Utility of Clostridium difficile toxin B for inducing anti-tumor immunity.

Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity

Cell death of TcdB-intoxicated CT26 cells.

<p>CT26 cells were exposed to 500 ng/ml of TcdB for different time, and cell viability was measured by the PI staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110826#s2" target="_blank">Material and Methods</a>. Propidium iodide-positive cells were analyzed by Flow cytometry. The data shown represent one of three independent experiments. ***represents P<0.001 vs. control (unpaired two-tailed <i>t-</i>test). Error bars, SEM.</p

Anti-tumor immunity induced by dying CT26 cells.

<p>Mice were subcutaneously immunized with PBS or TcdB-exposed CT26 cells (TcdB). In some experiments, mice were injected with lysate of TcdB-treated CT26 cells (TcdB-lysate). Mice were then challenged with 10⁵ live CT26 cells on the opposite side of the groin and tumor growth was monitored. (A) Tumor volume was calculated using the formula: length×width²×π/6. The data represent one of five independent experiments (n = 5∼8). **, P<0.01 vs. PBS; ***, P<0.001 vs. PBS (paired two-tailed <i>t-</i>test). Error bars, SEM. (B) The percentage of tumor-free mice was measured. The data in (B) represent a pool from five independent experiments (n = 5∼8 for each experiment).</p

Induction of an anti-tumor immune response by TcdB-intoxicated B16-F10 cells.

<p>Mice were immunized once with PBS or 2×10⁵ TcdB-exposed B16-F10 cells per mouse before challenge with lethal B16-F10 cells. Tumor volume (A) and the percentage of tumor-free mice (B) were measured. Representative data from one of three experiments are shown (n = 10 for each experiment). **, P<0.01 vs. PBS; ***, P<0.001 vs. PBS (paired two-tailed <i>t-</i>test). Error bars, SEM.</p

IFN-γ production induced by BMDCs loaded with TcdB-treated tumor cells.

<p>Autologous splenocytes were co-cultured with bone marrow DCs (BMDCs) preloaded with live or TcdB-intoxicated CT26 cells for two weeks, and the supernatant was collected to measure IFN-γ production by ELISA. Splenocytes incubated with mere DCs or mere TcdB-treated tumor cells were set as controls. The data represent the mean of three independent determinations ± SEM. ***represents P<0.001 (unpaired two-tailed <i>t-</i>test).</p

T-cell proliferation, IL-2 secretion, and specific CTL activity of splenocytes from immunized mice.

<p>Mice were immunized twice with PBS or TcdB-intoxicated CT26.CL25 (TcdB), and splenocytes were harvested 5 days after the second immunization. (A and B) splenocytes were restimulated with OVA, CT26.CL25 lysate, CT26 lysate, and β-galactosidase. (A) T-cell proliferation was determined by BrdU cell proliferation assay. (B) IL-2 production was measured by ELISA. The data in (A) and (B) represent the mean of three independent experiments ± SEM. *represents P<0.05 (one-way ANOVA). (C) Specific CTL induction. Splenocytes restimulated with CT26.CL25 lysate were tested for cytolytic activity against CT26.CL25 cells, parental CT26 cells, or myeloma p3x63Ag8.653 cells using cytotoxicity detection kit (LDH) assay. Representative data from one of three independent experiments are shown.</p

Specific and long-lasting anti-tumor immunity induced by TcdB-treated CT26 cells.

<p>(A and B) Protection against pre-injected tumors. Four hours after lethal CT26 tumor cell challenge, mice were injected with PBS, TcdB-exposed CT26 cells (TcdB), or CT26 lysate (TcdB-lysate). (A) Tumor volume was measured. The data represent one of four independent experiments (n = 5∼8). ***represents P<0.0001 vs. PBS (paired two-tailed <i>t-</i>test). Error bars, SEM. (B) The percentage of tumor-free mice was determined. The data presented is a pool from four independent experiments (n = 5∼8 for each experiment). (C and D) Mice vaccinated with TcdB intoxicated CT26 cells are not protected against myeloma p3x63 cells. Mice were challenged with lethal myeloma p3x63 cells and then immunized with TcdB-intoxicated CT26 cells (TcdB) or vehicle control (PBS) at a different site 4 h later. (C) Mouse tumor volume was measured (n = 8). (D) The percentage of tumor-free mice was measured (n = 8). (E and F) The anti-tumor immunity induced by TcdB-intoxicated tumor cells is long lasting. Mice surviving the first challenge with CT26 cells after either prophylactic or therapeutic vaccination with TcdB-treated tumor cells were rechallenged with 10⁶ (10 times the LD100) CT26 cells 3 months after the first challenge. The age-matched naive mice were challenged with 10⁶ CT26 cells as control. Tumor volume (E) and the percentage of tumor-free mice (F) were evaluated. The data shown represent one of three independent experiments. ** in e, P<0.01 between prophylactic group or therapeutic group vs. PBS (paired two-tailed <i>t-</i>test); *** in f, P<0.0001 vs. PBS (Log-rank test). Error bars, SEM.</p