Class b cpg oligodeoxynucleotides

CD19⁺ CD27⁺ memory B (MB) cells were isolated using the EasySep Human Memory B Cell Isolation Kit (STEMCELL Technologies, Canada) according to the manufacturer’s instructions. CD8⁺T cells were isolated using a CD8⁺ T Cell Isolation Kit (Miltenyi, Germany). MB cells (1×10⁵ /mL) from SLE and Healthy donors were cultured alone or in the presence of autologous CD8⁺ T cells (4×10⁵ /mL) at 1:4 ratio for 3 days at 37°C. Co-cultured cells were incubated in complete RPMI 1640 supplemented with 10% fetal bovine serum (Invitrogen) supplemented with 25 mM HEPES (Invitrogen), 2 mM L-glutamate (Invitrogen) and 1% non-essential amino acids (Life Technologies). During incubation, cells were stimulated with class B CpG oligodeoxynucleotides (InvivoGen) at 5 µg/mL, 1 µg/mL anti-CD3/CD28 (BD Biosciences, USA).

Recombinant lentiviral overexpression of PD-L1 was a gift from Wuhan University. The viral supernatant and medium were mixed in a 1:1 ratio, and the total volume was 1 mL. Polybrene was added at a concentration of 8 µg/mL. MB cells (1×10⁵ cells from patients with SLE) were resuspended in this mixture and inoculated into 24-well culture plates for 24 hours. The expression of PD-L1 on the surface of MB cells was detected by flow cytometry. Transfected MB cells (1×10⁴ transfected MB cells) were cultured with CD8⁺T cells from healthy donors or patients with SLE at a ratio of 1:4 for 72 hours. Class B CpG oligodeoxynucleotides (InvivoGen) at 5 µg/mL, 1 µg/mL anti-CD3/CD28 (BD Biosciences, USA) were added to the culture as stimulants. IFN-γ expression in CD8⁺ T cells was measured using flow cytometry.