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. 2013 Jun 17:11:148.
doi: 10.1186/1479-5876-11-148.

Impact of anti-CD25 monoclonal antibody on dendritic cell-tumor fusion vaccine efficacy in a murine melanoma model

Chunrui Tan  1 , Varun Reddy, Jens Dannull, Enyu Ding, Smita K Nair, Douglas S Tyler, Scott K Pruitt, Walter T Lee
Affiliations

Impact of anti-CD25 monoclonal antibody on dendritic cell-tumor fusion vaccine efficacy in a murine melanoma model

Chunrui Tan et al. J Transl Med. .

Abstract

Background: A promising cancer vaccine involves the fusion of tumor cells with dendritic cells (DCs). As such, a broad spectrum of both known and unidentified tumor antigens is presented to the immune system in the context of the potent immunostimulatory capacity of DCs. Murine studies have demonstrated the efficacy of fusion immunotherapy. However the clinical impact of DC/tumor fusion vaccines has been limited, suggesting that the immunosuppresive milieu found in patients with malignancies may blunt the efficacy of cancer vaccination. Thus, novel strategies to enhance fusion vaccine efficacy are needed. Regulatory T cells (Tregs) are known to suppress anti-tumor immunity, and depletion or functional inactivation of these cells improves immunotherapy in both animal models and clinical trials. In this study, we sought to investigate whether functional inactivation of CD4+CD25+FoxP3+ Treg with anti-CD25 monoclonal antibody (mAb) PC61 prior to DC/tumor vaccination would significantly improve immunotherapy in the murine B16 melanoma model.

Methods: Treg blockade was achieved with systemic PC61 administration. This blockage was done in conjunction with DC/tumor fusion vaccine administration to treat established melanoma pulmonary metastases. Enumeration of these metastases was performed and compared between experimental groups using Wilcoxon Rank Sum Test. IFN-gamma ELISPOT assay was performed on splenocytes from treated mice.

Results: We demonstrate that treatment of mice with established disease using mAb PC61 and DC/tumor fusion significantly reduced counts of pulmonary metastases compared to treatment with PC61 alone (p=0.002) or treatment with control antibody plus fusion vaccine (p=0.0397). Furthermore, IFN-gamma ELISPOT analyses reveal that the increase in cancer immunity was mediated by anti-tumor specific CD4+ T-helper cells, without concomitant induction of CD8+ cytotoxic T cells. Lastly, our data provide proof of principle that combination treatment with mAb PC61 and systemic IL-12 can lower the dose of IL-12 necessary to obtain maximal therapeutic efficacy.

Conclusions: To our knowledge, this is the first report investigating the effects of anti-CD25 mAb administration on DC/tumor-fusion vaccine efficacy in a murine melanoma model, and our results may aide the design of future clinical trials with enhanced therapeutic impact.

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Figures

Figure 1
Figure 1
FACS analysis of DCs and subsequent confirmation of DC-D5LacZ fusion hybrids. (A) Electrofusion of DCs and D5lacZ cells. (Top panel) Phenotype of DCs at day 0 (black histograms) and day 8 (grey histograms) of culture. (Bottom panel) Prior to fusion, D5LacZ were intracellularly and covalently labeled with CFSE. 24 hours after fusion, adherent cells were harvested and stained with DC markers as indicated. Numbers in the upper right quadrant represent percentages of double-positive fusion hybrids. (B) MHC class I (H2-kb) and MHC class II (I-Ab) expression of DCs and DC-tumor fusion cells.
Figure 2
Figure 2
Effects of Treg-inactivation on the efficacy of DC-tumor cell fusion vaccination. Lung metastases were established in C57BL/6 mice via tail vein injection of D5lacZ cells. After 2 days, mice received intraperitoneal injections of saline (−), Y13 mAb (Y13), or PC61 mAb (PC61). On day 3, DC-tumor fusion cells (FC) were delivered intranodally. IL-12 was administered intraperitoneally daily for 4 days, starting on the day of vaccination. Mice were sacrificed and lung metastases were enumerated after 21 days. P-values were calculated using the two-tailed Wilcoxon rank sum test. The number (n) of animals used in each group is indicated. Results are presented as box plots in which the median, the 25 percentile, the 75 percentile, as well as minimum and maximum of each group are shown.
Figure 3
Figure 3
IFN-γ ELISPOT analysis. (A) IFN-γ- stimulated D5lacZ tumor cells were used as stimulators in ELISPOT assays. Up-regulation of MHC class I (H2-kb) and class II (I-Ab) was confirmed by FACS. Isotypic controls (solid lines), MHC expression in the absence of IFN-γ (filled grey histograms), and MHC expression of D5lacZ tumor cells that had been treated for 72 hours with IFN-γ at 1,000 U/ml of media (dotted lines) are presented in our FACS analyses. (B) Frequencies of IFN-γ-secreting CD4+ and CD8+ T-effector cells in response to D5lacZ tumor stimulation. CD4+ and CD8+ T cells were isolated from spleens that were harvested from naïve mice (naïve), mice treated with control mAb and DC/D5lacZ fusion cells (Y13+FC), mice treated with PC61 mAb and fusion cells (PC61+FC), and mice treated with systemic IL-12 and fusion cells (IL-12+FC). IFN-γ secreting cells were enumerated without stimulation (−) or after stimulation with D5lacZ cells (stimulators). 2.5×105 T cells (responders) per well were stimulated at a responder to stimulator ratio of 5:1. ELISPOT assays were performed in triplicates and averaged data of 3 independent experiments are shown.
Figure 4
Figure 4
Combination treatment with PC61 mAb and mIL-12. Lung metastases were established in C57BL/6 mice via tail vein injection of D5lacZ cells. After 2 days, mice received intraperitoneal injections of 250 μg/0.5 ml PC61 mAb (PC61 only, Fusion + PC61 + ½ dose IL-12). On day 3, DC-tumor fusion cells (Fusion) were delivered intranodally. IL-12 was administered intraperitoneally daily for 4 days at 0.2 μg/0.5 ml (full dose) or 0.1 μg/0.5 ml (1/2 dose), respectively, starting on the day of vaccination. Mice were sacrificed after 21 days and lung metastases were enumerated.
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