Enhancement of dendritic cell-tumor fusion vaccine potency by indoleamine-pyrrole 2,3-dioxygenase inhibitor, 1-MT

Abstract

Purpose: Dendritic cell (DC)-based cancer vaccines are currently being evaluated as novel anti-tumor vaccination strategies, but in some cases, they are demonstrated to have poor clinical efficacies than anticipated. A potential reason is immune tolerance due to the immunosuppressive enzyme, indoleamine-pyrrole 2,3-dioxygenase (IDO). The aim of this study was to determine whether blocking the activity of IDO might improve the anti-tumor efficacy of DC/Lewis lung carcinoma (LLC) fusion vaccine applied to the mouse LLC model.

Methods: To prepare the DC/LLC fusion vaccine, DCs were fused with LLC using polyethylene glycol (PEG) as described. The IDO expression in the DC/LLC fusion vaccine and in the vaccinated mice was detected by western blot (WB) and/or immunohistochemical (IHC) analysis. This fusion vaccine, as a single agent or in combination with 1-methyl-tryptophan (1-MT, an IDO inhibitor), was administered to LLC mice. The anti-tumor efficacy in different treatment was determined by regular observation of tumor development and the level of splenic cytotoxic T lymphocyte (CTL) response, which was examined by lactate dehydrogenase (LDH) release.

Results: In the LLC mice, we observed that IDO-positive cells were extensively accumulated in tumor draining lymph nodes (TDLNs). Furthermore, WB and IHC analysis results showed that vaccination with fusion DC/LLC cells alone caused significant up-regulation of IDO in spleens. 1-MT enhanced the anti-tumor efficacy elicited by DC/LLC fusion vaccine via delaying the tumor development and inducing stronger splenic CTL responses.

Conclusions: Our results indicate an IDO-mediated immunosuppressive mechanism might be involved in weakening the anti-tumor efficacy elicited by DC/LLC fusion vaccine, and specific inhibition of IDO activity might be required for development of cancer vaccines.

Fig. 1

IDO expression in TDLNs but not LLC cells. a Tumor tissues (Tumor), tumor-draining lymph nodes (TDLNs) and distant lymph nodes (DLNs) were removed 10 days after mice were s.c. injected 1 × 10⁶ LLC, and were detected for IDO expression by WB analysis with an anti-IDO antibody. b LLC tumor cell line was cultured for 2 days with or without mouse IFN-γ (50 ng/ml) in the final 18 h culture. The cell lysates of tumor cells were analyzed for IDO expression and TDLN described above was used as positive control (PC)

Fig. 2

IDO is expressed by mononuclear cells infiltrating LLC tumors and tumor draining lymph nodes. Tissue sections from LLC tumor (a, b), distant lymph node (c), and tumor draining lymph node (d) were detected for IDO expression by IHC with a specific anti-IDO antibody. The slides were rinsed in distilled water and counterstained with haematoxylin. The insets in (b, d) show a higher magnification (400×) of a representative area in the panel

Fig. 3

Flowcytometer analysis of DCs, LLC cells, fusion cells and their fusion rates. a Prior to PEG treatment, expression of CD11c on DCs and DiOC18 fluorescence on LLC cells were detected. The fusion rate was determined by double staining of CD11c with DiOC18 on DCs and LLC fusion cells (DC/LLC). The double-positive cells were 22.1%. b Analysis of surface phenotypes of DCs, LLC and DC/LLC. The cells were analyzed using flowcytometery. The histograms show cells stained with the indicated molecule specific mAbs

Fig. 4

DC/LLC fusion cells did not express IDO, but induced IDO up-regulation in the spleens of vaccinated mice. a DCs, DC + LLC mixture (fusion mock) and DC/LLC fusion cells were lysed in a lysis buffer, and subjected to WB analysis with a specific anti-IDO antibody. b After second vaccination with or without DC/LLC fusion cells for 1 week, the spleens were removed and subjected to IHC analysis with a specific anti-IDO antibody. 1 Spleen from unvaccinated mice as a control; 2 Spleen from vaccinated mice with DC + LLC cell mixture; 3 Spleen from vaccinated mice with DC/LLC fusion cells. The inset in b shows a higher magnification (400×) of a representative area in the panel

Fig. 5

Combination treatment with 1-MT promots DC/LLC fusion vaccine-elicited host resistance against LLC tumor challenge. Mice were s.c. immunized twice with DC/LLC fusion vaccine at a 7-day interval as a single agent or in combination with 1-MT, the PBS was as a control. One week after the final immunization, mice were challenged by s.c. injection of 10⁶ LLC tumor cells. Tumor growth and tumor-free time in each group of mice were recorded. a Schematic representation of the treatment schedule in the LLC mice. b Average tumor growth over time for indicated treatment groups. Asterisks and double asterisks are an indication that significantly different from control on day 27 after tumor implantation (*P < 0.05; **P < 0.001, by Student’s t-test). c The percentage of mice that remained tumor-free over time for indicated treatment groups. The difference between the combination and the DC/LLC fusion vaccine alone treated groups was shown to be statistically evident using the Log-Rank test (P < 0.05)

Fig. 6

1-MT enhances DC/LLC fusion vaccine-induced tumor specific CTL responses. Splenocytes were collected on day 7 after second DC/LLC immunization and incubated at indicated E:T ratio. CTL activity was determined by LDH releasing assay. All of the experiments were performed in triplicated, and the results were calculated as the mean ± SD. Significantly different between DC/LLC fusion vaccine group and combination treatment group was determined by Student’s t-test (* P < 0.01)