PD-L1 expression levels on tumor cells affect their immunosuppressive activity

Abstract

Programmed cell death 1 (PD-1) is an immuno-checkpoint receptor which is primarily expressed on T cells, monocytes, natural killer cells and macrophages. Programmed death-ligand 1 (PD-L1) is the primary ligand of PD-1 and is constitutively expressed on antigen presenting cells, mesenchymal stem cells and bone marrow-derived mast cells. In addition, PD-L1 is also expressed on a wide range of tumor cells, including lung cancer, breast cancer and melanoma. PD-1 and PD-L1 are important members of the immunoglobulin super-family and participate in immune regulation. In the present study, the immune-suppressive effects of a number of tumor cell lines were determined. The breast tumor cell lines MCF-7 and MDA-MB-231 displayed the largest inhibitory effects on T-cell activation and cytokine secretion in a co-culture system. The HepG2, A549 and A375 cells displayed limited inhibitory effects. MCF-7 and MDA-MB-231 cells expressed the highest level of PD-L1 among the cells used, which may explain their higher immuno-suppressive effects. Compound A0-L, a small molecule inhibitor of the PD-1/PD-L1 interaction, restored T cell functions. Additionally, it was demonstrated that the tumor cells with higher levels of PD-L1 expression suppressed signaling pathways involved in T-cell activation, such as the T-cell receptor- zeta chain of T cell receptor associated protein kinase ZAP70-RAS-GTPase-extracellular-signal-regulated kinases and CD28-PI3K-Akt serine/threonine kinases pathways. These findings suggest that tumor cells with higher expression levels of PD-L1 may exhibit higher immuno-suppressive activity, and that drugs targeting the PD-1/PD-L1 interaction may have improved therapeutic effects on tumors expressing higher levels of PD-L1.

Keywords: co-culture; immunosuppression; programmed cell death 1; programmed death-ligand 1; signaling pathway.

Figure 1.

Immunosuppression by cancer cells. (A) Jurkat cells transfected with PGL3-NFAT-TA-Luciferase plasmid were co-cultured with various cancer cell lines and stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml). Luciferase activity was measured 24 h after stimulation. **P<0.01, ***P<0.001 vs. control (−). (B) Jurkat cells transfected with PGL3-NFAT-TA-Luciferase plasmid were cultured in various cancer cell-conditioned media and stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml). Luciferase activity was measured 24 h after stimulation. (C) Human PBMCs were co-cultured with various cancer cell lines and then stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml). **P<0.01, ***P<0.001 vs. control (−). (D) Human PBMCs cultured in various cancer cell-conditioned media were stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml). hPBMC, human peripheral blood mononuclear cells; IL-2, interleukin-2; IFN-γ, interferon-γ.

Figure 2.

Expression of immune checkpoint markers in various cancer cell lines. mRNA expression levels of (A) PD-L1, (B) PD-L2, (C) CD80, (D) CD86, (E) HVEM, (F) CD70, (G) CD137 and (H) OX40L in tumor cell lines. *P<0.05, **P<0.01, **P<0.001. (I) mRNA expression levels of immune checkpoint receptors in Jurkat cells. Gene expression was normalized to GAPDH in the same sample. (J) FACS analysis of PD-L1 in various cancer cell lines. APC-conjugated anti-human-PD-L1 was used as a binding antibody to cell-surface PD-L1 protein (red line, isotype control staining; blue line, PD-L1 staining). Cell count has been normalized to the peak height at the mode of the distribution, such that absolute count is presented as a percent of the total count. (K) Quantification of the FACS analysis shown in (J). **P<0.01, ***P<0.001 vs. 293. APC, allophycocyanin.

Figure 3.

PD-1/PD-L1 inhibitor restores the function of lymphocytes. (A) Jurkat cells transfected with PGL3-NFAT-TA-Luciferase plasmid cocultured with various cancer cell lines were treated with A0-L (10 µM) and subsequently stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml). Luciferase activity was measured 24 h later. **P<0.01, ***P<0.001. hPBMCs were co-cultured with MCF-7 or MDA-MB-231 in the presence of A0-L (10 µM), subsequently stimulated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml), and the levels of (B) IL-2 and (C) IFN-γ were measured. ***P<0.001. PD-1, programmed cell death 1; PD-L1, programmed death ligand 1; IL-2, interleukin-2; IFN-γ, interferon-γ; hPBMC, human peripheral blood mononuclear cells.

Figure 4.

Activation of AKT and ERK in Jurkat cells co-cultured with cancer cells. (A) Jurkat cells cultured alone or in the presence of MCF-7 or MDA-MB-231 cells were activated by anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml). Jurkat cells were collected and lysates were prepared, and the amounts of the indicated proteins were determined by western blotting. (B) Densitometry analysis of the phosphorylation of AKT and ERK presented in (A). For each time point, fold changes in the amounts of the indicated proteins in activated Jurkat cells that were stimulated through anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml) were compared with cells that were not activated. *P<0.05, **P<0.01, ***P<0.001 vs. 0 min. unstimulated cells; #P<0.05, ##P<0.01, ###P<0.001 vs. Jurkat cells that were stimulated with anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml). (C) Jurkat cells cultured with MCF-7 in the absence or presence of A0-L (10 µM) were activated by anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml). Jurkat cells were collected and lysates were prepared, and the amounts of the indicated proteins were examined by western blotting. (D) Densitometry analysis of the phosphorylation of AKT and ERK presented in (C). Fold changes in the amounts of the indicated proteins in activated Jurkat cells that were suppressed by MCF-7. *P<0.05, **P<0.01, ***P<0.001 vs. Jurkat cells that were stimulated through anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml) at 5 min. unactivated Jurkat cells that were reversed by A0-L; #P<0.05, ##P<0.01, ###P<0.001 vs. Jurkat cells that were stimulated with anti-CD3 (1 µg/ml), anti-CD28 (1 µg/ml) and co-cultured with MCF-7. AKT, protein kinase B; ERK, extracellular-signal regulated kinase; p, phospho; Ab, antibody.