Igg4 isotype

For allogeneic mixed lymphocyte reaction (MLR), monocytes were magnetically isolated using CD14 Microbeads (Miltenyi Biotec, Germany) and cultured for 5 days in the presence of IL-4 and GM-CSF (all Miltenyi Biotec, Germany). T cells from BCL patients and healthy controls were incubated with allogeneic mo-DCs in the presence or absence of either 0.1 µM HSP90 inhibitor (geldanamycin, GA) (Abcam, USA), 10 µg/mL PD-1 inhibitor (Nivolumab) (Opdivo, Bristol-Myers Squibb), or a combination of both drugs for 4 days. PD-1 inhibitor (Pembrolizumab) (10 µg/mL, Keytruda, MSD, USA) and IgG4 isotype (10 µg/mL, BioLegend, USA) were used as positive and negative controls, respectively. The CTLA-4 inhibitor (Ipilimumab) (10 µg/mL, Yervoy, Bristol-Myers Squibb, USA) was also used for the comparison.

Anti-PD-1 mAb nivolumab (purity 99.50%) and ALK inhibitor crizotinib (purity ≥98% (HPLC) were purchased from Selleckchem Co., Ltd. (Houston, TX). Crizotinib was dissolved in dimethyl sulfoxide (DMSO). DMSO was used as a control for crizotinib and 20 μg/mL IgG4 isotype (Biolegend, San Diego, CA)as a control for nivolumab. Concerning antibodies (Abs): The fluorochrome-conjugated FITC anti-human CD3 antibody (Clone OKT3), brilliant violet 421 anti-human CD8 antibody (RPA-T8), APC anti-human CD4 antibody (Clone OKT4), PE-CD56 (Clone 5.1 H11), APC anti-human PD1 (Clone NAT105), PE anti-human PD-L1(Clone 29E-2A3), APC anti-human CTLA-4 antibody (Clone L3D10), PE anti-human GITR antibody (Clone 621), brilliant violet 421 anti-human CD134 (OX40) antibody (Clone Ber-ACT35), and FITC anti-human/mouse granzyme B antibody (GB11) were purchased from Biolegend (San Diego, CA). For APC-anti-human PD-1 detection, its isotype control is mouse IgG1k, which is not compatible to nivolumab as an IgG4 antibody. Anti-Human Foxp3 Staining Set FITC kit (eBioscience, San Diego, CA) was used to identify Foxp3 CD4⁺CD25⁺/CD4⁺CD25^- cells according to the manufacturer’s instructions. For Western blot: Anti-FasL (Biolegend, San Diego, CA) and beta Tubulin Loading Control Monoclonal Antibody (BT7R) (Thermo Fisher Scientific, San Diego, CA) were used.