Ifn γ elisa

BMDCs generated in the presence or absence of Ac₅3F_axNeu5Ac (250 µM) were washed in 1 × PBS and pulsed with OVA protein (Endograde, Hyglos GmbH, Germany) for 3 h or OVA H-2 Kb peptide (257–264, AS-60193, Tebu-bio) for 1 h at 37 °C. After the incubation, the BMDCs were washed three times, and 50.000 cells were seeded per well into a round bottom plate in culture medium. The CD8⁺ OT-I T cells were labeled with 3 µM CFSE according to the manufacturer’s instructions. 50.000 CFSE-labeled CD8⁺ OT-I T cells were added to the BMDCs (1:1 ratio) in culture medium. The co-cultures were incubated for 3 days at 37 °C in the presence of 0 or 10 ng/ml IL-2. After 3 days, the cells were analyzed by flow cytometry. Interferon gamma (IFNγ) levels in the supernatants were analyzed using an IFNγ ELISA (Thermo Fisher Scientific) following the manufacturer’s protocol.

Stimulator cells (3 × 10⁴ cells/well or as indicated) were co-incubated with the CD4⁺ T-cell clone (5 × 10³ cells/well or as indicated) overnight at 37°C in 96-well plates in duplicates. For peptide loading, stimulator cells were incubated with indicated peptide concentrations for 2 h at 37°C before T-cell clones were added. For shutdown of the tet-off system, cells were cultured in the presence of 50 μg/ml doxycycline and washed twice before co-incubation with the T-cell clone. Cytokine release was measured after overnight incubation in 100-μl supernatants by IFN-γ ELISA following the instructions of the manufacturer (Thermo Fisher Scientific).

Spleens were harvested from mice, crushed in a filter of 100 microns and lysed in ACK lysis buffer (ThermoFisher Cat. #A1049201) for 5 min at room temperature to remove red blood cells. Splenocytes were plated in 24-well dish at 4 × 10⁶ cells/well with either 10 μg peptide or 1 × 10⁵ EO771-B7.1 cells in the presence of 20 units IL-2. Cells were then collected after 7 days and challenged with either peptide or EO771-B7.1 cells for either 3 days for flow cytometry proliferation assays or 24 h for LDH cytotoxic assays (Biolegend Cat. #426401) and IFNγ ELISA (ThermoFisher, Cat. #88-7314-22) following manufacture’s protocol.

Novel HLA-C CMV peptide sequences were kindly provided by Stanley Riddell² or obtained from the literature and described in Table 1. All peptides were purchased from “Peptide2.0” synthesized with crude purity (20–80%), resuspended in 100% dimethyl sulfoxide and stored at −20°C. Novel HLA-C peptides were identified after coculture of RV798 strain infected fibroblasts with PBMCs from seropositive donors (33 (link)). The protein antigens to which expanded CD8⁺ T-cells were specific for were identified after coculture with RV798 strains deleted for single CMV genes and loss of IFN-γ production (data not shown). The RV798 strain of HCMV is based on the AD169 strain backbone but carries a deletion of the US2-11 immune evasion proteins (28 (link)). The peptide specificity of the CD8⁺ T-cell clones was determined after coculture with COS7 cells transfected with overlapping peptide fragments from the determined cognate CMV protein (Riddell, unpublished data). The HLA restriction of the peptides was identified as either HLA-Cw*0702- or HLA-Cw*1601-restricted using the SYTHPETHI database. This was subsequently confirmed by IFN-γ ELISA (Thermo Scientific) after incubation with EBV-transformed LCL lines expressing a single HLA allele from the donor (HLA-Cw*0702 UL28-derived FRC HLA-restriction data provided in Figure S1 in Supplementary Material).

Supernatants from whole PBMC (matched samples from the Nanostring gene expression analyses described above) that were stimulated with BCG for either 4h or 18h were collected and stored at -80C until use. The levels of IFN-gamma protein was not detectable after 4 hours (not shown), and therefore the 18 hour data was evaluated here (both undiluted and at a 1:10 dilution). All samples were thawed on ice and assayed in duplicate. The IFN-γ ELISA (ThermoFisher) was performed according to the manufacturer’s protocol. Optical densities at 450nm and 540nm were measured, and the concentration of protein in each sample was calculated by averaging the optical density of the duplicate samples and plotting against a standard curve.