Article Figures & Data
Figures
- Figure 1.
GCC-specific CD4+ T-cell and antibody tolerance. A, GCC-specific CD4+ T-cell responses in Control-AV– or GCC-AV–immunized GCC+/+ (+/+) and GCC−/− (−/−) C57BL/6 mice, measured by IFNγ ELISpot following restimulation with recombinant GCC-6xHis protein (*, P < 0.05, two-sided Student's t test on values at 50 μg/mL). B, AV-specific CD4+ T-cell responses in GCC-AV–immunized GCC+/+ (+/+) and GCC−/− (−/−) C57BL/6 mice, measured by IFNγ ELISpot following restimulation with AV particles (#, P > 0.05, two-sided Student's t test on values at 1 × 108 IFU/mL). Data in A and B indicate pooled analysis of n = 2–3 mice per group and are representative of four independent experiments. C, ELISA analysis of GCC-specific IgG antibody responses in GCC+/+ (+/+) or GCC−/− (−/−) C57BL/6 mice 14 d after immunization with GCC-AV or Control-AV. Columns, mean of n = 3 mice per group at reciprocal serum dilutions of 25, 50, 100, and 200 (***, P < 0.001, two-way ANOVA). Representative of three independent experiments. D, ELISA analysis of AV-specific IgG antibody responses in naïve GCC+/+ (+/+) or GCC−/− (−/−) C57BL/6 mice or 10 to 14 d after immunization with AV. Columns, mean of n = 4 AV-immunized mice per genotype or pooled samples of three naïve mice per genotype at reciprocal serum dilutions of 100, 200, 400, and 800 (***, P < 0.001; #, P > 0.1, two-way ANOVA). Bars, SD (A–D).
- Figure 2.
GCC-specific CD8+ T-cell responses. A, GCC-specific CD8+ T-cell responses in GCC+/+ C57BL/6 mice following LacZ-AV (control) or GCC-AV immunization, measured by IFNγ ELISpot using GCC-expressing colorectal cancer cells as stimulators. B, β-galactosidase–specific CD8+ T-cell responses in GCC+/+ C57BL/6 mice following LacZ-AV or GCC-AV (control) immunization, measured by IFNγ ELISpot using β-galactosidase–expressing colorectal cancer cells as stimulators. Data in A and B indicate pooled analysis of n = 2 mice per group and are representative of six independent experiments (***, P < 0.001, two-sided Student's t test). C, GCC-specific CD8+ T-cell responses in GCC+/+ (+/+) and GCC−/− (−/−) C57BL/6 mice following GCC-AV immunization, measured by IFNγ ELISpot as in A. Data indicate pooled analysis of n = 2 mice per group and are representative of two independent experiments (**, P < 0.01, two-sided Student's t test). D, SIINFEKL-specific CD8+ T-cell responses in GCC+/+ (+/+) and GCC−/− (−/−) C57BL/6 mice following NP/SIINFEKL-AV immunization, measured by IFNγ ELISpot on restimulation with SIINFEKL-expressing stimulator cells. Representative of three independent experiments examining SIINFEKL- or β-galactosidase–specific responses (#, P > 0.9, two-sided Student's t test). Bars, SD (A–D).
- Figure 3.
GCC-specific immunotherapy against colon cancer metastases in lung. BALB/c mice were immunized with Control-AV or GCC-AV and then challenged with 5 × 105 CT26-GCC cells by tail vein injection 7 d later. A, on day 14 after challenge, metastases were visualized by PET/microCT. Images indicate merged PET and microCT images and lungs are outlined for clarity. Images are representative of n = 5–7 mice per immunization. B, lungs were removed and stained with India ink to visualize lung nodules. C, lung nodules were enumerated on visual inspection. Columns, mean of n = 12 mice per immunization; bars, 95% confidence intervals (***, P < 0.001, two-sided Student's t test).
- Figure 4.
GCC immunization does not intensify mucosal autoimmunity in experimental colitis. A, female C57BL/6 mice were immunized with Control-AV or GCC-AV and boosted sequentially with RV and VV at 28-d intervals. Four days later, mice were treated with a 7-d course of 4% DSS ad libitum in the drinking water, followed by normal water. Mouse weights were monitored daily. Points, mean; bars, 95% confidence intervals (P > 0.05, Bonferroni's multiple comparison's test on area under the curve values, control versus GCC-immunized DSS treatment groups; Supplementary Fig. S2). B to D, some mice were euthanized on day 9 for examination of disease markers including diarrhea (B), fecal blood (C), and histology (D). D1, representative sections from treated mice; D2, histologic scores from treated mice. B to D, columns, mean of n = 5 mice per group; bars, SD (#, P > 0.05, Bonferroni's multiple comparison test).
- Figure 5.
GCC-specific immunization does not amplify tumorigenesis in genetic and inflammatory models of colorectal cancer. A to C, APCmin/+ C57BL/6 mice were immunized with Control-AV or GCC-AV and boosted sequentially with RV and VV at 28-d intervals beginning at 4 wk of age. Two weeks after the final immunization, intestines were collected and examined by histology (A), and tumor burden was quantified in small (B) and large (C) intestines. Bars, 95% confidence intervals (#, P > 0.05, two-sided Welch's t test). D to F, female C57BL/6 mice were immunized with Control-AV or GCC-AV and boosted sequentially with RV and VV at 28-d intervals beginning at 6 wk of age. The procarcinogen axozymethane was administered 3 d before the final immunization. Seven days later, three cycles of DSS were initiated with 14 d of water between each cycle. D, colons were collected 10 d after the final cycle and examined. Tumor number (E) and tumor size (F) were determined under a dissecting microscope. Bars, 95% confidence intervals (#, P > 0.05, two-sided Welch's t test).
Volume 69, Issue 8
