Tumor-mediated inhibition of human dendritic cell differentiation and function is consistently counteracted by combined p38 MAPK and STAT3 inhibition

Abstract

Targeting dendritic cells (DC) through the release of suppressive factors is an effective means for tumors to escape immune control. We assessed the involvement of downstream signaling through the JAK2/STAT3 and p38 MAPK pathways in tumor-induced suppression of human DC development. Whereas the JAK2/STAT3 pathway has been pinpointed in mouse studies as a key regulator of myeloid suppression, in human DC this is less well established. We studied the effects of STAT3 inhibition on the suppression of monocyte-derived DC differentiation mediated by a short-list of four predominant suppressive factors and found that pharmacological STAT3 inhibition could only counteract the effects of IL-6. Accordingly, in testing a panel of supernatants derived from 11 cell lines representing various types of solid tumors, STAT3 inhibition only modestly affected the suppressive effects of a minority of supernatants. Importantly, combined interference in the STAT3 and p38 pathways completely prevented inhibition of DC differentiation by all tested supernatants and effected superior DC function, evidenced by increased allogeneic T cell reactivity with elevated IL-12p70/IL-10 ratios and Th1 skewing. Combined STAT3 and p38 inhibition also afforded superior protection against the suppressive effects of primary glioma and melanoma supernatants and induced a shift from CD14(+) cells to CD1a(+) cells in metastatic melanoma single-cell suspensions, indicating a potential for improved DC differentiation in the tumor microenvironment. We conclude that combined interference in the STAT3 and p38 MAPK signaling pathways is a promising approach to overcome tumor-induced inhibitory signaling in DC precursors and will likely support clinical immunotherapeutic strategies.

Figure 1. The tumor-derived factors IL-10, IL-6, VEGF, TGF-β and PGE-2 inhibit differentiation of MoDC; the inhibitory effects of IL-6 alone can be prevented by pharmacological STAT3 inhibiton. (A) Expression (in percentage positive cells) of the monocytic marker CD14 and the DC marker CD1a by MoDC differentiated with or without the indicated suppressive factors. Shown are mean ± SD from 5 experiments. * = p < 0.05 compared with GM4 differentiation. (B) FACS analysis of surface expression of the markers CD1a, CD14, CD40, HLA-DR and B7-H4 on MoDC, differentiated in the presence or absence of suppressive factors. Markers indicate fluorescence of IgG isotype controls and mean fluorescence intensities are listed. Results are representative of three experiments. (C) Representative western blot analyses of pSTAT3, STAT3 and β-actin expression in MoDC, differentiated in the presence or absence (-) of the indicated suppressive factors. (D) Expression of CD14 and CD1a by DC, differentiated with or without suppressive factors and in the presence or absence of the JAK2/STAT3 inhibitor AG490. Shown are mean ± SD from six experiments. * = p < 0.05 compared with the paired condition without AG490. (E) Representative western blot analyses of pSTAT3, STAT3 and β-actin expression in MoDC differentiated in the presence of IL-6 and treated with the STAT3 inhibitor AG490 (the latter two conditions shown for two separate donor MoDC).

Figure 2. STAT3 inhibition only partially interferes with MoDC-inhibitory effects of tumor-derived supernatants and in a minority of tested cell lines only. Expression of CD14 and CD1a (in % positive cells) by DC differentiated with or without tumor supernatants and in the presence or absence of AG490 (STAT3i). IL-6 levels in the tested supernatants are listed. * = p < 0.05 compared with the paired condition without AG490. Shown are mean ± SD from three experiments.

Figure 3. Combined STAT3 and p38 MAPK inhibition consistently prevents suppression of human MoDC differentiation by supernatants from a panel of tumor cell lines. Eleven supernatants derived from the indicated tumor cell lines representing 8 different tumor types were added during differentiation. The percentages of CD1a⁺ and CD14⁺ cells after normal DC differentiation with GM-CSF and IL-4 are indicated by the dotted lines. Shown are mean ± SD from 3 experiments. * = p < 0,05 compared with differentiation without inhibitors.

Figure 4. Blocking both the STAT3 and the p38 MAPK pathway improves the T cell stimulatory functions of human MoDC differentiated in the presence of U373 glioma supernatant. (A) Capacity of DC differentiated in the presence of U373 supernatant with or without the STAT3 and/or p38 MAPK inhibitors to stimulate allogeneic T-cell proliferation, means ± sd are shown, n = 4; *p < 0.05 between indicated conditions. Photographic inserts show proliferating T cell aggregates for the different conditions (magnification 400x). (B) Secretion levels of IL-12p70 and IL-10 by MoDC differentiated in the presence or absence of U373 supernatant with or without the STAT3 and/or p38 MAPK inhibition; expressed in pg/ml or as IL-12p70:IL-10 ratios. Shown are mean ± SD from 4 experiments (C) Secretion of Th1/Th2/Th17 cytokines at day four in the MLR cultures described under (A). Shown are mean ± SD from four experiments; *p < 0.05 between the indicated conditions.

Figure 5. Combined p38 MAPK and STAT3 inhibition blocks the suppressive effects of supernatants from primary tumor supernatants and improves the differentiation of monocytic DC precursors in melanoma cultures from metastatic lesions. (A) Relative CD14 and CD1a expression levels on MoDC differentiated in the presence of primary glioma (n = 4) or melanoma supernatants (n = 3) in the presence or absence of STAT3 and p38 MAPK inhibitors (percentages of medium controls were set at 1). Shown are the means ± SD; *p < 0.05 compared with condition without inhibitors. (B,C) Single-cell suspensions from human metastatic melanomas were cultured for 6 d without additives or with GM-CSF and IL-4 with or without additional STAT3 and/or p38 MAPK inhibitors. (B) FACS analyses of CD14 and CD1a expression (percentages listed) from a representative experiment and (C) mean ± SD from 3 independent experiments with different melanoma samples are shown. *p < 0.05 compared with the medium condition.