Soluble cd28 cd28.2

Syngeneic immature DC and NK cells or nicDC and nicNK cells (1×10⁵ DC: 2×10⁵ NK/well) exposed to Alum or TLR agonists for 8 hrs were subsequently co-cultured in 48-well plates (Falcon, Franklin Lakes, NJ) at a 1:10 ratio with CFSE labeled allogeneic naïve T cells isolated from PBMC using T cell isolation kit (StemCell Technologies, Vancouver, Canada). On day 5, the proliferating cells were transferred into new plates and rested in IL-2-containing medium (5 ng/ml) up to 10 days. The cells were subsequently collected and stained with CD4 (L200), CD8 (SK1), and CCR7 (3D12) (primary co-cultures). The remaining T cells were transferred to plates pre-coated with 10 μg/ml mAbs CD3 (UCHT1) and 2 μg/ml soluble CD28 (CD28.2) (BD Biosciences, San Diego, CA) for 72 hrs. The T cells were then stimulated for 4–6 hrs with leukocyte activation cocktail (BD Biosciences) containing Brefeldin A before staining with CD4 (L200), CD8 (SK1), CCR7 (3D12), and intracellular IFN-γ (4S.B3) (BD Biosciences, San Diego, CA) (secondary co-cultures). The frequency and amount of IFN-γ were further analyzed using Flow Cytometry and ELISA.

Syngeneic immature DCs and NK cells (1×10⁵ DC: 2×10⁵ NK/well) exposed to Alum or TLR agonists for 8hrs were subsequently co-cultured in 48-well plates (Falcon, Franklin Lakes, NJ) at a 1:10 ratio with CFSE labeled allogeneic naïve T cells isolated from PBMCs using T cell isolation kit (StemCell Technologies, Vancouver, Canada). The purity of the enriched T cells was >95% based upon prevalence of CD3+CD56- phenotypes. On day 5, the proliferating cells were transferred into new plates and rested in IL-2-containing medium (5ng/ml) up to 10 days. The cells were subsequently collected and stained with CD4 (L200), CD8 (SK1), and CCR7 (3D12) (primary co-cultures). The remaining T cells were transferred to plates pre-coated with 10μg/ml mAbs CD3 (UCHT1) and 2μg/ml soluble CD28 (CD28.2) (BD Biosciences, San Diego, CA) for 72hrs. The T cells were then stimulated for 4–6hrs with leukocyte activation cocktail (BD Biosciences) containing Brefeldin A before staining with CD4 (L200), CD8 (SK1), CCR7 (3D12), and intracellular IFN-γ (4S.B3) (BD Biosciences, San Diego, CA) (secondary co-cultures). The frequency and amount of IFN-γ were further analyzed using Flow Cytometry and ELISA.