Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity. in treating polyposis. 2. MATERIALS AND METHODS 2.1. Mice MIN (C57BL/6J-Apcwith the MUC1 TR peptides (APGSTAPPA, SAPDTRPAP) and the hepatitis B computer virus core antigen pan helper peptide (TPPAYRPPNAPIL), each at 20 ng/mL, in the presence of 100 U/mL IL-2 for six days. Media, peptides and IL-2 were replenished on Day 3. Cytolytic activity was assessed on day 6 using 51Cr-labeled MUC1-expressing murine colon carcinoma L-Mimosine cells (MC38.MUC1)  as targets. Mock-transduced L-Mimosine cells (MC38.neo), not expressing MUC1, were labeled with 51Cr and used as negative control targets. Approximately 1106 effector cells per well were plated in a 96 well U-bottom microtiter Pdgfrb plate made up of 1104 51Cr-labeled MC38.MUC1 or MC38.neo tumor cells per well. Microtiter plates were centrifuged at 100 g for 5 min, and then incubated at 37C for 6 h. At the end of the incubation period, a 100 L aliquot of the supernatant fluid was removed from each well, and the released 51Cr (experimental) was quantified using a Beckman LS6500 scintillation counter (Beckman Coulter, Inc. Fullerton, CA). The total 51Cr amount incorporated in tumor cells was determined by lysing 1104 51Cr-labeled cells with Triton-X 100 (total) and counting a 100L aliquot. Spontaneous 51Cr release was measured by counting 100l of 1104 51Cr-labeled cells that were incubated with media alone (spontaneous). The percentage of specific tumor cell lysis was calculated using the following formula: %Cytotoxicity=[(experimental) ? (spontaneous)]/[(total) ? (spontaneous)]100. Spontaneous release did not exceed 10%. 2.11. Statistical Analysis Data were expressed as mean and standard deviation. Sizes and numbers of adenomas were evaluated using Poisson regression to analyze the difference between peptide- and non-peptide-based vaccination. IFN- data were analyzed using the Wilcoxon signed-rank non-parametric test. The L-Mimosine cytotoxicity data were analyzed using Students re-stimulation with the MUC1 peptides. MUC1.Tg/MIN mice that were vaccinated with the peptide-based vaccine secreted significantly more IFN- (357.5181.4 pg/mL) than MUC1.Tg/MIN mice vaccinated with the non-peptide-based vaccine (45.831.0 pg/mL) (p 0.001) (Physique 4A). Unstimulated splenocytes did not produce detectable levels of IFN- (data not shown). The increased IFN- production by splenocytes from the peptide-vaccinated mice was also correlated with cytolysis of MUC1-expressing tumor cells (Physique 4B). Of note is that analysis of sera from these mice revealed the absence of detectable anti-MUC1 antibodies (data not shown) suggesting that a cellular rather than a humoral immune response is usually elicited using this peptide-based vaccine cocktail. Open in a separate window Physique 4 Peptide-based vaccine elicits a MUC1-specific immune response(A) MUC1.Tg/MIN mice were vaccinated three times with either the peptide-based vaccine (peptides + CpG-ODN + GM-CSF) or the non-peptide vaccine (CpG-ODN + GM-CSF) at two week intervals. Splenocytes were then isolated and stimulated with the MUC1 TR peptides and the hepatitis B computer virus core antigen pan helper peptide for 48 h. Subsequently, supernatant was evaluated for IFN- production by ELISA. Splenocytes from individual mice from the peptide vaccine group (n=4) and the non-peptide vaccine group (n=3) were evaluated in triplicate. Data represent mean SD. (B) Cytolytic activity is usually MUC1-specific. Splenocytes from vaccinated MUC1.Tg/MIN mice were isolated and stimulated with the MUC1 TR peptides and the hepatitis B computer virus core antigen pan helper peptide for six days in the presence of 100 U/mL IL-2. Target cells were 51Cr-labeled MC38.MUC1 (murine colon carcinoma L-Mimosine cell line expressing human MUC1) or MC38.neo (mock-transfected cell line not expressing human MUC1) plated at 5104 cells/well. Effector to target cell ratios (E:T) and p values of statistical evaluations at each L-Mimosine E:T ratio are indicated around the x-axis. 4. DISCUSSION Apc/ em MIN /em /+ (MIN) mice are characterized by a mutation in the APC tumor suppressor gene that results in the development of multiple intestinal adenomas. Since these adenomas do not progress to invasive carcinoma, the MIN model is considered an appropriate model for.
Vaccination with irradiated tumor cells engineered to secrete murine granulocyte-macrophage colony-stimulating factor stimulates potent, specific, and long-lasting anti-tumor immunity