Enhancing radiotherapy response via intratumoral injection of the TLR9 agonist CpG to stimulate CD8 T cells in an autochthonous mouse model of sarcoma

Abstract

Radiation therapy is frequently used to treat cancers including soft tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to radiation therapy (RT) in transplanted tumors, but the mechanism(s) remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and two doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to RT + CpG, we performed bulk RNA-seq, single-cell RNA-seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and interferon-γ. CpG + RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG + RT, TCR clonality analysis suggests an increase in clonal T-cell dominance. Collectively, these findings demonstrate that RT + CpG significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft tissue sarcoma.

Fig 1.. Increased tumor growth delay after treatment with CpG ODN and radiation therapy in autochthonous p53/MCA sarcomas.

A. Primary sarcoma initiation by intramuscular injection of Adeno-Cas9-sgp53 and MCA. B. Autochthonous sarcomas develop at the injection site about 7–11 weeks after injection. Mice were treated with CpG ODN or control GpC dinucleotides and 0 or 20 Gy when tumors reached >70 mm³. C. Mice with p53/MCA sarcomas received control GpC dinucleotides with 0 Gy (solid black, n = 26), CpG ODN alone (solid green, n = 23), control GpC dinucleotides with 20 Gy (solid blue, n = 21), or CpG ODN with 20 Gy (solid red, n = 20). Time to tumor quintupling (days) after the indicated treatment. D. Mice with p53/MCA sarcomas received control GpC dinucleotides with 0 Gy (solid black, n = 26), CpG ODN alone (solid green, n = 23), control GpC dinucleotides with 20 Gy (solid blue, n = 21), or CpG ODN with 20 Gy (solid red, n = 20). Kaplan-Meier analysis with tumor quintupling as the endpoint. Kruskal-Wallis Test was used for comparison across the groups, while the Wilcoxon test was selected for the pair-wise comparisons. * shows the significance of the P Value (***: p <= 0.001, ****: p <= 0.0001)

Fig 2.. CyTOF and IHC staining demonstrates enhanced intratumoral infiltration of activated CD8 T cells after combination treatment.

A. Treatment schedule and tumor processing schematic. B. UMAP plot of CyTOF data clustering for all CD45 high cells from all tumors and treatment groups. C. Frequency of CD8+ cells/live CD45+ cells by CyTOF. Data show mean ± SEM, analyzed by three-way ANOVA. D. Average number of CD8+ cells/0.2 mm² in IHC slides. E. Representative IHC staining with CD8 Ab. Scale bar = 100 μm. F. CD8 expression in CyTOF CD45 high UMAP plot. G. Granzyme B expression in CyTOF CD45 high UMAP plot.

Fig 3.. Single cell RNA-seq shows increased CD8 T cell infiltration into the tumor after RT + CpG.

A. Treatment schedule and tumor processing schematic. B. Bubble plot of top 3 differentially expressed genes in each of the T cell subclusters. The shades of color are correlated with levels of expression. The sizes of circles are correlated with percentage of cells in that cluster that express the gene of interest. C. UMAP plot of T cells and NK cells scRNA-seq subclustering. D. UMAP plot of T cells and NK cells subclustering colored by treatment groups. E. UMAP plot of lymphocytes subclustering colored by CD4 (red), CD8 (blue), NK/ILC (black), and γδT (yellow) cells. Mice with p53/MCA sarcomas received control GpC dinucleotides with 0 Gy (n = 5), CpG ODN alone (n = 5), control GpC dinucleotides with 20 Gy (n = 5), or CpG ODN with 20 Gy (n = 5).

Fig 4.. Treatment with RT + CpG promotes tumor-antigen specific clonal expansion of T cells and tumor immune microenvironment remodeling.

A. p53/MCA sarcoma develops at the injection site about 7–11 weeks after induction. Mice were treated with CpG ODN or GpC dinulcotides control and 0 or 20 Gy when tumors reached >180 mm³. Sarcomas received control GpC dinucleotides with 0 Gy (n = 5), CpG ODN alone (n = 5), control GpC dinucleotides with 20 Gy (n = 5), or CpG ODN with 20 Gy (n = 5). Shannon entropy calculated from the abundance of TCR sequences captured by TCR sequencing and stratified by treatment group. Increasing entropy indicates reduced uniformity of TCR sequences. P values were calculated using two-sided Wilcoxon tests. B. TCR evenness is Shannon entropy normalized by species richness. C. Immune-based classification of murine primary sarcomas. Sample sizes: muscle control (n=3), tumor control (n=5), CpG ODN (n=5), RT (n=5), RT+CpG ODN (n=5).

Fig 5.. Treatment with RT + CpG promotes intratumoral myeloid cell remodeling.

A. UMAP plot of CyTOF clustering for CD45hi cells from Ctl (GpC dinucleotides) and CpG+RT treatment groups. MHC-I, MHC-II, and CD11c expression are highlighted in red. B. Immune cell composition of all treatment groups from Bulk RNA-seq. C. UMAP plot of dendritic cells subclustering D. Bubble plot of top 3 differentially expressed genes in each of the DC subclusters. The shades of color are correlated with levels of expression. The sizes of circles are correlated with percentage of cells in that cluster that express the gene of interest. Sample sizes: tumor control (n=5), CpG ODN (n=5), RT (n=5), RT+CpG ODN (n=5).

Fig 6.. Lymphocytes mediate the anti-tumor effects of the combination treatment RT + CpG.

A. Primary sarcoma initiation by intramuscular injection of Adeno-Cas9-sgp53 and MCA. B. Autochthonous sarcoma develops at the injection site about 7–11 weeks after induction. Mice were treated with CpG ODN or control GpC dinucleotides and 0 or 20 Gy when tumors reached >70 mm³. C. Heterozygous Mice (Rag2+/−;yc+ or Rag2+/−;yc+/−) with p53/MCA sarcomas received control GpC dinucleotides with 0 Gy (black, n = 9), CpG ODN alone (green, n = 17), control GpC dinucleotides with 20 Gy (blue, n = 17), or CpG ODN with 20 Gy (red, n = 18). Figure shows time to tumor quintupling (days). D. Heterozygous mice (Rag2+/−;yc+ or Rag2+/−;yc+/−) with p53/MCA sarcomas received control GpC dinucleotides with 0 Gy (black, n = 9), CpG ODN alone (green, n = 17), control GpC dinucleotides with 20 Gy (blue, n = 17), or CpG ODN with 20 Gy (red, n = 18). Figure shows time to tumor quintupling (days). E. Homozygous mice (Rag2−/−;yc- or Rag2−/−;yc−/−) with p53/MCA sarcomas received GpC dinucleotides control with 0 Gy (black, n = 14), CpG ODN alone (green, n = 12), GpC dinucleotides control with 20 Gy (blue, n = 12), or CpG ODN with 20 Gy (red, n = 12). Figure shows time to tumor quintupling (days). F. Homozygous mice (Rag2−/−;yc- or Rag2−/−;yc−/−) with p53/MCA sarcomas received GpC dinucleotides control with 0 Gy (black, n = 14), CpG ODN alone (green, n = 12), GpC dinucleotides control with 20 Gy (blue, n = 12), or CpG ODN with 20 Gy (red, n = 12). Figure shows time to tumor quintupling (days). Kruskal-Wallis Test was used for the group comparison, while the Wilcoxon test was selected for the pair-wise comparisons. * shows the significance of the P Value (***: p <= 0.001, ****: p <= 0.0001).

Fig 7.. CD8 T cells are required for the treatment effects of RT + CpG.

A. Primary sarcoma initiation by intramuscular injection of Adeno-Cas9-sgp53 and MCA. Autochthonous sarcoma develops at the injection site about 7–11 weeks after induction. Mice were treated with CpG ODN or control GpC dinucleotides and 0 or 20 Gy when tumors reached >70 mm³. Mice received intraperitoneal CD8 isotype control or CD8 depletion antibodody (Ab) on the same day tumors received RT. CD8 isotype control or CD8 depletion Ab are repeated every 3.5 days until tumor size reached humane end point. B. 129/SvJ mice with p53/MCA sarcomas, injected with CD8 isotype control Ab, received GpC dinucleotides control with 0 Gy (black, n = 13), CpG ODN alone (green, n = 12), GpC dinucleotides control with 20 Gy (blue, n = 15), or CpG ODN with 20 Gy (red, n = 14). C. 129/SvJ mice with p53/MCA sarcomas, injected with CD8 isotype control Ab, received GpC dinucleotides control with 0 Gy (black, n = 13), CpG ODN alone (green, n = 12), GpC dinucleotides control with 20 Gy (blue, n = 15), or CpG ODN with 20 Gy (red, n = 14). D. 129/SvJ mice with p53/MCA sarcomas, injected with CD8 depleting Ab, received GpC dinucleotides control with 0 Gy (black, n = 16), CpG ODN alone (green, n = 14), GpC dinucleotides control with 20 Gy (blue, n = 13), or CpG ODN with 20 Gy (red, n = 11). F. 129/SvJ mice with p53/MCA sarcomas, injected with CD8 depletion Ab, received GpC dinucleotides control with 0 Gy (black, n = 16), CpG ODN alone (green, n = 14), GpC dinucleotides control with 20 Gy (blue, n = 13), or CpG ODN with 20 Gy (red, n = 11). Figure shows time to tumor quintupling (days). Kruskal-Wallis Test was used for the group comparison, while the Wilcoxon test was selected for the pair-wise comparisons. * shows the significance of the P Value (***: p <= 0.001, ****: p <= 0.0001).