Demonstration of the ability of intratumoral administration of AdmCD40L-transduced DCs to traffic to the spleen and induce T-cell activation.
The expression of CD40L mRNA by the AdmCD40L-modified DCs (panel A) was used to tract the genetically modified DCs, and immunocytochemistry for the IL-2 receptor α chain used to identify activated T cells in the local milieu. (A) AdmCD40L-mediated CD40L expression assessed by RT-PCR in DCs or control CL7 cells transduced with AdmCD40L. The RT-generated cDNA from DCs transduced with AdmCD40L or AdNull, or CL7 cells transduced with AdmCD40L was amplified with primers for exogenous mCD40L or control GAPDH mRNA. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. (B) Detection of DCs expressing AdmCD40L-mediated CD40L by nested RT-PCR in spleens from mice treated with intratumoral administration of AdmCD40L-transduced DCs. Balb/c mice were injected subcutaneously in the right midflank with 2 × 105 CT26 cells (day 0). On day 8, tumor-bearing mice were treated by intratumoral injection of 2 × 106 AdmCD40L- or AdNull-transduced DCs or AdmCD40L-transduced CL7 cells. On day 11, the RT-generated cDNA from the spleens was amplified with outer and inner primer pairs for exogenous mCD40L by nested PCR. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. PCR for the control GAPDH mRNA is shown in the lower part of the panel. (C) Immunohistochemical evaluation of spleens in panel B for CD25 (IL-2 receptor α chain). On day 11, spleens were dissected, and frozen spleen sections were stained using 10 μg/mL rat antimouse CD25 mAb, followed by the visualization with 10 μg/mL antirat IgG Oregon Green antibody. Specific immunoreactivity showed the presence of CD25-expressing cells in spleens from mice treated with AdmCD40L-modified DCs, whereas only a low level of tissue autofluorescence was evident in control spleens (AdNull/DCs and AdmCD40L/CL7).
