PD-L1 and PD-L2 regulate in cell-autonomous and N-glycosylation-dependent manners the pro-tumorigenic characteristics and functions of human cancer-associated fibroblasts.
BACKGROUND Tumor progression is regulated by cancer-associated fibroblasts (CAFs), pro-inflammatory cytokines (e.g., TNFα, IL-1β, IFNγ) and acquired immunity (such as inhibitory immune checkpoints: PD-L1, PD-L2). Here, we determined the interactions between these elements by analyzing the impact of pro-inflammatory cytokines on the proportions of PD-L1 + PD-L2-expressing CAFs; the cell-autonomous regulation of pro-tumorigenic characteristics and functions of CAFs by PD-L1/PD-L2; and the control of PD-L1/PD-L2 activities in CAFs by their N-glycosylation. METHODS CAFs were obtained from breast and lung cancer patients (BC-CAFs, Lung-CAFs). PD-L1/PD-L2 surface and whole-cell expression were determined by flow cytometry and Western blot, respectively. CAFs were transduced to express WT-PD-L1, WT-PD-L2, WT-PD-L1 + WT-PD-L2, PD-L1/PD-L2 N-glycosylation mutants or vector controls, followed by determination of the pro-tumorigenic factors osteopontin (qRT-PCR) and CXCL8 (IL-8)/CCL2 (MCP-1)(ELISA). The effects of factors produced by CAFs expressing WT-PD-L1/WT-PD-L2/their N-glycosylation mutants on tumor cell migration were analyzed by wound-healing assays. RESULTS TNFα + IL-1β potently increased the expression of CXCL8, whereas IFNγ stimulation up-regulated the proportions of BC-CAFs and Lung-CAFs expressing PD-L1 + PD-L2 at cell surface. Combined TNFα + IL-1β + IFNγ stimulation has prominently increased intracellular pools of N-glycosylated PD-L1 and PD-L2, in BC-CAFs and Lung-CAFs. WT-PD-L1 induced in cell-autonomous manners pro-tumorigenic characteristics and functions in BC-CAFs: elevated the expression of osteopontin, CXCL8 and CCL2 and induced the production of factors that promoted tumor cell migration. WT-PD-L2 also acted in a cell-autonomous manner in BC-CAFs, generally down-regulating these phenotypes and activities. When co-expressed in BC-CAFs, the cancer-supporting effects of WT-PD-L1 dominated the anti-tumorigenic impacts of WT-PD-L2. The cell-autonomous functions of PD-L1 and PD-L2 were regulated, in context- and function-dependent manners, by their N-glycosylation sites, primarily N35/N192 in PD-L1 and N37/N163 in PD-L2. CONCLUSIONS Chronic inflammation can up-regulate the proportions of PD-L1 + PD-L2 expressing CAFs. PD-L1 and PD-L2 act in cell-autonomous manners to control the pro-tumorigenic characteristics and functions of CAFs, with dominance of the tumor-promoting effects of PD-L1 over the tumor-inhibiting roles of PD-L2. The cell-intrinsic functions of PD-L1 and PD-L2 in CAFs depended on their N-glycosylation. These observations emphasize the need to consider the expression of PD-L1 and PD-L2 in stromal cells when designing treatments with immune checkpoint blockades in patients.