Immune tolerance of transplants induced by the combination of methoxy polyethylene glycol and anti-OX40L monoclonal antibody

Feng Sa-ran, Huang Yi-hong, Du Bing, Pan Xiu-ying, Xu Kai-lin

Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou   221002, Jiangsu Province, China

Feng Sa-ran★, Studying for master’s degree, Attending physician,
fengqiongxin@ 163.com

Correspondence to: Huang Yi-hong, Master, Professor, Chief physician, Tutor of master, Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou   221002, Jiangsu Province, China
yhcs7758@163.com

Supported by: the Outstanding Youth Teacher Program of Higher Learning School of Jiangsu Province, No. sjs200512*

Received: 2007-12-01
Accepted: 2008-05-21

Abstract
BACKGROUND: Methoxy-polyethylene glycol (mPEG) can have a stereospecific blockade, which can shield surface antigen of T lymphocytes. OX40 and its ligand OX40L is a pair of important costimulator. To block the signal pathway can induce T cells in an inexcitable incapacitation, resulting in immune tolerance.
OBJECTIVE: To investigate the effect of combined usage of methoxy-polyethylene glycol-succinimidyl-propionic acid ester (mPEG-SPA) and anti-OX40L monoclonal antibody (McAb) on proliferation, phenotypes of T lymphocytes and secretion of cytokines in vitro to obtain an ideal immune tolerance.
DESIGN, TIME AND SETTING: The in vitro control experiment was performed at the Department of Hematology, Affiliated Hospital of Xuzhou Medical College from December 2006 to June 2007.
MATERIALS: Totally 12 clean inbred line C57BL/6(H-2b) male and BALB/c(H-2d) female adult mice each were selected. Prepared splenic lymphocytes were used as responder cells and stimulator cells. mPEG-SPA and rat anti-mouse OX40L monoclonal antibody were respectively purchased from Kaizheng, China and eBioscience, USA.
METHODS: Responder cells were regulated to a density of 4×109 L-1, and modified by 3, 6, 12, 15, 18 g/L mPEG-SPA for 1 hour. Blank control cells were treated with an equal volume of phosphate buffer saline. In addition, responder cells were given 15 g/L mPEG-SPA for 1, 24, 48, 96 and 120 hours. Expression of CD3+ T cells was detected by flow cytometer. In one-way mixed lymphocyte culture, cells in the cell control group received stimulator cells and responder cells. Cells in the anti-OX40L McAb group underwent 10 mg/L anti-OX40L McAb. Cells in the mPEG-SPA group were subjected to 15 g/L mPEG-SPA. Cells in the combination group were treated with anti-OX40L McAb and mPEG-SPA.
MAIN OUTCOME MEASURES: CD3 expression; Outcome of mPEG-SPA screening splenic lymphocytes; Proliferation and phenotype of lymphocytes; Cytokine content in supernatant.
RESULTS: After modification of mPEG-SPA, expression of CD3+ T cells significantly reduced compared with the blank control group, especially cells treated with 15, 18 g/L mPEG-SPA (P < 0.01). No significant difference in expression of CD3+ T cells was detected after treatment of 15 g/L mPEG-SPA at different time points (P > 0.05). Compared with the cell control group, the inhibitory rate of lymphocyte proliferation significantly increased in the anti-OX40L McAb group, mPEG-SPA group and combination group, especially in the combination group (P < 0.05). CD4+CD25+/CD8+CD25+ ratio did not significantly change in the anti-OX40L McAb group and mPEG-SPA group (P > 0.05), but decreased in the combination group (P < 0.05). Interferon-γ contents were significantly lower in the anti-OX40L McAb group, mPEG-SPA group and combination group than in the cell control group, and the significant reduction was detected in the combination group (P < 0.05). Interleukin-4 levels were significantly higher in the combination group than in the cell control group (P < 0.05).
CONCLUSION: mPEG can significantly camouflage surface antigen of T lymphocytes, and the effect of camouflage is durable. The combined use of mPEG-SPA and anti-OX40L McAb can block T-cell activation antigen and co-stimulatory pathway, regulate the differentiation of T cells and induce the immune shift of Th0 cells towards Th2 cells.

Feng SR, Huang YH, Du B, Pan XY, Xu KL.Immune tolerance of transplants induced by the combination of methoxy polyethylene glycol and anti-OX40L monoclonal antibody.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(29):5663-5667
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