Integrating IL-12 mRNA nanotechnology with SBRT eliminates T cell exhaustion in preclinical models of pancreatic cancer

Abstract

Pronounced T cell exhaustion characterizes immunosuppressive tumors, with the tumor microenvironment (TME) employing multiple mechanisms to elicit this suppression. Traditional immunotherapies, such as immune checkpoint blockade, often fail due to their focus primarily on T cells. To overcome this, we utilized a proinflammatory cytokine, interleukin (IL)-12, that re-wires the immunosuppressive TME by inducing T cell effector function while also repolarizing immunosuppressive myeloid cells. Due to toxicities observed with systemic administration of this cytokine, we utilized lipid nanoparticles encapsulating mRNA encoding IL-12 for intratumoral injection. This strategy has been proven safe and tolerable in early clinical trials for solid malignancies. We report an unprecedented loss of exhausted T cells and the emergence of an activated phenotype in murine pancreatic ductal adenocarcinoma (PDAC) treated with stereotactic body radiation therapy (SBRT) and IL-12mRNA. Our mechanistic findings reveal that each treatment modality contributes to the T cell response differently, with SBRT expanding the T cell receptor repertoire and IL-12mRNA promoting robust T cell proliferation and effector status. This distinctive T cell signature mediated marked growth reductions and long-term survival in local and metastatic PDAC models. This is the first study of its kind combining SBRT with IL-12mRNA and provides a promising new approach for treating this aggressive malignancy.

Keywords: IL-12; MT: Delivery Strategies; T cell immunity; antitumor immune response; lipid nanoparticles; mRNA; pancreatic cancer; stereotactic body radiation therapy.

Graphical abstract

Figure 1

Combination treatment of SBRT + IL-12mRNA eliminates pancreatic tumors and promotes survival (A) Illustration of PDAC mouse model where 2.5 × 10⁴ KP2-luciferase-expressing tumor cells were injected orthotopically (red arrow) and flanked with titanium fiducial clips (white arrows) for SBRT targeting. The tumors were treated with or without SBRT (6 Gy/d over 4 consecutive days) followed by an intratumoral injection of IL-12mRNA or control scr-mRNA 1 day after the final fraction of radiation. (B) Tumor burden for primary disease was assessed by weekly BLI measurements for each treatment. (C) Survival was tracked out to 300 days post tumor injection. (D) BLI growth curve for systemic rechallenge of cured mice versus aged-matched naive mice (E) Illustration of the metastatic model developed to test the systemic effects SBRT/IL-12mRNA treatment: pancreas and hepatic portal vein were injected concurrently to establish primary (pancreas) and metastatic (liver) tumors. (F) BLI growth curve for each treatment for the metastatic PDAC model. (G) Survival curve for the metastatic model was tracked out to 150 days. n = 15/group from three independent experiments in (B) and (C). n = 5 for cured mice and n = 9 aged-matched naive mice in (D). n = 6–8 in (F) and (G). For BLI growth curves, mean values for each time point are presented for each treatment group. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 2

T cells undergo distinct transcriptomic changes following SBRT/IL-12mRNA treatment (A) Unsupervised clustering of intratumoral cells following scRNA-seq could identify four distinct cell subsets: Tumor/Stromal cells, Myeloid cells, B cells and T/NK cells. (B) Separating this clustering to treatment-specific UMAPs could identify significant transcriptomic changes to T cells and myeloid, in particular, following SBRT/IL-12mRNA treatment (see red and black highlighted regions). See materials and methods for further details.

Figure 3

Modulations to macrophages after SBRT/IL12mRNA treatment (A) Combined UMAP for all macrophages across the four treatment groups. (B) Expression and relative proportion levels of key genes to classify classically and alternatively activated macrophages. (C) UMAP of macrophage populations for each treatment group. (D) Relative proportions of classically and alternatively activated macrophages for each treatment group. (E) Principal-component analysis for macrophages based on protein expression of IFNγ, arginase-1, CD163, CD206, MHCII, MHCI, and PDL1 using flow cytometry on tumors from day 13 (3 days post IL-12mRNA/scr-mRNA injection). Ovals indicate groups of similar principal component scores. (F) Protein expression of CD206 on macrophages. (G) Protein expression of IFNγ on macrophages. n = 5 for (E), (F), and (G). Analyzed by one-way ANOVA followed by Tukey’s test in (F) and (G). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. Lines represent mean±standard error of the mean (SEM) in (F) and (G).

Figure 4

SBRT/IL-12mRNA treatment remodels the CD4 and CD8 T cell response in tumors (A and B) Subclustered UMAPs and relative proportions of CD4 and CD8 T cell subpopulations following each treatment. (C and D) Ratios of activated/cycling to exhausted CD4 and CD8 T cells. (E) Ratios of activated/cycling to regulatory CD4 T cells. (F and G) Co-expression heatmaps for Mki67/Lag3 for CD4 T cells and Prf1/Ifng for CD8 T cells for each treatment. (H and I) Trajectory analysis for CD4 and CD8 T cells with Naive T cells utilized as the root population in each case. Black circles indicated branch nodes and white circles signify potential terminal outcomes along the trajectory. See materials and methods for further details.

Figure 5

SBRT/IL-12mRNA induces proliferation in tumor CD4 and CD8 T cells (A and B) Cell cycle score for CD4 and CD8 T cells using genes associated with G1, S, and G2/M phases of cell cycle. (C and D) Representative flow graphs for BrdU staining in CD4 and CD8 T cells at 3 days post IL-12mRNA/scr-mRNA injection. (E and F) Quantification of BrdU+ CD4 and CD8 T cells. (G and H) CD4 and CD8 T cell tumor levels at day 1, day 3 and day 7 post IL-12mRNA/scr-mRNA injection. n = 10/group from two independent experiments in (C)–(H). One-way ANOVA followed by Tukey’s test. Significance against untreated and between treatments are reported. ∗p < 0.05; ∗∗∗∗p < 0.0001. Lines represent mean ± SEM for (G) and (H).

Figure 6

SBRT reshapes the TCR repertoire in tumors (A) Illustration of experimental protocol for TCR sequencing analysis following SBRT/IL-12mRNA treatment: KP2 tumors are injected on day 0 and SBRT/IL-12mRNA treatment is given as described previously. Three days after IL-12mRNA/scr-mRNA injection, tumors are harvested, CD4 and CD8 T cells sorted, and their receptors sequenced. (B) Chao1 diversity index plot indicating the number of unique clonotypes for each treatment for both CD4 and CD8 T cells. (C) Bubble plots for the top clonotypes for each treatment group for CD4 T cells. (D) Bar charts depicting the average number of clonotypes for each treatment for CD4 T cells. (E) Bubble plots for the top clonotypes for each treatment group for CD8 T cells. (F) Bar charts depicting the average number of clonotypes for each treatment for CD8 T cells. See materials and methods for further details.

Figure 7

IFNγ expression is induced by SBRT/IL-12mRNA and is essential to treatment efficacy (A) Tumor IFNγ concentration measured by luminex at 24 h and 96 h post IL-12mRNA injection. (B) IFNγ+ CD4 and CD8 T cells at 24 h and 72 h post IL-12mRNA injection. (C) UMAPs for CD4 and CD8 T cells for each treatment group depicting levels of IFNγ gene expression. (D) SBRT/IL-12mRNA efficacy in IFNγ KO vs. wild-type mice. (E) Proliferation and frequency of CD4 and CD8 T cells in IFNγ KO vs. wild-type mice following SBRT/IL-12mRNA treatment. n = 5/group from two independent experiments in (A). n = 10/group from two independent experiments. n = 5/group in (D) and (E). One-way ANOVA followed by Tukey’s test. Significance against untreated and between treatments are reported. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. Lines represent mean ± SEM for (A), (B), (D) and (E).