Dendritic cell based genetic immunization stimulates potent tumor protection dependent on CD8 CTL cells in the absence of autoimmunity

Abstract

Although antibodies (Abs) produced by B cells can treat cancer in certain models, T cells have been accountable for the major effector to control cancer. Immune recognition toward tyrosinase-related protein-1 (TRP-1), a melanoma associated antigen up-regulated on the surface of B16F10 melanomas, generally leads to tumor protection mediated by Abs. In this study, immunization with dendritic cells ex vivo transduced with adenovirus encoding TRP-1 stimulates immune activation and potent tumor protection mediated by CD8 T cells in the absence of autoimmune consequence. Transfer of CD8 T cells from immunized mice also leads to tumor protection. The immune activation and CD8 T cell mediated tumor protection rely on the CD4 T cell help. Thus DC based genetic immunization targeting TRP-1, an antigen usually causes Ab predominant immune recognition, is capable of stimulating potent tumor protection dependent on CD8 T cells in the absence of autoimmunity.

Fig. 1

IFN-γ secretion. Mice were immunized with DC/AdTRP-1 and DC/AdBHG, respectively. After 7 days splenocytes were taken out and restimulated with irradiated (100 Gy) F10 and MCA207 cells at the ratio of 20–1, respectively. After 48 h, supernatant was gathered for ILISA to measure IFN-γ secretion. The result is the summary of more than three experiments

Fig. 2

CD8 CTL cytotoxicity. Mice were immunized with DC/AdTRP-1 and DC/AdBHG, respectively. After 7 days splenocytes were taken out and restimulated with irradiated (100 Gy) F10 and MCA207 cells at the ratio of 20–1 for 5 days, respectively. The restimulated cells were measured for CTL cytotoxicity with ⁵¹Cr release assay against F10 cells. In one experiment, anti-CD8 antibody and rabbit complement were added for 1 h at 37°C to remove CD8 T cells before ⁵¹Cr release assay. This is the summary of at least three experiments

Fig. 3

Vaccination induced tumor protection. Mice were immunized with DC/AdTRP-1 and DC/AdBHG, respectively. After 7 days 2 × 10⁴ F10 cells were injected subcutaneously (s.c.) into the mice, they were followed until the tumors grew. The result is the summary of at least three experiments

Fig. 4

Enhanced protection after DC based genetic immunization in the i.v. challenge model. The mice were challenged with 1 × 10⁶ F10 cells in 200 μl through i.v. injection 7 days after the immunization. After 3 weeks the mice were sacrificed and the tumor nodules in the lungs were enumerated under the microscope

Fig. 5

CD8 T cell dependent tumor protection. Mice were immunized with DC/AdTRP-1. On day 7, 2 × 10⁴ F10 cells were injected s.c. into the mice, they were followed until the tumors grew. In some experiments, 100 μl CD8 or CD4 Ab was injected intraperitoneally (i.p.) on days 5 and 7 to remove CD8 T cells and CD4 T cells, respectively. The result is the summary of at least three experiments

Fig. 6

The transfer of CD8 T cell but not CD4 T cell leads to tumor protection. Mice were immunized with DC/AdTRP-1. After 7 days splenocytes were taken out and sorted by MACS. Then 5 million CD8 or CD4 T cells were injected intravenously (i.v.) into the recipient naive mice which had been pretreated with 5.5 Gy irradiation. One day later, 2 × 10⁴ F10 cells were injected s.c. into the recipient mice, they were followed until the tumors grew. The result is the summary of at least three experiments

Fig. 7

CD8 T cell cytotoxicity requires CD4 T cell activation. Mice were immunized with DC/AdTRP-1. After 7 days splenocytes were taken out and restimulated with irradiated (100 Gy) F10cells at the ratio of 20–1 for 5 days, respectively. The restimulated cells were measured for CTL cytotoxicity with ⁵¹Cr release against F10 cells. In some experiments, 100 μL anti-CD4 Ad was injected i.p. into the mice on day 2, 0 (before immunization) or on day 4, 6 (after immunization). The result is the summary of at least two experiments

Fig. 8

CD8 T cell dependent tumor protection requires CD4 T cell activation. Mice were immunized with DC/AdTRP-1. 2 × 10⁴ F10 cells were injected s.c. into the mice, they were followed until the tumors grew. In some experiments, 100 μL anti-CD4 Ad was injected i.p. into the mice on day 2, 0 (before immunization) or on day 4, 6 (after immunization). The result is the summary of at least two experiments