Clone mab11

To assess the effect of relatlimab on cytokine production, PBMCs from patients with CLL were treated with anti-LAG-3 blocking mAb or control IgG (10 µg/mL) alone or in combination with lenalidomide (10 µM) or DMSO for 72 h. Then, immune subset identification followed by intracellular cytokine staining and flow cytometry analyses were performed as previously described by our group [36 (link)]. Briefly, treated PBMCs were stimulated with 50 nM PMA and 1 µg/mL ionomycin for 4 h. After 1 h incubation with PMA/ionomycin, brefeldin A (Biolegend) was added. Afterwards, BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, BD Biosciences, San Jose, CA, USA) was employed according to the manufacturer’s instructions. Percentage of positive T lymphocytes for IL-2 (clone MQ1-17H12, Biolegend), IFN-γ (clone 4S.B3, Biolegend) and TNF-α (clone Mab11, Biolegend) staining was determined. For evaluation of intracellular Bcl-2 protein levels, PBMCs from patients were treated with relatlimab or control IgG (10 µg/mL) for 72 h or 7 days. Thereupon, cells were stained with anti-CD19-APC for leukemic cell identification and BD Cytofix/Cytoperm Kit was used to fix and permeabilize cells. Bcl-2 expression was evaluated using anti-Bcl-2-PE (clone 100, Biolegend).

The chimeric α-ganglioside GM2 monoclonal antibody (KM966) was a kind gift from Kyowa Hakko Kirin Co., Ltd. The chimeric α-CD20 (rituximab) and the humanized α-HER2 (trastuzumab) were purchased from Roche Pharmaceuticals. The TCR-like antibodies were generated in laboratory of Antonio Bertoletti. α-CD64 (clone 10.1, BioLegend), α-CD32 (clone AT10, Abcam), α-CD32a (clone IV.3, StemCell Technologies), α-CD32b (polyclonal, Abcam), α-CD16 (clone DJ130c, AbD Serotec), α-CD18 (Clone TS1/18, Biolegend), α-CD11a (Clone TS1/22, Thermo Scientific), α-CD11b (Clone CBRM1/5, eBioscience), α-CD11c (Clone 3.9, Biolegend), α-CD29 (Clone P5D2, R&D systems), α-TNFα (Clone Mab11, Biolegend), α-TNFR (Clone mab625, R&D systems) and an isotype-matched control antibody (MOPC21, BioLegend) were used for blocking experiments.

Functional avidity of WT and PDCD1-edited Melan-A-specific T cell clones was evaluated after coculture with TAP-deficient T2 cells loaded with a range of Melan-A_A27L (ELAGIGILTV) peptide at the effector/target ratio 1/2, through the measurement of CD107a mobilization and cytokine production. CD107a mobilization was measured after 3 hours of coculture at 37°C in the presence of a CD107a-specific mAb (H4A3 clone, BioLegend). T lymphocytes were then stained with anti-CD8 antibodies (Clone RPA-T8, BioLegend) and analyzed by flow cytometry. Cytokine production was determined after a 5-hour stimulation period at 37°C, in the presence of Brefeldin A at 10 µg/mL (Sigma, B7651). T cell clones were labeled with anti-CD8 mAb (Clone RPA-T8, BioLegend), fixed with PBS 4% paraformaldehyde (VWR, 100504-858) and stained for cytokine production using anti-tumor necrosis factor (TNF)-α (Clone MAB11, BioLegend), anti-interferon (IFN)-γ (Clone B27, BioLegend) and anti-IL2 (Clone MQ1-17H12, BioLegend) mAbs. All the cytometric analyses were performed on a Facs Canto II (BD Biosciences).