Human ifn γ elispot set

In vitro priming of T cell responses and evaluation of peptide immunogenicity was carried out as described. Briefly, monocytes were purified from PBMC obtained from HLA-A*02:01 healthy donors by using CD14 microbeads (Miltenyi) and cultured for one day in complete RPMI medium containing rhGM-CSF (Miltenyi; 10 ng/ml) and rhIL-4 (Inmunotools; 2 ng/ml) for their differentiation into dendritic cells (DC). Next, rhTNF (Miltenyi; 10 ng/ml), IL-1β (Immunotools; 10 ng/ml) and PGE2 (Sigma-Aldrich; 1 µM) were added for DC maturation. One day later, cells were collected, loaded with peptide (10 µg/mL) for 1 hour and washed 3 times. DC were co-cultured (ratio 1:30) with CD8 T cells purified from the CD14^- fraction using CD8 microbeads (Miltenyi), in complete medium containing anti-human CD28 mAb (Biolegend; 0.5 µg/mL). Cells were fed on days 3, 7, 10, 14 and 17 with culture medium containing IL-2 (Proleukin, 20U/ml).
NeoAg-specific responses were evaluated at day 21 of co-culture by using a human IFN-γ ELISPOT set (BD-Biosciences) following manufacturer’s instructions. Expanded CD8 T cells (5 x 10⁴/well) were stimulated with peptides (10 µg/mL) and DC (10⁴/well) for 24 h. Next, wells were washed, incubated with conjugate antibody and developed. Spot forming cells were counted as described above.

Briefly, an ELISPOT plates (Human IFN-γ ELISPOT set, BD, USA) were coated with IFN-γ capture antibody and incubated overnight at 4°C. The plates were washed 3 times with phosphate-buffered saline (PBS) and then blocked with RPMI 1640. After 2h the blocking, the plates were washed with PBS. The 5×10⁵ CD4⁺ T cells were co-incubated with peptide-pulsed or not 5×10⁴ aAPCs for 20h. The cells were removed and the plates were washed 3 times with 0.05% Tween 20/PBS using microplate washer (405LSR; BioTek). Biotinylated detection antibody for IFN-γ was added and incubated for 2h at 37°C. After 4 times washes with 0.05% Tween 20/PBS, avidin-horseradish peroxidase was added and plates were incubated for 1h at 37°C. The plates were washed 4 times with 0.05% Tween 20/PBS, followed by 2 washes with PBS and the addition of AEC substrate (BD) per well. The reaction was stopped by washing with deionized water, and the plates were dried overnight. The spot forming units were counted using an AID ELISPOT Reader System (AID Diagnostika GmbH). The frequencies of an HLA allotype-restricted CD4⁺ T cell response to antigens were calculated as [(response to aAPCs expressing HLA pulsed with peptide pools) − (response to aAPCs expressing HLA)] − [(response to aAPCs pulsed with peptide pools) − (response to aAPCs)], as previously described (32 (link), 33 (link)).

We performed an ELISPOT assay using Human IFN-γ ELISpotPRO kit (MABTECH, 3420-2HST) or Human IFN-γ ELISPOT set (BD Biosciences, cat# 551849). Briefly, we pulsed C1R cells expressing a single HLA-A allele with each respective peptide for 16–24 h at 37 °C under 5% CO₂. We pre-treated T cells with 30 IU/mL of IL-2 for 16 h and then co-cultured with the peptide-pulsed C1R cells (2 × 10⁴ cells/well) at 37 °C for 24 h in a 96-well plate. We captured and analyzed spots with an automated ELISPOT reader, ImmunoSPOT S6 (Cellular Technology Ltd., OH, USA). We used anti-CD3 antibody (clone CD3-2) or 50 ng/mL of phorbol myristate acetate (PMA, cat# 162-23591)/1 μg/mL ionomycin (Sigma-Aldrich, cat# 10634) as positive control wells. To measure tumor necrosis factor (TNF)-α secretion, we used an IFN-γ/TNF-α Human T cell ELISpot kit (Cellular Technology Ltd.).
We performed ELISA to measure the secreted IFN-γ levels in the supernatant using the OptEIA Human IFN-γ ELISA set (BD Biosciences). Similar to the ELISPOT assay, we co-cultured T cells with the peptide-pulsed C1R-A0206 cells in 96-well plates, and then measured cytokine concentration in the supernatants according to the manufacturer’s instructions.