Anti ifn γ antibody

IFN-γ ELISPOT was performed as described previously (28 (link)). Briefly, frozen PBMCs were thawed, washed and 0.5 million cells were added to ELISpot^PLUS plates pre-coated with anti-IFN-γ antibodies (Mabtech, catalog no. 3420-4AST-P1-1). The cells were stimulated with SARS-CoV-2 peptides from the S1 region of the spike protein at a concentration of 2 μg/mL along with CD28-stimulating antibody at a concentration of 0.1 μg/mL for 48 hours in a humidified incubator (37°C with 5% of CO₂). Monoclonal CD3-stimulating antibody was used as a positive control and unstimulated PBMCs were used as negative controls. The detection antibody (7-B6-1-Biotin) was then added and the plate and incubated for two hours at room temperature. Afterwards, Streptavidin-ALP and then its substrate solution (BCIP/NBT-plus) were added. The reaction was left to develop until distinct spots appeared, after which it was stopped by extensive washing with tap water. After air drying, the plate was read using KS ELISpot reader (Zeiss, Thornwood, NY) with software version KS ELISpot 4.9.16 (29 (link)). Results are reported as spots per 10⁶ PBMCs. An increase of ≥32 spots per 10⁶ PBMCs from baseline was defined as a positive result. The cut-off was determined as an increase of 3 standard deviations of the negative controls as was done in previous studies (13 (link)).

Enumeration of antigen-specific IFN-γ secreting cells was done by ELISpot assay, as described before [3 (link),5 (link),10 (link),25 (link)]. Briefly, spleen cells (4 × 10⁵/well) were added to ELISpot plates coated with an anti-IFN-γ antibody (Mabtech Inc., Cincinnati, OH, USA), followed by incubation in the presence of appropriate antigen-specific stimulant at a concentration of 2 µg/mL for 20 h at 37 °C, 5% CO₂. A peptide corresponding to CD⁸⁺ T cell epitope OVA_257-264: SIINFEKL (JPT Peptide Technologies GmbH, Berlin, Germany) was used as a stimulant. To measure background responses, cells were incubated in the absence of any stimulants. The plates were developed according to the manufacturer’s instructions. AEC substrate (Becton Dickenson, Franklin Lakes, NJ, USA) was added to visualize the spots. Spots were counted using an automated ELISpot plate reader (Cellular Technology LTD, Beachwood, OH, USA).

CD19 CAR T cells were rested for 3 days after expansion in low‐dose IL‐2 (20 IU/ml) before they were cocultured with Daudi target cells at ratio of 1:1 for 20–24 h. IFN‐γ secretion from stimulated T cells was measured in supernatants by ELISA using an anti‐IFN‐γ antibody (Mabtech, Nacka Strand, Sweden) and the T cells were stained for expression of the degranulation marker CD107a using a FITC‐labeled anti‐CD107 antibody (BD Biosciences, Cat: 555800) and evaluated by flow cytometry.

Enumeration of antigen-specific IFN-γ secreting cells was performed by ELISpot assay as previously described [15 (link),17 (link),21 (link)]. Briefly, spleen cells (at a final cell density of 4 × 10⁵/well) were added to ELISPOT plates coated with an anti-IFN-γ antibody (Mabtech Inc., Cincinnati, OH, USA), and incubated in the presence of appropriate antigen-specific stimulant at a concentration of 2 µg/mL for 20 h at 37 °C, 5% CO₂. For OVA protein-immunized animals, CD8 T cell epitope OVA_257–264: SIINFEKL or CD4 T cell epitope OVA_323–339: ISQAVHAAHAEINEAGR peptides (JPT Peptide Technologies GmbH, Berlin, Germany) were used as stimulants. Cells were also incubated without any stimulants to measure background responses. The plates were then incubated, washed and developed according to the manufacturer’s instructions. AEC substrate (Becton Dickenson, Franklin Lakes, NJ, USA) was used to visualize the spots. Spots were counted using an automated ELISpot plate reader (BIOSYS, Miami, FL, USA).

IFN-γ ELISpot assays were performed to measure Spike-specific T-cell responses^{64 (link)}. ELISPOT plates (S5EJ044I10; Merck Millipore, Darmstadt, Germany) pre-wetted with 30 µl of 70% ethanol for a maximum of 2 min, washed with sterile PBS and coated overnight at 4 °C with anti-IFN-γ antibody (10 µg/ml in PBS; clone 1-D1K; Mabtech, Nacka Strand, Sweden). Following overnight incubation, plates were washed with PBS and blocked with R10 (RPMI supplemented with penicillin–streptomycin, L-Glutamine, and 10% FBS) for a minimum of 2 h at 37 °C. The cells were then plated at 2 × 10⁵ cells/well and cultured with overlapping peptide pools at 2 μg/ml or PHA (Sigma Aldrich, St Louis, MO) at 5 µg/ml as a positive control for 16–18 h at 37 °C. Plates were washed four times with 0.05% Tween/PBS (Sigma Aldrich) followed by two washes with PBS and then incubated for 2 h at room temperature with biotinylated anti-IFN-γ (1 μg/ml; clone mAb-7B6-1; Mabtech). Next, plates were washed and incubated with alkaline phosPHAtase-conjugated streptavidin (Mabtech) at 1:1000 dilution for 1 h. After six further washes, plates were developed using VECTASTAIN® Elite ABC-HRP according to the manufacturer’s instructions (Mabtech). All assays were performed in duplicate. Spots were counted using an automated ELISpot Reader System.

The generation of Melan-A_{aa26–35*A27L} peptide-specific CD8⁺ cells was investigated using the IFN-γ ELISpot assay. Expanded CD8⁺ cells were purified using immunomagnetic beads (MACS-system, Miltenyi Biotec, Bergisch Gladbach, Germany) and were incubated as effector cells (2 × 10⁴/well) with peptide-loaded T2 cells as targets (1 × 10⁵/well) at an effector/target ratio of 1:5 for 48 h in 96-well nitrocellulose-plates (Millipore, Eschborn, Germany) precated with anti-IFN-γ antibody (Mabtech AB, Nacka, Sweden) in a final volume of 200-μl culture medium. To obtain T2 cells loaded with Melan-A_{aa26–35*A27L} peptide or a HLA-A2-restricted control peptide, T2 cells were preincubated for two hours with 10 μg/mL of the respective peptide. After detection with biotinylated anti-cytokine-antibodies (Mabtech AB, Nacka, Sweden) and conjugation with Avidin ALP (Sigma, Deisenhofen, Germany), BCIP/NBT substrate (Sigma, Deisenhofen, Germany) was added. The spots of the IFN-y-secreting cells were counted with a computer-controlled microscope (Tecan sunrise™, Crailsheim, Germany).