Optimization of Dendritic Cell-Mediated Cytotoxic T-Cell Activation by Tracking of Dendritic Cell Migration Using Reporter Gene Imaging.

PURPOSE: The aim of this study is to optimize the dendritic cell (DC)-mediated T-cell activation using reporter gene imaging and flow cytometric analysis in living mice.

PROCEDURES: A murine dendritic cell line (DC2.4) co-expressing effluc and Thy1.1 genes were established by transfection with retroviral vectors. Thy1.1 positive cells were sorted by magnetic bead separation system (DC2.4/effluc). Cell proliferation assay and phenotype analysis to determine the effects of gene transduction on the function of dendritic cells between parental DC2.4 and DC2.4/effluc were performed. To optimize the DC-mediated immune response by cell number or frequency, different cell numbers (5 × 105 , 1 × 106 , and 2 × 106  DC2.4/effluc) or different frequencies of DC2.4/effluc (first, second, and third injections) were injected in the right footpad of mice. The migration of the DC2.4/effluc into the draining popliteal lymph node of mice was monitored by bioluminescence imaging (BLI). Flow cytometric analysis was performed with splenocytes to determine the cytotoxic T-cell population after injection of DC2.4/effluc.

RESULTS: Parental DC2.4 and DC2.4/effluc exhibit no significant differences in their proliferation and phenotype. BLI signals were observed in the draining popliteal lymph node at day 1 after injection of DC2.4/effluc in 1 × 106 and 2 × 106 cells-injected groups. The highest BLI signal intensity was detected in 2 × 106 cells-injected mice. On day 11, the BLI signal was detected in only 2 × 106 cell-injected group but not in other groups. Optimized cell numbers (2 × 106 ) were injected in three animal groups with a different frequency (first, second, and third injection groups). The BLI signal was detected at day 1 and maintained until day 7 in the first injection group, but there is low signal intensity in the second and the third injection groups. Although the expression levels of Thy1.1 gene in the first injection group were very high, there reveals no expression of Thy1.1 gene in the second and the third injection groups. The number of tumor-specific CD8+ T-cells in the spleen significantly increased, as the number of DC injections increases.

CONCLUSIONS: Successful optimization of DC-mediated cytotoxic T-cell activation in living mice using reporter gene imaging and flow cytometric analysis was achieved. The optimization of DC-mediated cytotoxic T-cell activation could be applied for the future DC-based immunotherapy.