Interferon-α Up-Regulates the Expression of PD-L1 Molecules on Immune Cells Through STAT3 and p38 Signaling

Abstract

Interferon-α (IFNα) has one of the longest histories of use amongst cytokines in clinical oncology and has been applied for the treatment of many types of cancers. Due to its immune-activating properties, IFNα is also an attractive candidate for combinatory anti-cancer therapies. Despite its extensive use in animal tumor models as well as in several clinical trials, the different mechanisms underlying patient responses and affecting desirable clinical benefits are still under investigation. Here we show that in addition to its immune-activating properties, IFNα induces the expression of a key negative regulator, immunosuppressive PD-L1 molecule, in the majority of the specific immune cell populations, particularly in the dendritic cells (DC). DC can modulate immune responses by a variety of mechanisms, including expression of T-cell regulatory molecules and cytokines. Our results showed that treatment of DC with IFNα-2b led to pronounced up-regulation of surface expression of PD-L1 molecules, increased IL-6 and decreased IL-12 production. Moreover, we present evidence that IFNα-treated DC exhibited a reduced capacity to stimulate interferon-γ production in T cells compared to control DC. This T-cell response after treatment of DC with IFNα was recovered by a pre-treatment with an anti-PD-L1 blocking antibody. Further analyses revealed that IFNα regulated PD-L1 expression through the STAT3 and p38 signaling pathways, since blocking of STAT3 and p38 activation with specific inhibitors prevented PD-L1 up-regulation. Our findings underline the important roles of p38 and STAT3 in the regulation of PD-L1 expression and prove that IFNα induces STAT3/p38-mediated expression of PD-L1 and thereby a reduced stimulatory ability of DC. The augmentation of PD-L1 expression in immune cells through IFNα treatment should be considered by use of IFNα in an anti-cancer therapy.

Keywords: IFNα; PD-L1 (B7-H1); STAT3 signaling; cancer immunotherapy; dendritic cell; immunosuppression.

Figure 1

IFNα up-regulates PD-L1 expression on different myeloid immune cell populations ex vivo. FACS analysis of PD-L1 expression on the surface of different myeloid immune cells from splenocytes of healthy mice. Splenocytes were isolated, treated for 24 h with 1,000 U/ml IFNα and investigated by flow cytometry. The results are presented as interleaved bars (A,B), as box and whiskers plots (C,E) or column bar graphs (D,F) and statistically analyzed using two-way ANOVA (A,B) or unpaired T-tests (C–F), n = 4, ^**p < 0.01, and ^***p < 0.001.

Figure 2

IFNα up-regulates PD-L1 expression on different T-cell populations ex vivo. FACS analysis of PD-L1 expression on the surface of different lymphoid immune cells from spleens of healthy mice. Splenocytes were isolated, treated for 24 h with 1,000 U/ml IFNα and investigated by flow cytometry. The results are presented as interleaved bars (A) or as box and whiskers plot or column bar graph (B) and statistically analyzed using two-way ANOVA or unpaired T-tests, n = 4, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 3

IFNα up-regulates PD-L1 expression on DC in vivo. FACS analysis of PD-L1 expression on the surface of DC from spleens of healthy mice with or without treatment with IFNα. Splenocytes were isolated and examined using flow cytometry. The results are presented as percent of positive cells in a column bar graph and statistically analyzed using unpaired T-test, n = 10, ^***p < 0.001. A representative FACS histogram (red-control, blue-IFNα) is shown.

Figure 4

IFNα up-regulates PD-L1 expression on human dendritic cells. (A) FACS analysis of PD-L1 expression on the surface of mDC. Myeloid DC were isolated, treated for 24 h with IFNα and analyzed with flow cytometry. The results are presented as column mean, error bars and mean connected and statistically analyzed using ordinary one-way ANOVA with the Tukey's multiple comparisons post-test, n = 2, ^*p < 0.05 and ^**p < 0.01. (B) FACS analysis and Immunocytochemistry (ICH) of PD-L1 expression on the surface of MoDC. MoDC were treated for 24 h with 1,000 U/ml IFNα and analyzed with flow cytometry. Alternatively, cytospin slides were produced and ICH was performed using an anti-PD-L1 primary antibody followed by a biotinylated secondary antibody. FACS histograms (red-control, blue-IFNα) and ICH pictures are representative for independent experiments. (C) Cytokines in the supernatant of the IFNα-treated DC analyzed with Luminex assays. The results are presented as column bar graphs and statistically analyzed using unpaired T-test, n = 2.

Figure 5

PD-L1 controls IFNγ release from CD4 T cells stimulated by DC. Allogeneic CD4 cells were isolated and cocultured with DC in the presence or the absence of anti-PD-L1 blocking antibody or vehicle control. The level of IFNγ in supernatants was determined by Luminex assay. The results are presented as column bar graphs and statistically analyzed using ordinary one-way ANOVA with the Tukey's multiple comparisons post-test, n = 3–6, ^*p < 0.05, ^***p < 0.001, and ^****p < 0.0001.

Figure 6

IFNα up-regulates PD-L1 expression in mDC in p38/STAT3-dependent manner. (A,B) FACS analysis of PD-L1 expression on mDC activated for 24 h with 1,000 U/ml IFNα with or without 1 h pre-incubation with signal transduction inhibitors of p38, STAT3 (ST3), PI3K, ERK, p38, and Jak. The results are presented as column bar graphs and statistically analyzed using ordinary one-way ANOVA with the Dunnett's multiple comparisons post-test, n = 8–10, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001. (C) Western blot analysis of p38, STAT3, and ERK phosphorylation (pp38, pERK, and pSTAT3 for phosphorylated form) in mDC before and after activation for indicated period of time with IFNα.