Manipulation of T cell costimulatory and inhibitory signals for immunotherapy of prostate cancer

Abstract

The identification of potentially useful immune-based treatments for prostate cancer has been severely constrained by the scarcity of relevant animal research models for this disease. Moreover, some of the most critical mechanisms involved in complete and proper antitumoral T cell activation have only recently been identified for experimental manipulation, namely, components involved in the costimulatory pathway for T cell activation. Thus, we have established a novel syngeneic murine prostate cancer model that permits us to examine two distinct manipulations intended to elicit an antiprostate cancer response through enhanced T cell costimulation: (i) provision of direct costimulation by prostate cancer cells transduced to express the B7.1 ligand and (ii) in vivo antibody-mediated blockade of the T cell CTLA-4, which prevents T cell down-regulation. In the present study we found that a tumorigenic prostate cancer cell line, TRAMPC1 (pTC1), derived from transgenic mice, is rejected by syngeneic C57BL/6 mice, but not athymic mice, after this cell line is transduced to express the costimulatory ligand B7.1. Also, we demonstrated that in vivo antibody-mediated blockade of CTLA-4 enhances antiprostate cancer immune responses. The response raised by anti-CTLA-4 administration ranges from marked reductions in wild-type pTC1 growth to complete rejection of these cells. Collectively, these experiments suggest that appropriate manipulation of T cell costimulatory and inhibitory signals may provide a fundamental and highly adaptable basis for prostate cancer immunotherapy. Additionally, the syngeneic murine model that we introduce provides a comprehensive system for further testing of immune-based treatments for prostate cancer.

Figure 1

Percentage of pTC1 cells expressing MHC class I as a function of cell passage. pTC1 cells, in vitro, were harvested and then evaluated by flow cytometry after staining the cells with an anti-MHC class I antibody followed by FITC-conjugated goat anti-rat immunoglobulin (Caltag) second-step reagents. For each histogram, the pTC1 cell passage number is given first and is followed by the percentage of pTC1 cells that stain positive for MHC class I molecules.

Figure 2

(A and B) mRNA analysis for SV40 large tumor T antigen (Tag) of pTC1, v-TC1, or B7-TC1 grown in vitro (A) or in vivo (B) in BALBc nu/nu mice. (A) Total RNA was isolated from cells in culture, DNase treated, and reverse transcribed; PCR was performed; and the products were visualized by ethidium bromide staining in agarose gel. L-19 was used as an internal control for the PCR. Tag* is the above gel transferred to membrane and probed with a ³²P-labeled internal oligonucleotide sequence. Lanes: 1, late-stage TRAMP tumor from TRAMP mouse; 2, molecular weight markers; 3, TC1; 4, vTC1; 5, B7-TC1. (B) Same analysis as above using RNA from: 1, late-stage TRAMP tumor; 2, molecular weight markers; 3, TC1 tumor; 4, vTC1 tumor; and 5, B7-TC1 tumor.

Figure 3

(A and B) Rejection or growth of B7-TC1, and growth of pTC1 and vTC1 tumors, in male syngeneic C57BL/6 mice (A) or C57BL/6 nu/nu mice (B). (A) Male C57BL/6 mice received subcutaneous injections to the back on day 0 with 5 × 10⁶ cells of p-, v-, or B7-TC1. (B) Male C57BL/6 nu/nu mice received similar injections on day 0 with 1 × 10⁶ pTC1 cells or its derivatives. Data are presented as the mean tumor base area (mm²) ± SD from a single representative experiment that was repeated three times. Each group contained 5–7 mice. The fraction of animals in each group that formed tumors is provided in parentheses next to corresponding marker designations. Animals were euthanized when their tumors achieved 250 mm².

Figure 4

(A and B) Effects of intraperitoneal anti-CTLA-4 administration on growth of early-passage pTC1 (A, passages 9, 10, and 11) and late passage pTC1 (B, passages >15). C57BL/6 mice were injected with 2.0 × 10⁶ (B) or 2.5 × 10⁶ (A) wild-type pTC1 cells. Data are mean tumor areas from a single, representative experiment in which groups of five mice received either 100 μg of a control hamster antibody (Ctrl Ab) or 100 μg of anti-CTLA-4 antibody (aCTLA-4) on days 7, 10, and 13 following tumor injection. The fraction of animals in each group that formed tumors is provided in parentheses next to corresponding marker designations. Experiments in A were performed three times, and experiments in B were performed two times.