Objectives

Blockade of co-stimulatory pathways is currently being tested as a new immuno-modulatory strategy for the treatment of graft failure. CD40L (the ligand of CD40) on the activated CD4+T-cells can interact with CD40, leading the proliferation and activation of B-lymphocytes.

Methods

Islet cells were transfected with Ad-CD40L at multiplicity of infection (MOI) =5, 10, and 30, respectively. The transfection efficiency was determined by using fluorescence microscope. The stimulation index was calculated by the following formula: stimulation index= insulin response to high glucose/the insulin response to low glucose. PKH67 labeled allergenic naÏve splenocytes Subsequently, the labeled splenocytes were co-cultured with islets of each group for 3 days. PKH67-labelled splenocytes (1.0×10⁶) that were not co-cultured with islets were used as negative control group. Streptozocin-induced diabetic Wistar rats were transplanted intraportally with 2000 IEQ islets isolated from Sprague-Dawley rats. The islet graft mean survival time (MST), insulin expression of islet grafts and the levels of cytokines in peripheral blood were measured for the animals in each group.

Results

After 72 h of infection, EGFP was expressed in 97.69±0.94% of islet cells transfected with Ad-sCD40L at an MOI of 10, was significantly higher than that in control and MOI of 5 groups (P<0.05). Significant homospecific hyporesponsiveness was observed in Ad-CD40L-transfected group (P<0.01).

MST of the diabetic rats transplanted with non-treatment group of islet cells was 7.1±1.16 days; Ad-sCD40L-transfected was 47.8±9.02 days. The expression of insulin in the control group was undetectable. Less inflammatory cell infiltration and stronger insulin staining were detected in the Ad-sCD40L group. We also found that Adenoviral-mediated gene transfer was capable of inducing sustained gene expression after 2 and 4 weeks of transplantation. The result showed that the levels of IL-1Β, TNF-Α and IFN-Γ in the Ad-CD40L group were significantly lower than other groups. (P<0.01)

Conclusion

We developed a relatively simple and safe method of adenovirus-mediated gene transfection to islet cells in vitro to induce systemic immune tolerance and reduce inflammation, which significantly prolonged the survival of islet grafts.

This work was supported by grants from National Nature Science Foundation of China (Nos.81270548) and 973 Program (No.2009CB522400).

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