Cpg odn2395

IFN-γ production by NK cells was evaluated as previously reported [59 (link)]. Briefly, PBMCs from patients with CLL were treated with anti-BTLA blocking antibody, control IgG for 72 h, plate-coated agonistic anti-BTLA antibody (clone MIH26, Biolegend, San Diego, CA, USA) or proper isotype control (all at 10 µg/mL) for 24 h. Afterwards, PBMCs from each condition were stimulated with 50 nM PMA and 1 µg/mL ionomycin for 4 h. After the first 1 h of incubation, brefeldin A (Biolegend) was added to avoid the release of cytokines to the cell culture media. Following incubation, cells were stained with anti-CD56-APC and anti-CD3-FITC to identify NK cells. For intracellular cytokine staining, BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences, San Jose, CA, USA) was used according to the manufacturer’s instructions. NK cells were stained with anti-human IFN-γ-PE (clone 4S.B3, Biolegend) and IFN-γ+ NK cells were evaluated by flow cytometry. For IL-10 evaluation, PBMCs from patients with CLL were cultured unstimulated or stimulated with 200 ng/mL CpG (ODN2395, Miltenyi Biotec, Bergisch Gladbach, Germany) alone or in combination with blocking anti-BTLA mAb for 48 h. IL-10+CD19+ leukemic cells were determined as described above.

Histones (0–20 μg/mL, Sigma Cat# H9250), PAM3CSK4 (1 μg/mL, Invivogen, San Diego, CA, USA Cat# tlrl-pms), PAM2CSK4 (1 μg/mL, Invivogen, Cat# tlrl-pm2s-1), PHAD (1 μg/mL, Avanti Polar Lipids, Alabaster, AL, USA, Cat 699800) or CpG ODN2395 (1 μg/mL, Miltenyi Cat# 130-100-282) diluted in complete IMDM were added to naive T cells at the time of culture. For stimulation with NETs, NET preparations in MCDB 131 media or MCDB 131 media alone were similarly added at the start of the culture period. For experiments inhibiting histones/NETs, the inhibitor β-methyl-cellobioside sulfate (mCBS) (100 μg/mL) or PBS was added simultaneously with the addition of histones or NETs to cultures. Unless otherwise stated, in all experiments, histones/TLR agonists/NETs/mCBS were added at the start of the culture period and remained in the media for the duration of the cell culture. TLR4 activity of PHAD was confirmed using HEK-Blue™ mTLR4 Cells (Invivogen, Cat# hkb-mtlr4) as described by the manufacturer.