A mouse model that is immunologically tolerant to reporter and modifier proteins

Abstract

Reporter proteins have become an indispensable tool in biomedical research. However, exogenous introduction of these reporters into mice poses a risk of rejection by the immune system. Here, we describe the generation, validation and application of a multiple reporter protein tolerant 'Tol' mouse model that constitutively expresses an assembly of shuffled reporter proteins from a single open reading frame. We demonstrate that expression of the Tol transgene results in the deletion of CD8⁺ T cells specific for a model epitope, and substantially improves engraftment of reporter-gene transduced T cells. The Tol strain provides a valuable mouse model for cell transfer and viral-mediated gene transfer studies, and serves as a methodological example for the generation of poly-tolerant mouse strains.

Fig. 1. Generation and validation of the Tol mouse model.

a Top left: cartoon depicting the strategy for reporter gene fragmentation, applied for each RP/MP gene included in the Tol ORF. Top right: reporter proteins included in the Tol ORF. Bottom: schematic overview of gene fragment placement in the Tol ORF. Note that the Tol ORF encodes the HPV E7_49–57 epitope in the COOH-terminal region of the chimeric Tol protein. b Cartoon depicting the targeting strategy of the Tol ORF into the Col1a1 locus through recombinase-mediated cassette exchange. c Differential gene expression analysis of indicated organs of Tol mice relative to WT mice. Scatterplots indicate average log2 fold changes and average counts per million. Genes with a statistically significant change in expression in Tol mice are indicated in gray, and the Tol transcript is indicated in blue. d Expression levels of the Tol transcript in thymi of Tol and WT mice. Data shown (c, d) is aggregated from n = 4 (Tol) and n = 4 (WT) mice. RP reporter protein, MP modifier protein, RMCE recombinase-mediated cassette exchange, PURO-R puromycin resistance gene, TK thymidine kinase gene, CPM counts per million.

Fig. 2. Complete loss of E7_49–57 immunogenicity in Tol mice.

a Schematic overview of vaccination and monitoring strategy. b Left: Representative flow cytometry plots depicting E7_49–57-specific CD8⁺ T cells at day 13 post DNA vaccination in WT and Tol mice. Right: E7_49–57-specific CD8⁺ T-cell response in blood of WT (gray) and Tol (blue) mice at the indicated timepoints post first DNA vaccination. Lines indicate individual mice. Cells are gated on IR-dye^–CD8⁺ lymphocytes. Depicted data are representative of two independent experiments, including at least 5 mice per group. P values were determined by Repeated Measures two-way ANOVA with Sidak’s multiple comparisons test to test the significance at each timepoint. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3. Enhanced engraftment of Kaede- and Katushka-expressing cells in Tol mice.

a Schematic overview of experimental design. b Percentage of Kaede⁺ OT-I T cells in the blood of WT (gray) and Tol (blue) mice at the indicated timepoints post first DNA vaccination. c Percentage of Kaede⁺ (left) or Kaede⁻ (right) OT-I cells in WT and Tol mice at day 57 after first DNA vaccination. d Left: representative flow cytometry plots depicting Kaede expression in transferred (Ly5.1⁺) cells at day 57 after first DNA vaccination. Right: median fluorescence intensity (MFI) of Kaede⁺ OT-I cells in WT and Tol mice at day 57 post first DNA vaccination. e Percentage of Katushka⁺ (red) and Katushka⁻ (black) OT-I cells in WT animals at the indicated timepoints after first DNA vaccination. f Percentage Katushka⁺ OT-I cells in WT and Tol mice at the indicated timepoints after first DNA vaccination. Cells (b–f) are gated on IR-dye⁻CD8⁺ lymphocytes, and plots depict n = 5 (Tol) and n = 5 (WT) mice. Depicted data are representative of one (b–d) or two (e, f) independent experiments. Box-plots (c, d) depict interquartile range, with whiskers representing minimum and maximum values. Dots (c, d) and lines (b, f) indicate individual mice. Error bars (e) represent SD. P values were determined by either two-tailed Mann–Whitney signed rank test (c, d) or Repeated Measures two-way ANOVA with Sidak’s multiple comparisons test (e, f). *p < 0.05; **p < 0.01; ***p < 0.001, ns not significant.