (BD Biosciences) at −80 °C until use. The production of mouse IFN-γ in the culture supernatant fluid was assayed using a mouse cytokine EIA kit (BD Biosciences).

Mouse splenocytes and lung cells were stimulated with antigens and assayed for production of IFN-γ as we published previously (6 (link)). Briefly, a single cell suspension of 1.0 × 10⁵ splenocytes or lung cells per well was seeded in U-bottom 96-well plates and incubated with T cell medium (Complete Advanced RPMI 1640 [Invitrogen] supplemented with 1 mM HEPES [Cellgro, Mediatech], 2 mM l-alanyl-l-glutamine [GlutaMAX Supplement, Gibco], 50 μM 2-mercaptoethanol [Sigma-Aldrich], 100 U/mL penicillin/100 μg/mL streptomycin, and 2% fetal bovine serum) alone or T cell medium supplemented with 2 μg/mL of N protein, 2 μg/mL of N peptide pool, or 2 μg/mL of M protein of SARS-CoV-2 or 5 × 10⁶ HI-LVS for 3 or 6 days. Afterwards, the culture supernatant fluid was collected, cell debris removed by centrifugation, and the supernatant fluid diluted 5-fold and stored in assay diluent (BD Biosciences) at −80°C until use. The production of mouse IFN-γ in the culture supernatant fluid was assayed using a mouse cytokine EIA kit (BD Biosciences) per the manufacturer’s instructions.

A single cell suspension of 1.0 × 10⁵ splenocytes or lung cells per well was seeded in U-bottom 96-well plates and incubated with T-cell medium alone, or T-cell medium supplemented with 2 µg/mL of recombinant PA, LF, F1, LcrV, or F1-LcrV monomer for three days. After a 3-day incubation, the culture supernatant fluid was collected, cell debris removed by centrifugation, and the supernatant fluid stored in assay diluent (BD Biosciences) at −80 °C until use. The production of mouse IFN-γ and IL-4 in the culture supernatant fluid was assayed using a mouse cytokine EIA kit (BD Biosciences)²⁴ .