Cyclic-AMP Is an Actionable Novel Regulator of PD-L1 Expression in Untransformed Lymphocytes and Diffuse Large B Cell Lymphomas

Antigen-specific T lymphocytes can recognize and eliminate aberrant cells. Cancer cells halt this process by hijacking a system of immune checkpoints, the programmed cell death 1 (PD-1) and its ligands (PD-L1/2) pathway, which physiologically regulates the quantity and activity of T cells, establishing peripheral T cell tolerance and limiting tissue damage. PD-L1-expressing cancer cells interact with and inhibit PD-1 positive T cells, thus abrogating anti-cancer immunity, which can be restored by checkpoint inhibitors (CPI).

Improved understanding of the regulation of PD-L1 expression will shed further light on how cancer cells escape immune surveillance, and it may help in the design of combinatorial therapeutic strategies that expand the activity of CPI. Oncogenes (e.g., MYC, STAT3, HIF1 and NF-KB) have been shown to directly induce PD-L1 transcription. In addition, pro-inflammatory cytokines, notably IFN-γ, via the JAK/STAT pathway, also increase PD-L1 expression, an intuitive counteracting regulatory axis that prevents unchecked inflammation and auto-immunity. The second messenger cyclic-AMP (cAMP) is a classical mediator of anti-inflammatory and immunosuppressive inputs. However, its putative role in PD-L1 regulation is unknown. Addressing this knowledge gap is especially relevant because this signaling node can be modulated with a class of FDA-approved agents, the phosphodiesterase 4 (PDE4) inhibitors.

We have recently reviewed the pleiotropic roles that cAMP/PDE4 plays in diffuse large B-cell lymphoma (DLBCL) biology (BloodPMID: 27756749). Thus, to examine if cAMP modulates PD-L1 expression, we first used DLBCL cell lines (n=10). Raising the levels of intracellular cAMP readily induced PD-L1 expression (measured by WB and FACS) in ABC-DLBCLs but not in GCB-DLBCLs. This cAMP-mediated induction of PD-L1 occurred also at RNA level; however, using reporter assays we found that the canonical cAMP-PKA-CREB pathway does not directly activate the PD-L1 promoter. The immune modulatory activity of cAMP is mediated, at least in part, by transcriptional activation/secretion of cytokines. Thus, we considered that cAMP induction of PD-L1 in DLBCL may be driven by an autocrine loop. In agreement with this idea, cAMP promoted JAK/STAT activation and culturing DLBCL cell lines in conditioned media (CM) from cAMP-high models induced PD-L1 expression. These assays pointed to secreted factor(s) as intermediaries in the cAMP/PD-L1 axis. Therefore, we screened a panel of 105 cytokines to identify those secreted by DLBCL cell lines following cAMP up-modulation - in most models, we detected a significant cAMP-driven increase in IL-6, IL-8, IL-10 and IL-1α secretion. For validation, we focused on IL-10 because this was the most commonly cAMP-induced cytokine across the DLBCL models. We found that recombinant IL-10 induced PD-L1, albeit this induction was significantly less marked than that observed following an increase in intra-cellular cAMP. Concordantly, antibody-based blocking of the IL-10 signals, and pharmacologically inhibiting the JAK/STAT pathway, only partially abrogated the cAMP-mediated induction of PD-L1. We concluded that IL-10 and JAK/STAT signals relay part, but not all, of the cAMP effects on PD-L1 expression in DLBCL. Next, we utilized the Pde4b null mouse model to examine if these observations were present in an organismal level and in non-immortalized immune cells. In these assays, spleens of Pde4b WT, +/- and -/- mice (8-16 weeks old, male and female, n=8) were collected and analyzed by WB and FACS. Spleen cells from Pde4b deficient mice had markedly higher expression of PD-L1 (WB). By FACS, we found that the increase in PDL1 expression in Pde4b null mice derived from T cells, B cells, but from the smaller non-B/T cell population (CD19/CD3 negative). Finally, we found that the PDE4 inhibitor roflumilast used as a single agent in vitro robustly induced PD-L1 expression in DLBCL cell lines. In summary, we identified cAMP as an "actionable" novel regulator of PD-L1 expression in normal and malignant immune cells. Mechanistically, cAMP drives an autocrine loop enacted by cytokines and transduced in part by JAK/STAT. This finding supports the clinical testing of roflumilast to induce PD-L1 expression, a strategy that may improve the activity of checkpoint inhibitors in DLBCL and related tumor types.