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. 2012;7(12):e52926.
doi: 10.1371/journal.pone.0052926. Epub 2012 Dec 21.

TNF-α and tumor lysate promote the maturation of dendritic cells for immunotherapy for advanced malignant bone and soft tissue tumors

Shinji Miwa  1 , Hideji Nishida, Yoshikazu Tanzawa, Munetomo Takata, Akihiko Takeuchi, Norio Yamamoto, Toshiharu Shirai, Katsuhiro Hayashi, Hiroaki Kimura, Kentaro Igarashi, Eishiro Mizukoshi, Yasunari Nakamoto, Shuichi Kaneko, Hiroyuki Tsuchiya
Affiliations

TNF-α and tumor lysate promote the maturation of dendritic cells for immunotherapy for advanced malignant bone and soft tissue tumors

Shinji Miwa et al. PLoS One. 2012.

Erratum in

  • PLoS One. 2013;8(10). doi:10.1371/annotation/7c0f14e6-cfba-4f5f-b93d-1a149eaeec96

Abstract

Background: Dendritic cells (DCs) play a pivotal role in the immune system. There are many reports concerning DC-based immunotherapy. The differentiation and maturation of DCs is a critical part of DC-based immunotherapy. We investigated the differentiation and maturation of DCs in response to various stimuli.

Methods: Thirty-one patients with malignant bone and soft tissue tumors were enrolled in this study. All the patients had metastatic tumors and/or recurrent tumors. Peripheral blood mononuclear cells (PBMCs) were suspended in media containing interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF). These cells were then treated with or without 1) tumor lysate (TL), 2) TL + TNF-α, 3) OK-432. The generated DCs were mixed and injected in the inguinal or axillary region. Treatment courses were performed every week and repeated 6 times. A portion of the cells were analyzed by flow cytometry to determine the degree of differentiation and maturation of the DCs. Serum IFN-γ and serum IL-12 were measured in order to determine the immune response following the DC-based immunotherapy.

Results: Approximately 50% of PBMCs differentiated into DCs. Maturation of the lysate-pulsed DCs was slightly increased. Maturation of the TL/TNF-α-pulsed DCs was increased, commensurate with OK-432-pulsed DCs. Serum IFN-γ and serum IL-12 showed significant elevation at one and three months after DC-based immunotherapy.

Conclusions: Although TL-pulsed DCs exhibit tumor specific immunity, TL-pulsed cells showed low levels of maturation. Conversely, the TL/TNF-α-pulsed DCs showed remarkable maturation. The combination of IL-4/GM-CSF/TL/TNF-α resulted in the greatest differentiation and maturation for DC-based immunotherapy for patients with bone and soft tissue tumors.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Preparation of DC from peripheral blood monocytes. A. Basic DC-preparation Protocol.
DCs were generated from peripheral blood (50 ml) collected from study patients. Mononuclear cells were isolated by density gradient centrifugation on Lymphoprep. Interphase cells were removed, washed twice in phosphate-buffered saline (PBS), and suspended in CellGro. After adherence for 2 hours, adherent cells were cultured in media containing IL-4 (50 ng/ml) and GM-CSF (100 ng/ml). On days 4 and 5, the cultures were additionally supplemented with or without tumor lysate (TL) (100 μg/ml) and OK-432 (0.1 KE/ml). B. Modified DC-preparation Protocol. TNF-α was added to the protocol of tumor lysate and OK432. Similar to the first DC preparation protocol, the cells were cultured in a medium containing IL-4 and GM-CSF. On days 4 and 5, the cultures were additionally supplemented with or without tumor lysate (TL) (100 μg/ml), rhTNF-α (100 ng/ml), and OK-432 (0.1 KE/ml).
Figure 2
Figure 2. Comparison of %CD80, %CD83, and %CD86 by DC maturation cocktails.
TL: tumor lysate, TNF: tumor necrosis factor-α, ** P<0.01, n.s.: not significant A. Basic DC-preparation Protocol. Cells treated with tumor lysate (TL) showed slightly higher maturation than control cells (not significant). Cells treated with OK-432 showed greater maturation (P<0.01). B. Modified DC-preparation Protocol. Cells treated with a combination of tumor lysate (TL) and TNF-α (TNF) showed markedly higher maturation than cells treated with TL (P<0.01).
Figure 3
Figure 3. Serum IFN-γ and serum IL-12 before and after DC therapy.
* P<0.05, ** P<0.01, n.s.: not significant.
Figure 4
Figure 4. Kaplan-Meier analysis of overall survival and progression free survival distributions for 31 patients with advanced malignant bone and soft tissue tumors.
See this image and copyright information in PMC

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