Fig. 5
Modulation by rs822336 allele-specificity of the in vitro activity of anti-PD-1 nivolumab on NSCLC cells co-cultured with HLA-matched PBMCs. EGFRmut HCC827G/G, H1975G/G and EGFRwt H460G/G, A549G/G, H1299C/C and H1437C/C cells were seeded into 24-well plates at a density of 2 × 105 cells per well. Following a 24 h incubation at 37 °C in a 5% CO2 atmosphere, cells were co-cultured with HLA-matched PBMCs in a 1:10 ratio and incubated with nivolumab (10 µg/ml). HLA-A*11, HLA-A*01, HLA-A*24, HLA-A*25, HLA-B*40 and HLA-A*03 haplotypes were used for matching EGFRmut HCC827G/G, H1975G/G and EGFRwt H460G/G, A549G/G, H1299C/C and H1437C/C cells, respectively, with PBMCs. HLA-matched PBMCs were activated utilizing an anti-CD3 (1 µg/mL) and an anti-CD28 (1 µg/mL) T Cell TransAct (T- Act). Non-activated HLA-matched PBMCs were used as controls. Purified human IgG4 was used as a control for nivolumab. (A) Following a 48 h incubation, cancer cell viability was determined by cell counting kit-8 (CCK-8) assay. Data are expressed as mean percentages of the viability of nivolumab treated cells ± SD as compared to cells incubated with purified human IgG4. The mean percentage of cell viability and SD were calculated from three independent experiments; each of them was performed in triplicate. (B) Following a 48 h incubation, apoptosis induction was determined by Annexin V/PI assay by flow cytometry. Data are expressed as mean percentages of apoptotic cell population of nivolumab treated cells ± SD as compared to cells incubated with purified human IgG4. The mean percentage of apoptotic cells and SD were calculated from three independent experiments; each of them was performed in triplicate. (C-E) Following a 48 h incubation, LDH (C), IFN-γ (D) and TNFα (E) levels in the medium harvested from cultures of HLA-matched PBMCs with cancer cells were measured by LDH assay kit, ELISA Max Deluxe Set Human IFN-γ kit and human TNFα ELISA, respectively. Data are expressed as means of LDH, IFN-γ and TNFα levels ± SD of the results obtained in three independent experiments; each of them performed in triplicate. (ns: not significant; *P ≤ 0.05; ***P ≤ 0.001)