FIGURE 1 from Overcoming Xenoantigen Immunity to Enable Cellular Tracking and Gene Regulation with Immune-competent “NoGlow” Mice

Tolerance to foreign antigens is animal model specific. A and B, Mice expressing GFP and Luc in the pituitary (GH; n = 5), full-body GFP, and Luc (CAG Luc-GFP; n = 5), in FoxP3⁺ cells (Foxp3-GFP; n = 4), or wild-type (WT) C57Bl/6 (n = 4) were vaccinated with adenovirus (Ad) encoding GFP/Luc (2.6 × 10¹⁰ vp/mouse) and anti-Luciferase (A) or anti-GFP (B) in the serum were evaluated by ELISA after 2 weeks. Only CAG Luc-GFP animals, which express both GFP and Luc, display humoral tolerance to both upon vaccination. P values shown are at 1:50 dilution by two-way ANOVA with Tukey multiple comparisons test. C and D, E0771 GFP-Luc cells were implanted into the MFP of female WT C57Bl/6, GH, or CAG Luc-GFP mice (n = 10 each) and subsequently vaccinated with Ad-GFP/Luc or Ad-LacZ as control on day 3 (n = 5/group each). Growth of individual tumors for the duration of the experiment (C) or average tumor volume until time of first euthanasia (D) comparing control or antigen-specific Ad demonstrate that E0771 GFP-Luc cells grow significantly larger in tolerant CAG Luc-GFP mice. P values shown are at day of first euthanasia by two-way ANOVA with Bonferroni multiple comparisons test. E, Female WT (n = 3) or mice expressing human HER2 under the whey acid protein (WAP) promoter (WAP HER2; n = 5) or MMTV Cre–driven CAG promoter (MMTV CAG HER2; n = 3) were vaccinated with Ad-Human Her2 and splenocytes were analyzed for anti-HER2 responses by ELISPOT after 3 weeks. WAP HER2 mice displayed reduced anti-HER2 responses compared with WT animals; however, MMTV CAG HER2 animals produced a limited anti-HER2 T-cell response compared with both WT and WAP HER2. Groups were compared by two-way ANOVA with Tukey multiple comparisons test. F, Doxycycline-inducible GFP animals were given doxycycline (dox)-containing chow at the indicated time points, vaccinated at 9 weeks old, and euthanized after two weeks. G, Serum anti-GFP responses in animals from F reveal that GFP remains highly immunogenic in mice with late (8 weeks old) versus early (4 weeks old/at birth) expression of GFP via doxycycline. P values shown are at 1:50 dilution by two-way ANOVA with Tukey post hoc analysis. All P values represent mean ± SEM.