Tolerance Induction by Cd40 Silenced Dendritic Cells Through Antisense



Karimi MH^{1, 5} ; Ebadi P² ; Pourfathollah AA¹ ; Soheili ZS³ ; Samiee SH⁴ ; Ataee Z⁴ ; Tabei SZ⁵ ; Nadali F⁶ ; Moazeni SM¹ Show Affiliations
Source: Iranian Red Crescent Medical Journal Published:2009

Abstract

Background: One of the valuable tools for inhibiting the specific gene expression is antisense technique. To determine T cell responses, co-stimulatory molecule expression on the antigen presenting cells is important. In the present study, the effects of high affinity antisense against CD40 mRNA on the function and phenotype of DCs (dendritic cells) were investigated. Methods: The DCs were separated from the mice spleens and then cultured in vitro. By means of lipofectamine 2000, the antisense was delivered into the cells and the efficacy of transfection was estimated by flow cytometry. Also, the mRNA expression and protein synthesis were assessed by real time PCR and flow cytometry, respectively. The DCs were transfected with 6 μM antisense and 2 μl lipofectamine 2000. Results: The percentage of CD40 expression in DCs was 38%. The results showed that CD40 expression is reduced in DCs to 22% and 24%. By annexine V and propidium iodine staining, we could evaluate the viability of the transfected cells. The inhibition of CD40 gene expression was associated with the increase in IL-4 secretion. This shifted the DCs to stimulate Th2 cytokine production from the allogenic T cells. In addition, in the MLR, the DCs without CD40 expression showed poor allostimulatory effects. This finding is valuable in the study of the costimulatory molecules of DCs. Conclusion: These data demonstrate that direct interference of the cell surface expression of CD40 at transcriptional level by antisense confers tolerogenecity potential of DCs. This approach is a useful tool through which DCs become tolerogenic and can be studied as a potential therapeutic option for the autoimmune diseases and allograft rejection. © Iranian Red Crescent Medical Journal.