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. 2020 Jun 22;9(1):1771925. doi: 10.1080/2162402X.2020.1771925

Figure 2.

Figure 2.

Activated poly-functional Th1-like CD4+ T cells against the immunodominant vaccine-encoded CD4 neoantigen are induced in the spleen of CD4 neoantigen vaccine/LRT treated mice. (a–c) CT26 tumor growth (a) and survival (b) of BALB/c mice (n = 7–10/group) treated with 12 Gy at a mean tumor volume of 45 mm3 and immunized with different RNA-LPX vaccines. RNA-LPX vaccines included CT26 PME1; CT26 ME1, the most immunogenic CT26 PME1 neoantigen; Pctrl. encoding mutations not expressed in CT26 and control RNA-LPX, encoding no antigens at all. (c) Gp70-AH1 tetramer+ CD8+ T cells in treated mice (n = 7–10/group). (d, e) Phenotypic analysis of enriched splenic CD4+ T cells from mice immunized with CT26 PME1 or control RNA-LPX (n = 5/group). (d) Differential expression of ICOS, CD69, PD-1, CD62L, T-bet, CD25 on ME1-specific (ME1tet+) and -nonspecific (ME1tet−) CD4+ T cells as determined by flow cytometry. Representative pseudocolor plots of ME1 tetramer staining are shown. (e) Supernatant cytokine secretion after 48 h co-culture of CD4+ T cells with ME1-peptide-loaded BALB/c BMDC. (f) IFNγ intracellular cytokine staining after ex vivo re-stimulation of enriched splenic CD4+ T cells from CT26-tumor-bearing mice, locally 12 Gy irradiated at a mean tumor volume of 60 mm3 and immunized with CT26 PME1 or control RNA-LPX, with ME1 peptide-loaded BMDC (n = 6/group). Significance was determined using (b) Mantel-Cox log-rank test, (c, d, f) one-way ANOVA, Tukey’s multiple comparison test and (e) unpaired, two-tailed Student’s t-test. (a) Tumor growth is displayed on a log2-scale. Ratios depict frequency of mice with complete tumor responses (CR). Mean±SEM.