Ifn γ elispot assay

CEA.Tg mice bearing MC38-CEA tumors were fed an SX-682-containing diet starting on day 7; on days 14 and 21, mice received i.p. injections of bintrafusp alfa, with a priming vaccine dose of s.c. Ad-CEA administered on day 7 and boosting doses of Ad-CEA/N-803 vaccine on days 14 and 21. Control mice were left untreated and fed a base diet without SX-682. Splenocytes were harvested from control versus treated mice and assayed ex vivo on day 24 for antigen-dependent cytokine secretion using an IFNγ ELISPOT assay (BD Biosciences), according to the manufacturer’s instructions. Briefly, 0.5 × 10⁶ splenocytes were incubated overnight with 10 μg/mL of CEA_526–533, p15e_604–611, the MC38 neoepitope PTGFR, or a negative control peptide [10 (link)]. Spot-forming cells were quantified using an ImmunoSpot analyzer (Cellular Technology, Ltd, Shaker Heights, OH, USA). The amount of CD8⁺ T cells added per well was calculated by flow cytometry analysis. Data were adjusted to the number of spots/0.5 × 10⁵ CD8⁺ T cells present in the assay, subtracting the number of spots in paired wells containing the control peptide.

The procedures used in this experiment have been published previously [37 (link)]. Briefly, CD8⁺ T cells were purified using the MACS CD8⁺ T Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. The purified CD8⁺ T cells were cocultured with the DCs pulsed with the OVA peptide (SIINFEKL) for 20 h. An IFN-γ ELISPOT assay was then conducted according to the manufacturer’s instructions (BD Biosciences). The number of spots was manually counted.

IFN-γ-producing CD8⁺ T cells were measured by IFN-γ ELISPOT assay (BD Biosciences)^{76 (link)}. Briefly, 96 well Millipore Immunospot M200 plates were coated overnight with purified anti-mouse IFN-γ antibody (1:200). Purified splenocytes were resuspended in complete RPMI and seeded for overnight stimulation with peptide epitopes (described below) at 37 °C. After stimulation, plates were washed with water once and PBS-Tween twice followed by a 2 h incubation with biotinylated anti-mouse IFN-γ antibody (1:250) at room temperature. After washing, streptavidin-horseradish peroxidase (HRP) (1:100) was added to wells and incubated for 1 h at room temperature. Following the final washes, 3-amino-9-ethyl-carbazole (AEC) chromogen substrate (BD Biosciences) was added to the wells and developed at room temperature for 30 min to 1 h. Plates were rinsed with water and dried before proceeding with spot quantification. Spot formation was quantified using an automated spot counter (Immunospot, Cellular Technology Ltd).