Chemotherapeutic agents in noncytotoxic concentrations increase antigen presentation by dendritic cells via an IL-12-dependent mechanism

Abstract

Antineoplastic chemotherapeutic agents may indirectly activate dendritic cells (DCs) by inducing the release of "danger" signals from dying tumor cells. Whereas the direct cytotoxic or inhibitory effect of conventional chemotherapy on DCs has been reported, modulation of DC function by chemotherapeutic agents in low noncytotoxic concentrations has not yet been investigated. We have tested the effects of different classes of antineoplastic chemotherapeutic agents used in low noncytotoxic concentrations on the Ag-presenting function of DCs. We revealed that paclitaxel, doxorubicin, mitomycin C, and methotrexate up-regulated the ability of DCs to present Ags to Ag-specific T cells. Stimulation of DC function was associated with the up-regulation of expression of Ag-processing machinery components and costimulatory molecules on DCs, as well as increased IL-12p70 expression. However, the ability of DCs treated with paclitaxel, methotrexate, doxorubicin, and vinblastine to increase Ag presentation to Ag-specific T cells was abolished in DCs generated from IL-12 knockout mice, indicating that up-regulation of Ag presentation by DCs is IL-12-dependent and mediated by the autocrine or paracrine mechanisms. At the same time, IL-12 knockout and wild-type DCs demonstrated similar capacity to up-regulate OVA presentation after their pretreatment with low concentrations of mitomycin C and vincristine, suggesting that these agents do not utilize IL-12-mediated pathways in DCs for stimulating Ag presentation. These findings reveal a new mechanism of immunopotentiating activity of chemotherapeutic agents-a direct immunostimulatory effect on DCs (chemomodulation)-and thus provide a strong rationale for further assessment of low-dose chemotherapy given with DC vaccines for cancer treatment.

FIGURE 1

Up-regulation of Ag-presenting function of DCs by chemotherapeutic agents. DC cultures were initiated from the bone marrow precursors and treated with low concentrations of selected chemotherapeutics on day 1 for 48 h. Day 6 DCs were loaded with OVA overnight and cocultured with B3Z CD8⁺ T cell clone specific for the H-2K^b-restricted SIINFEKL peptide of OVA. Production of IL-2, which reflects T cell activation upon recognition of the OVA epitope 257–264 in the context of the H-2K^b molecule, was determined by ELISA. *, p < 0.01 vs control, ANOVA. Shown are the results of one representative experiment of three independent experiments with similar results. Data are expressed as the mean ± SEM. Control, nontreated DCs.

FIGURE 2

Chemotherapeutic agents regulate expression of APM components in DCs. Bone marrow-derived DCs were treated with medium (control) or chemotherapeutic agents, collected, washed, fixed, permeabilized, and subjected to intracellular staining for different APM proteins as described in Materials and Methods. Staining for tapasin, TAP binding protein, which mediates the interaction between newly assembled MHC class I molecules and TAP, is shown as an example. Data from one representative experiment from three independent studies are shown; y-axis, event count; x-axis, fluorescence intensity; isotype control, open field. Chemotherapeutic agents were used in the following concentrations: methotrexate, 5 nM; doxorubicin, 10 nM; and bleomycin, 5 nM.

FIGURE 3

Phenotype of DCs treated with anticancer chemotherapeutic agents. Immature DCs were generated from the bone marrow hematopoietic precursors in cultures supplemented with GM-CSF and IL-4 as described in Materials and Methods. Chemotherapeutic agents were added on day 1 for 48 h in indicated concentrations. Treatment with medium served as a control. DCs were harvested and analyzed on day 6 by FASCan. The percentage of CD11c⁺MHC class II⁺ (A) and CD11c⁺CD86⁺ (B) DCs are shown. The data shown are representative of three independent experiments.

FIGURE 4

Regulation of IL-12 expression in DCs by chemotherapeutic drugs. DCs were differentiated from the bone marrow hematopoietic precursors in cultures supplemented with GM-CSF and IL-4 and treated with indicated concentrations of cytotoxic drugs for 48 h. DCs were collected on day 6 and intracellular staining for IL-12 was performed without any additional DC activators as described in Materials and Methods. The results are expressed as MFI. The data shown are the means ± SEM of three independent experiments. Control, nontreated DC; *, p < 0.05 vs control, ANOVA.

FIGURE 5

IL-12 mediates chemotherapy-induced up-regulation of OVA Ag presentation by DCs. Bone marrow hematopoietic precursors were isolated from IL-12 knockout and control wild-type mice and treated with chemotherapeutics on day 1 for 48 h. Day 6 DCs were washed, pulsed with OVA (1 mg/ml, overnight), and cultured with B3Z CD8⁺ T cell clone specific for the H-2K^b-restricted SIINFEKL peptide. Production of IL-2 by T cells, which reflects T cell activation, was assessed by ELISA. The levels of OVA presentation by DCs from wild-type and IL-12 knockout mice after pretreatment with chemotherapeutic agents were compared. The data are presented as the percentage of activation or inhibition of OVA presentation by chemotherapeutic agent-treated DCs relative to control untreated DCs. The results of one representative experiment out of three independent experiments are shown; *, p < 0.05 wild type vs IL-12 knockout (one-way ANOVA, n = 3).