Ifn γ capture antibody

Splenocytes were harvested from humanized mice at 24 days after CAdVEC injection, and human CD8⁺ T cells were isolated using CD8 isolation column (Miltenyi Biotec Inc.). The day before CD8⁺ T cell isolation, parental MCF-7 and SUM-159 cells were seeded in the presence of human IFN-γ (10 ng/ml), and single-cell suspensions were subjected to 100 Gy of irradiation the next day. CD8⁺ T cells (4 × 10⁵) were mixed with 4 × 10⁴ irradiated cancer cells (effector:target = 10:1) in 200 μl of complete medium and seeded in a 96-well Millipore ELISPOT plate (BD Biosciences) that was precoated with IFN-γ capture antibody (BD Biosciences) (24 (link)). Plates were cultured for 24 hours in a 37°C incubator and then developed according to the manufacturer’s protocol. Plates were scanned using Mabtech IRIS Elispot reader (Mabtech).

Spleens were harvested and processed individually into single-cell suspensions. 1 × 10⁶ splenocytes were plated onto 96-well plates previously coated with an IFNγ capture antibody (BD Cat# 551083). C57BL/6-CEA Tg splenocytes were stimulated with one of the following  H2-D^b- or H2-K^b-restricted peptides (10 µg/mL) for 18 hours: CEA_526-533 (EAQNTTYL), CEA_572-579 (GIQNSVSA), p15E (KSPWFTTL), and HIV-gag (SQVTNPANI). The CEA_526-533, CEA_572-579, p15E, and HIV-gag peptides were synthesized by CPC Scientific (Sunnyvale, California, USA). IFNγ spots were detected using the BD mouse IFNγ ELISPOT kit and developed using the BD ELISPOT AEC substrate set according to the manufacturer’s instructions (BD Biosciences; San Jose, California, USA). IFNγ spots were visualized and quantified using the CTL ImmunoSpot Analyzer (Cleveland, Ohio, USA).

Spleens were harvested and processed individually into single cell suspensions. 1×10⁶ splenocytes were plated onto wells of 96-well plates previously coated with IFN-γ capture antibody (BD Cat# 551083). C57BL/6-CEA Tg splenocytes were stimulated with one of the following H-2D^b- or H-2K^b-restricted peptides (10 µg/mL) for 18 hours: CEA_526-533 (EAQNTTYL), CEA_572-579 (GIQNSVSA), gp70 (KSPWFTTL), JAK1 (IVYLYVVCV), Ptgfr, (VITYFFGHL), HIV-gag (SQVTNPANI), while Balb/c splenocytes were stimulated with one of the following H-2L^d-restricted peptides: Twist (LYQVLQSDEL), AH1 (SPSYVYHQF), and β-gal (TPHPARIGL). The CEA_526-533, CEA_572-579, gp70, Twist, β-gal, and HIV-gag peptides were synthesized by CPC Scientific and the JAK1, Ptgfr, and AH1 peptides were generated by GenScript. IFN-γ spots were detected using the BD mouse IFN-γ ELISpot (enzyme-linked immunospot) kit and developed using the BD ELISPOT AEC substrate set according to the manufacturer’s instructions. IFN-γ spots were visualized and quantified using the CTL ImmunoSpot Analyzer.

ELISpot plate (BD Biosciences) was treated with sterile-filtered 70% ethanol before being washed three times with sterile PBS 1×. IFNγ capture antibody (BD Biosciences no. 551881) was plated and the plate was sealed and left to incubate at 4°C overnight. Positive control wells were also plated with anti-CD3ε (BioLegend no. 100340). The plate was washed three times with sterile PBS and blocked with 10% (v/v) FBS in PBS overnight at 4°C. IFNγ-stimulated 6694c2 or B16F10 cells were plated, except in the unstimulated and positive control wells. Pancreatic draining lymph nodes were excised from treated mice bearing orthotopic tumours, and lymph nodes were macerated to form a single-cell suspension. Lymph node cells (one-third of lymph node per well) were plated on top of pre-plated tumour cells with human IL-2 (PeproTech), and positive control wells also received anti-CD28 (BioLegend no. 102116). Plate was incubated at 37°C for 24 h before being washed with sterile water followed by PBST. IFNγ detection antibody (BD Biosciences no. 551881) was added, and plate was incubated for 2 h at room temperature. Wells were washed with PBST, and streptavidin-HRP (BD Biosciences) was added. Plate was incubated for 1 h at room temperature before being washed with PBST and PBS. AEC chromagen substrate (BD Biosciences) was added, and plate was developed. Plate was dried and analysed.

Interferon (IFN)-γ-secreting cell spots were determined by culturing splenocytes (5 × 10⁵ or 10⁶ cells/well) and lung cells (5 × 10⁵ or 3 × 10⁵ cells/well) for 72 h on multi-screen 96-well plates (MilliporeSigma, St. Louis, MO, USA) coated with IFN-γ capture antibody (BD Pharmingen) as described [28 (link)], in the presence of inactivated influenza A/Cal H1N1 (4 µg/mL) as an antigenic stimulator. The plates were then incubated with biotinylated mouse anti-IFN-γ antibody (BD Pharmingen), followed by incubation with alkaline phosphatase-labeled streptavidin antibody, and the IFN-γ-secreting T cells were visualized using color-developing 3,3′-diaminobenzidine substrate and counted using an ELISpot reader (BioSys, Miami, FL, USA).