Figure 2.

Optimal mRNA-LNPs induce qualitative T cell responses and strong antitumor efficacy

(A) Mice were immunized with optimal LNPs encapsulating 5 μg of E7 and 5 μg of TriMix mRNA at days 0, 7, 14, and 50. Blood CD8 E7-specific T cell responses were determined at days 5, 20, 49, and 55. (B and C) Differential expression of KLRG1 and CD127 on blood E7-specific (+) and non-specific (−) CD8 T cells at day 49 and day 55. Representative contour plots (B, day 49) and quantification (C, mean ± SD [n = 4]) of KLRG1 and CD127 expression are shown. (D) IFN-γ levels in serum were measured 6 h after each immunization (n = 4–6). (E and F) IFN-γ and TNF-α expression by splenic CD8 T cells after three immunizations with TBS or mRNA-LNPs (weekly intervals, 10 μg of E7-TriMix). Representative contour plots (E) and quantification (F: mean ± SD [n = 3 for TBS, n = 5 for LNPs]) is shown. (G) When TC-1 tumors reached a mean volume of 52 mm³, mice were injected i.v. with 5 μg of E7-TriMix mRNA-LNPs (weekly intervals). (H) TC-1 growth curves in mice. Data are shown as mean ± SEM (n = 6–7). (I) Kaplan-Meier survival curves of TC-1-bearing mice. (J and K) Representative contour plots (J) and graph (K, mean ± SD [n = 4] with individual data points shown in red) quantifying the infiltration of CD8 T cells in TC-1 tumors. (L) E7 specificity of CD8 TILs in TC-1 tumors. Data are shown as mean ± SD with individual data points in red (n = 4). Statistical differences were assessed using a Mantel-Cox log-rank test with Bonferroni multiple testing correction (I), one-way ANOVA with Tukey post hoc test (J), or two-way Student’s t test (L).∗∗p < 0.01, ∗∗∗p < 0.001; ns, not significant.