A novel method of modifying immune responses by vaccination with lipiodol-siRNA mixtures

Abstract

The dendritic cell (DC) possesses the ability to stimulate both T helper 1 (Th1) and Th2 responses depending on activation stimuli. Although it is known that chemically or genetically modified DC can be used therapeutically to steer immune responses towards either Th1 or Th2, cellular therapy with ex vivo manipulated DC is clinically difficult. Here we demonstrate a novel method of switching immune responses from Th1 to Th2 through in vivo immune modulation by administration of siRNA. We demonstrate that siRNA targeting of the IL-12p35 gene leads to a Th2 bias in vitro through an IL-10 dependent mechanism. In vivo administration of siRNA admixed with the oil-based contrast agent lipiodol in the presence of antigen and adjuvant induced a deviation in recall response to reduced production of IFN-gamma and augmented IL-4 response using either KLH or ovalbumin. This simple method of in vivo modification of immune response possesses therapeutic potential in Th1-mediated diseases such as multiple sclerosis and autoimmune diabetes.

Figure 1

Lipiodol can serve as a transfection reagent. Day 7 bone marrow derived DC were cultured alone, or with IL-12 p35 specific siRNA delivered by optimized GenePorter concentration (3 μl/culture) or 3 concentrations of lipiodol (1,2, or 3 μl/culture). Following a 24 hour incubation cells were plated at 1 × 10⁶ in 6 well culture dishes and activated for 24 hours with 10 ng/ml LPS and 10 ng/ml TNF-α. Supernatant was harvested and analyzed by ELISA for IL-12 p70 production.

Figure 2

Lipiodol transfected DC are immune modulatory. A. Day 7 bone marrow derived DC were cultured alone or transfected with mismatched siRNA, or IL-12p35-specific siRNA using 3 μl/culture lipiodol. Following a 24 hour incubation cells were plated at 1 × 10⁶ in 6 well culture dishes and activated for 24 hours with 10 ng/ml LPS and 10 ng/ml TNF-α. Supernatant was harvested and analyzed by ELISA for IL-10 production. B. C57/BL6 DC were transfected with mismatched siRNA, IL-12p35-specific siRNA or lipiodol alone, irradiated (3,000 rad) and seeded in triplicate at various concentrations in a flat-bottom 96-well plate. Splenic T cells from BALB/c mice were added as responders (5 × 10⁵ cells/well). The mixed lymphocytes were cultured for 72 h and proliferation was assessed by thymidine incorporation. C &D. IFN-γ and IL-4 concentrations, respectively, were assessed from MLR cultures at 48 hours of incubation.

Figure 3

IL-10 is mediates immune modulation by IL-12 silenced DC. A. MLR was performed with various concentrations of irradiated C57/BL6 stimulator DC transfected with siRNA to IL-12p35 or control transfected DC, and BALB/c responder T cells. 5 μl/ml of anti-IL-10 (JES5 2A5) antibody was added throughout the culture time. Supernatant was collected from 48-hour MLR cultures and assessed for IFN-γ or IL-4 (B) by ELISA.

Figure 4

Inhibition of recall response by lipiodol/siRNA vaccine. A. C57/BL6 mice were immunized intradermally at the interior side of both hind legs with 100 μl of KLH or ovalbumin (1 μg/μl) emulsified in CFA in the presence or absence of 10 nMol siRNA and 10% lipiodol. After 14 days mice were sacrificed and T cell recall responses were assessed culturing purified CD4+ T cells with irradiated syngeneic splenocytes in triplicate and mixed with serial dilutions of KLH or OVA (B) at concentrations ranging from 0–10 ug/ml. Following a 72-h incubation, proliferation was assessed by thymidine incorporation.

Figure 5

In vivo immune modulation lipiodol/siRNA vaccine. C57/BL6 mice were immunized intradermally at the interior side of both hind legs with 100 μl of KLH or ovalbumin (1 μg/μl) emulsified in CFA in the presence or absence of 10 nMol siRNA and 10% lipiodol. After 14 days mice were sacrificed and T cell cytokine responses were assessed culturing purified CD4+ T cells with irradiated syngeneic splenocytes in triplicate and mixed with serial dilutions of antigen. IFN-γ in KLH (A) and ovalbumin (C) cultures, and IL-4 in KLH (B) and ovalbumin (D) cultures was assessed by ELISA.