Amount of interleukin 12 available at the tumor site is critical for tumor regression.

The C26 colon carcinoma is resistant to systemic recombinant interleukin 12 (rIL-12) therapy. Transduction of C26 with genes encoding the two subunits of murine IL-12 to release 30-80 pg/ml resulted in delayed tumor onset after injection of 5 x 10(4) cells into syngeneic BALB/c mice and in 40% tumor regression after injection into CD4-depleted mice. Here, we analyzed the activity of rIL-12 (1 microgram/day) against C26 grown into CD4-depleted mice. Like in mice given injections of interleukin 12 (IL-12) gene-transduced C26 cells, depletion of CD4+ cells led to tumor regression in 6 of 14 mice, and immumocytochemical characterization of tumor-infiltrating leukocytes showed abundant infiltration by CD8+ T cells and asialoGM1+ natural killer cells, which were scanty in tumors from nondepleted mice. On the basis of the percentage of tumor regression and leukocyte infiltration we can conclude that, in the C26 system, systemic rIL-12 (1 pmicrogramg/day) produces the same results as 30-80 pg/ml IL-12 released at the tumor site. A new polycistronic retroviral vector was then used to increase the amount of IL-12 produced by C26-transduced cells. C26 cells releasing 5 ng/ml IL-12, nearly 100 times more than the above-mentioned transduced cells, were tumorigenic in less than 50% of the mice given injections of 5 x 10(4) cells. In mice given injections of 5 x 10(5) cells, an initial tumor take of 100% followed by a complete tumor regression. Tumor regression was associated with infiltration of CD8+ and asialoGM1+ cells, and mice that remained tumor free were immune to a rechallenge of nontransduced C26 cells. The results indicate that the amount of IL-12 made available at the tumor site may determine both the type and number of infiltrating leukocytes and the events leading to tumor regression as well as it may overcame host immunosuppression.

Interleukin 12 potentiates the curative effect of a vaccine based on interleukin 2-transduced tumor cells.

The purpose of these studies was to determine whether systemic administration of recombinant interleukin 12 (rIL-12) is able to potentiate an initial, but insufficient T-cell antitumor response. Mice challenged with carcinoma cells engineered to release interleukin 2 (IL-2) and displaying such a response received single or multiple i.p. injections of rIL-12. This combination of systemic rIL-12 and local IL-2 increased the percentage of mice that rejected two different IL-2 gene-transduced tumors. In another set of experiments more closely resembling a clinical situation, IL-2 gene-transduced tumors were used as vaccines in an attempt to cure mice bearing wild-type parental tumors. The combination of these vaccines with systemic rIL-12 cured mice more effectively than rIL-12 and IL-2 gene-transduced tumor vaccines alone.

CD4 T cells inhibit in vivo the CD8-mediated immune response against murine colon carcinoma cells transduced with interleukin-12 genes.

Retroviral-mediated cytokine gene transfer into tumor cells is a highly effective way of inducing tumor inhibition and immunity. We analyzed the tumorigenicity of C-26 murine colon carcinoma cells transduced with genes encoding the two subunits of murine interleukin-12 (IL-12) in a polycistronic retroviral vector and selected for resistance to G418 and for IL-12 production (30-80 pg/ml). BALB/c mice injected s.c., i.v. and intrasplenically with C-26/IL-12 cells from three different IL-12-producing clones showed delayed tumor onset as compared with mice injected with control NeoR-transduced or parental tumor cells. Although C-26/IL-12 tumor-bearing mice eventually died of lung metastasis, their survival time was twice as long as that of mice injected with control cells. In experiments with mice selectively depleted of natural killer (NK) cells before tumor cell injection, the time of tumor onset and survival of mice injected with C-26/IL-12 s.c. and i.v., respectively, was reduced. CD8+ T cell depletion had no effect on latency or survival, whereas removal of CD4+ T cells led to C-26/IL-12 tumor regression in about 40% of mice. Histological and immunocytochemical characterization of leukocytes infiltrating C-26/IL-12 tumors showed only slight infiltration with few T cells in non-depleted mice but abundant infiltration by CD8+ T cells and asialo-GM1+ NK cells in tumors of mice depleted of CD4+ T cells. The lack of CD8+ T cell infiltration is not due to a CD4-mediated suppression of their activation because irradiated C-26/IL-12 cells primed for the induction of a strong cytotoxic T lymphocyte response against C-26 parental cells and induced CD8+ effector cells that protected against C-26/IL-12 in a Winn assay. Rather, the results suggest that, although C-26/IL-12 cells injected in vivo stimulate both NK and CD8+ T cells, tumor infiltration by the latter is inhibited by CD4+ T cells.