Reprogramming of TLR7 signaling enhances antitumor NK and cytotoxic T cell responses

Abstract

Toll-like receptor (TLR) 7 agonists are effective in topical application for the immunotherapy of skin cancers, but their performance for the systemic treatment of solid tumors is limited by the development of TLR tolerance. In this study, we describe a novel strategy to overcome TLR tolerance and enhance TLR7-dependent antitumor immune responses through reprogramming of TLR signaling pathways. The sensitivity of TLR7 signaling in dendritic cells (DC) was increased by prior stimulation with the dsRNA poly(I:C) that mimics virally induced immune activation. Timing of the stimulations was important, as sequential stimulation with poly(I:C) and the TLR7 agonist R848 interspaced by 24 h induced higher MAPK and NFkB signaling in DC than the simultaneous application of the same ligands. DC activated by sequential poly(I:C)/R848 stimulation efficiently induced Th1 differentiation and primed NK-cell and cytotoxic T-cell responses. We have developed a treatment regimen taking advantage of TLR7 reprogram-ming that cured over 80% of large immunogenic tumors in mice by the action of NK cells and cytotoxic T cells. These results have direct implications for the use of these clinically established ligands in the immunotherapy of cancer.

Keywords: Cancer immunotherapy; RIG-I-like receptors agonists; Toll-like receptors; dendritic cells; poly(I:C).

Figure 1.

Sequential PRR stimulation with poly(I:C) and R848 enhances activation of immune cells. (A) Cytokine levels in supernatants of bone marrow cells or BMDC stimulated with combinations of poly(I:C) and R848 at a 24 h interval were measured by ELISA 18 h after the second stimulation. Mean + SEM of triplicates are shown. (B) Expression of CD40, CD69, and MHC-I on CD11c⁺ BMDC stimulated as in (A). Mean fluorescence intensities (MFI) + SEM of quadruples are shown. Data are representative of at least three independent experiments.

Figure 2.

Sequential PRR stimulation of BMDC enhances cytokine mRNA expression and NFκB and MAP kinase signaling. (A) Il-6, Il-12a, and Il-12b mRNA expression 4 and 8 h after the second stimulation in BMDC stimulated as in Fig. 1. Mean + SEM of four to eight independent experiments are shown. (B) Immunoblot analysis of p65 and MAPK in BMDC stimulated with combinations of poly(I:C) and R848, at 0, 20 and 40 min, after the second stimulation. Individual blots are indicated by rectangles and are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, and ****p < 0.0001, compared to the sequential PRR stimulation; one-way ANOVA, Dunetts post-test.

Figure 3.

Sequential PRR stimulation of BMDC increases activation of effector T cells and NK cells. (A) Expression of CD69 on CD4⁺ and CD8⁺ splenocytes added to sequentially stimulated BMDC 1.5 h after the second stimulation and cocultured for 24 h. Representative histograms and MFI + SEM of quadruples are shown. (B) Expression of CD69 on purified NK cells co-cultured with BMDC as in (A). (C) IFNγ levels in supernatants of splenocytes and purified NK cells co-cultured with BMDC as in (A) and (B). (D) Experimental protocol for the stimulation of BMDC and co-culture with splenocytes or purified NK cells. MFI + SEM of quadruples are shown. Data are representative of two independent experiments.

Figure 4.

Sequential application of PRR ligands enhances NK-cell activation and IFNγ production in vivo. (A) Expression of CD69 on NK1.1⁺/CD3⁻ splenic NK cells isolated from mice 4 h after two applications of poly(I:C) (100 µg i.p.), R848 (25 µg s.c.) or a combination of both at a 24 h interval. MFI + SEM of four to five mice/group are shown. (B) Percentage of IFNγ⁺ splenic NK cells from mice treated as in (A). Mean + SEM of 9 to 10 mice/group are shown. (C) Serum IFNγ levels in mice treated as in (A), 4 or 8 h after the second injection. Mean + SEM of four to five mice are shown. *p < 0.05 and ***p < 0.001 compared to the sequential PRR stimulation; one-way ANOVA, Dunetts post-test.

Figure 5.

Sequential PRR activation induces higher cytotoxic T-cell responses and Th1 cell differentiation. (A) Percentage of IFNγ⁺ CD8⁺ T cells and secreted IFNγ in 3d co-cultures of OT-I cells with antigen-pulsed BMDC stimulated as in Fig. 3A. Representative histograms and mean percentage + SEM of quintuplicates are shown. (B) Total numbers of IFNγ, IL-4, and IL-17-expressing CD4⁺ T cells and secreted IFNγ in 4-d co-cultures of OT-II T cells with BMDC stimulated as in (A). Mean + SEM of sextuplicates are shown. Data are representative of at least three independent experiments. (C) Specific lysis of target cells in mice immunized s.c. with different combinations of poly(I:C) and R848 together with OVA. Representative histograms show the percentage of antigen-pulsed target (CFSE^lo) and control (CFSE^high) cells. Specific lysis for individual mice (n = 3–6) is shown. ***p < 0.001 and ****p < 0.0001 compared to the sequential PRR stimulation; one-way ANOVA, Dunnetts post-test. (D) Frequencies of OVA-specific CD8⁺ T cells in individual mice (n = 3 to 4) immunized as in (C). *p ≤ 0.05 compared to the sequential PRR stimulation; one-way ANOVA, Dunnetts post-test.

Figure 6.

Sequential injections of TLR3/MDA5 and TLR7 ligands are more efficient than single agents for the treatment of established tumors. Growth of CT26 tumors in mice treated with combinations of poly(I:C) and R848 on two consecutive days, repeated twice (arrows). (A) Tumor size is shown for individual mice. (B) Mean + SEM of tumor size (n = 5) are shown. Data are representative of three independent experiments. (C) Tumor size for mice treated as in (A) combined with immune cell depletion. Mean + SEM of tumor size (n = 5) are shown. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, compared to the sequential PRR stimulation; two-way ANOVA, Bonferroni post-test.