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To enhance cancer immunotherapy, immune cells that have been weakened or inactivated in cancer patients need to be reinvigorated to respond specifically and dynamically to cancer cells that are alive, mutated and adapted in a complex and dynamic way1. Because the activation of resting antigen-presenting cells (APCs) is a crucial step in the initiation of adaptive antitumour immunity, nanotechnology-based material design with a ‘pathogen-mimicking’ strategy has utilized key features of the size, shape and surface-molecule organization of pathogens to control innate immunity in the context of cancer immunotherapy2,3,4,5,6,7,8,9,10,11,12,13,14. However, the maturation of APCs is regulated not only by the nature of the APC maturation stimuli but also by the duration, combination and timing of stimulation, which collectively affect subsequent T-cell responses6,15,16. Furthermore, although conventional innate immune stimuli, such as Toll-like receptor (TLR) agonists, contribute to immune activation, they ultimately induce exhausted APCs and T cells, resulting in suboptimal cancer immunotherapy; hence, the strategy of tailoring innate immunity is particularly important for achieving effective cancer immunotherapy17,18,19,20,21. In particular, how the kinetics of immune modulation of APCs can be controlled to achieve robust innate and adaptive immune responses without inducing exhausted immune cells remains unknown. Taking cues from pathogens that trigger combinatorial TLR stimulation, we sought to integrate multiple TLR stimuli within a defined ‘temporal window’ via physicochemical manipulation of TLR stimuli to induce effector/non-exhausted dendritic cells (DCs) that prolonged the secretion of interleukin (IL)-12(p70), which has been known to induce not only the effector functions of CD8+ T and natural killer (NK) cells but also the protection of CD8+ T cells from exhaustion22,23,24,25,26,27.

Kinetics of the immune responses induced by t-TLR7/8a

To address these issues, we designed and developed a nanoliposome-based K-nanoadjuvant that can simultaneously deliver TLR3a and timely activating TLR7/8 agonist (t-TLR7/8a) into APCs, thus achieving the optimal order, duration and time window of TLR activation in a synchronous way (Fig. 1a,b). We hypothesized that the sequential combination of stimuli in K-nanoadjuvant, which uses TLR3a-mediated immune activation as the first wave of stimulation and t-TLR7/8a activation as the second wave of stimulation within the optimal time window, would be more effective than a simultaneous combination of stimuli for the induction of the effector/non-exhausted DCs that could programme high-magnitude and persistent secretion of IL-12 (Fig. 1b). We also reasoned that the tailored immune activation initiated by K-nanoadjuvant could generate effector/non-exhausted CD8+ T cells and activated NK cells, leading to potent antitumour immunity as a monotherapy or combination therapy with anti-PD-L1 or liposomes (doxorubicin) (Fig. 1b).

Fig. 1: Design and characterization of t-TLR7/8a and K-nanoadjuvant and their time-dependent immunomodulatory activity.
figure 1

a, The mechanism of GILT-responsive recovery of cholesterol-conjugated TLR7/8a activity. b, Schematic of the strategy for K-nanoadjuvant-enabled synchronous and dynamic integration of innate immune stimuli for enhanced cancer immunotherapy. c,d, Representative cryo-transmission electron microscopy image (c) and particle size distribution of t-TLR7/8a measured by dynamic light scattering (d). e, Representative fluorescence imaging of draining lymph nodes (dLNs) (left) and sectioned dLNs (right) after administration of PBS, Cy5 (amine) or liposomes (Cy5-cholesterol). Scale bars, 400 μm. f, Left: representative fluorescence imaging of endocytosis-dependent uptake of liposomes (FITC–cholesterol) for 4 h in BMDCs preincubated with or without Dynasore (40 μM) for 1 h. Cell membrane, wheat germ agglutinin Texas Red (red); nuclei, Hoechst 33342 (blue). Right: endocytosis-dependent IL-12(p70) production in BMDCs (n = 3). g, GILT-dependent R848 recovery kinetics of t-TLR7/8a (n = 3). h, GILT expression in wild-type (siControl-treated) or GILT knockdown (siGILT-treated) RAW 264.7 cells measured by reverse-transcription polymerase chain reaction. i, TNF-α production in GILT-knockdown RAW 264.7 cells after t-TLR7/8a (3.18 μM) incubation for 12 h (n = 4). j, Left: IL-12(p70) concentration measured; right: IL-12 mRNA expression in BMDCs at the indicated times after treatment with R848 or t-TLR7/8a (n = 2). k, IL-12(p70) production in BMDCs at the indicated times after treatment with R848 or t-TLR7/8a for 12 h and washing (n = 3). l, IFN-γ and IL-4 in the cell culture supernatant of naive CD4+ T cells co-cultured for 3, 5 or 7 days with BMDCs treated with R848 or t-TLR7/8a for 12 h (n = 2). m, In vitro PD-1 expression in CD8+ T cells after treatment with R848 or t-TLR7/8a (3.18 μM) in co-culture of BMDCs and exhausted CD8+ T cells for 24 h (n = 3). All data are presented as the mean ± s.d. Statistical significance was evaluated by two-way ANOVA with Sidak’s multiple-comparisons test in f,g,i and by one-way ANOVA with Tukey’s multiple-comparison test in m. P values: NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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To develop a spatiotemporally activated t-TLR7/8a, we designed a novel TLR7/8a based on transient chemical inactivation of the putative active site in TLR7/8a (C4 amine, an amine moiety that is known to interact with TLR7/8 via hydrogen bonding)28 and recovery of activity via a chemical linker that was cleavable by γ-interferon-inducible lysosomal thiol reductase (GILT) within the endolysosome microenvironment (Fig. 1a and Supplementary Figs. 1–3). This study reports the use of GILT, which is the only enzyme known to catalyse disulfide bond reduction of exogeneous antigen in the endocytic pathway29,30, as the trigger for a quiescent immunostimulant for dynamic immunomodulation of APCs.

Cholesterol was chosen as a transient shielding blocker because of its capacity for immobilization in the lipid membrane, thus enabling facile incorporation of cholesterol–TLR7/8a conjugates into nanoliposomes with a high encapsulation efficacy (Supplementary Table 1)31. t-TLR7/8a exhibited a uniform size distribution at approximately 100 nm, which could promote direct drainage to and retention in the lymph nodes (LNs) after subcutaneous administration (Fig. 1c–e and Supplementary Figs. 4 and 5)32. Endocytosis-mediated internalization of t-TLR7/8a into APCs was required for the activation of t-TLR7/8a, as shown by fluorescence microscopy images of cellular uptake and cytokine production by bone marrow-derived DCs (BMDCs) treated with t-TLR7/8a with Dynasore (an endocytosis inhibitor) (Fig. 1f). We also observed that active TLR7/8a was generated during incubation with GILT-containing medium and cysteine mimicking the endolysosomal environment (Fig. 1g and Supplementary Fig. 6)33. Treatment with t-TLR7/8a induced tumour necrosis factor-α (TNF-α) production in wild-type RAW 264.7 cells, whereas TNF-α production was abrogated in GILT-knockdown RAW 264.7 cells (Fig. 1h,i, Supplementary Fig. 7 and Supplementary Table 2). Human-monocyte-derived dendritic cells (moDCs) expressed high levels of GILT and t-TLR7/8a induced IL-12(p70) release in human moDCs (Supplementary Figs. 8 and 9). We also confirmed that t-TLR7/8a activated mouse TLR7/8 using RAW-Blue mouse TLR-reporter cells (Supplementary Fig. 10). t-TLR7/8a-treated BMDCs started to secrete IL-12(p70), a polarizing cytokine that promotes T helper 1 (Th1) cell differentiation and mediates effective adaptive immune responses, after 4 h of treatment and exhibited sustained cytokine production over 24–32 h, while R848-treated BMDCs rapidly secreted IL-12(p70) followed by a decrease in secretion (Fig. 1j). When we measured residual cytokine production after washing BMDCs after 12 h of stimulation with R848 or t-TLR7/8a, the t-TLR7/8a-treated BMDCs continued to produce IL-12(p70) for the next 12–16 h, whereas the R848-treated BMDCs no longer produced IL-12(p70) (Fig. 1k). The levels of co-stimulatory markers (CD80 and CD86) in R848-treated BMDCs increased rapidly within 6 h, while t-TLR7/8a-treated BMDCs gradually increased their co-stimulatory marker expression over the course of 36 h (Supplementary Fig. 11). The secretion of interferon-γ (IFN-γ) from CD4+ T cells co-cultured with non-exhausted DCs (treated with t-TLR7/8a) was sustained and gradually increased over 7 days, while that from CD4+ T cells co-cultured with exhausted DCs (treated with R848) rapidly increased and then decreased with time (Fig. 1l). In contrast, the secretion of IL-4 from CD4+ T cells co-cultured with exhausted DCs (treated with R848) decreased with time and was higher than that from CD4+ T cells co-cultured with non-exhausted DCs (treated with t-TLR7/8a) (Fig. 1l). When R848 or t-TLR7/8a was used to treat co-cultures of BMDCs and exhausted OT-1 CD8+ T cells for 24 h in vitro, a further increase in PD-1 expression in OT-1 CD8+ T cells during TLR7/8a stimulation was efficiently inhibited in the t-TLR7/8a-treated group where the secretion of IL-12(p70) was sustained and durable, compared to the free R848-treated group in which the secretion of IL-12(p70) occurred in bursts and was transient (Fig. 1m and Supplementary Fig. 12). The experimental results suggested that the kinetic activation of DCs by t-TLR7/8a and sustained and durable production of IL-12(p70) could be assumed to be more effective than the bursts and rapid production of IL-12(p70) in DCs stimulated by conventional free R848 in supporting Th1 polarization of CD4+ T cells and inhibiting a further increase in PD-1 expression in CD8+ T cells during TLR7/8a stimulation (Fig. 1m and Supplementary Fig. 12)19,24,25,26,27. The kinetic modulation properties of t-TLR7/8a observed in BMDCs were also observed in RAW 264.7 cells (Supplementary Fig. 13).To prime antigen-specific Th1-type CD4+ T cells and CD8+ T cells, it is essential for DCs to efficiently home to lymphoid tissues and continuously secrete a large amount of IL-12(p70) after CD40–CD40L interactions (as an ‘immune amplifier’) in the LNs34,35 (Extended Data Fig. 1a,b). IFN-γ (an ‘immune conditioner’) and TLR7/8a treatment increased IL-12(p70) production in both BMDCs and human moDCs by up to 5- to 15-fold compared with TLR7/8a treatment alone (Extended Data Fig. 1a–c and Supplementary Fig. 14)36,37. After 12 h of treatment, the IFN-γ/R848-treated DCs did not secrete IL-12(p70) or respond to CD40L, while the IFN-γ/t-TLR7/8a-treated DCs secreted residual IL-12(p70) for 8 h and were responsive to CD40L (Extended Data Fig. 1d,e)38. Although the incorporation of prostaglandin E2 (PGE2) into the DC maturation protocol (IFN-γ/R848/PGE2-treated DCs) resulted in a migratory capacity higher than observed for IFN-γ/R848-treated DCs39,40, PGE2 treatment abrogated IL-12(p70) production and increased the production of immunosuppressive IL-10 (Extended Data Fig. 1f)41. In contrast, IFN-γ/t-TLR7/8a-treated DCs exhibited a strong chemotactic ability with high IL-12(p70) production (Extended Data Fig. 1f,g). Compared with an IFN-γ/R848-treated DC vaccine, subcutaneous injection of the IFN-γ/t-TLR7/8a-treated DC vaccine generated significantly enhanced effector memory and increased the percentage of IFN-γ-producing CD8+ T cells in splenocytes (Extended Data Fig. 1h and Supplementary Fig. 15). The IFN-γ/t-TLR7/8a-treated DC vaccine also efficiently inhibited tumour growth in both prophylactic and therapeutic settings in a B16OVA tumour model (Extended Data Fig. 1i).

Combinatorial codes of t-TLR7/8a and TLR3a or TLR4a

Through systematic in vitro experiments, we observed that the synergistic effect of dual TLR agonists on IL-12(p70) production was maximized when R848 was administered after LPS (TLR4a) or poly(I:C) (TLR3a) treatment of BMDCs within a 4 h ‘temporal window’, while R848 treatment before LPS or poly(I:C) treatment showed no synergistic effect (Fig. 2a,b). However, these mechanistic studies were limited to in vitro conditions because asynchronous bolus injection of TLR agonists in vivo fail to achieve co-delivery of different TLR agonists (Supplementary Fig. 16)15. Importantly, as the reactivation of t-TLR7/8a requires 4 h (Fig. 1j), this ideal ‘temporal window’ allowed us to achieve potent IL-12(p70) production by BMDCs via synchronous treatment with t-TLR7/8a and LPS or poly(I:C) (Fig. 2a,b). Co-treatment with t-TLR7/8a and LPS or poly(I:C) also led to the upregulation of co-stimulatory molecules as well as the synergistic production of various cytokines (IL-1α, IL-1β, TNF-α and IL-6) and chemokines (CCL2 and CCL5) (Fig. 2c,d and Supplementary Figs. 17 and 18). Moreover, we also observed that co-treatment with t-TLR7/8a and LPS or poly(I:C) prevented DC exhaustion, as demonstrated by the prolonged and continuous secretion of proinflammatory cytokines (IL-12(p70), IL-6 and TNF-α) by BMDCs (Fig. 2e,f). Finally, we observed that synergistic IL-12(p70) production of t-TLR7/8a and poly(I:C) was abrogated in IFNAR1−/− BMDCs (Fig. 2g) or BMDCs treated with a TBK1 inhibitor (Supplementary Fig. 19), consistent with the results of previous reports42,43.

Fig. 2: Combinatorial codes of t-TLR7/8a and TLR3a or TLR4a and characterization of K-nanoadjuvant.
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a,b, In vitro production of IL-12(p70) by BMDCs after the combination of TLR7/8a and TLR4a (n = 3) (a) or TLR3a (n = 3 for asynchronous treatment; n = 4 for synchronous treatment) (b) (R848, 3.18 μM; t-TLR7/8a, 3.18 μM; LPS, 2 ng ml−1; poly(I:C), 2 μg ml−1). Left: TLR stimuli (R848 plus LPS (a) and R848 plus poly(I:C) (b)) were added simultaneously (time interval, 0 h) or sequentially (dashed lines) at various time intervals (horizontal axes). ‘% of control’ represents the relative percentage value of IL-12(p70) for the sequential stimulation treatment at specific time intervals compared with that for the simultaneous stimulation treatment. Right: The concentration of IL-12(p70) after synchronous treatment with the indicated samples. c–f, CD80 and CD86 on CD11c+ BMDCs at 36 h (c,d) (n = 4), and cytokine secretion from BMDCs at 24 h and 36 h (e,f) after treatment with R848 or t-TLR7/8a with LPS (c,e) or poly(I:C) (d,f) (n = 5 for TNF-α; n = 4 for the others). g, IL-12(p70) production in IFNAR1−/− BMDCs (n = 3). h, Size and zeta potential of K-nanoadjuvant according to the mass ratio of t-TLR7/8a and poly(I:C) (n = 3). i, Gel retardation assays. j, Representative fluorescence image of BMDCs presenting intracellular stability of the FITC–cholesterol-loaded nanoliposomes. Lysosomes: Lysotracker deep red (brown), nuclei: Hoechst 33342 (blue). Scale bars, 10 μm. k,l, The in vivo kinetics of innate immune cell activation in the dLN: the total single-cell numbers of macrophages (CD11b+) and DCs (CD11c+) (k), and IL-12(p70) production at the indicated times (l) after injection of R848 + poly(I:C) or K-nanoadjuvant (R848, 79.5 μmol; t-TLR7/8a, 79.5 μmol; poly(I:C), 6.25 μg) with SIINFEKL (15 μg) (n = 3). All data are presented as the mean ± s.d. Statistical significance was evaluated by one-way ANOVA with Tukey’s multiple-comparison test in a–d,g and by two-way ANOVA with Sidak’s multiple-comparisons test in k and l. P values: NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Inspired by this combinatorial code, we developed a K-nanoadjuvant composed of t-TLR7/8a and poly(I:C) as a nanoliposome formulation, and the ratio of the two components in K-nanoadjuvant was optimized by measuring the strength of the electrostatic interaction for the stable conformation and the non-aggregating nanoparticulate formulation (Fig. 2h,i and Supplementary Fig. 20). Stable co-localization of the two components after cellular uptake into BMDCs (Fig. 2j) or RAW 264.7 cells was verified (Supplementary Fig. 21). A single injection of a vaccine formulation composed of K-nanoadjuvant and the SIINFEKL peptide antigen led to a highly increased number of innate immune cells (CD11b+ and CD11c+ cells) for over 14 days and IL-12(p70) production for over 72 h in the draining LNs (Fig. 2k,l). Mice vaccinated three times with K-nanoadjuvant exhibited robust numbers of central memory and effector memory CD8+ T cells in the spleen, compared with other groups (Supplementary Fig. 22).

Immune responses to K-nanoadjuvant in TDLNs and TME

We investigated whether K-nanoadjuvant could modulate immune responses within the tumour microenvironment (TME) and the tumour-draining lymph nodes (TDLNs) in favour of T- and NK-cell-based antitumour immunity in the B16OVA melanoma model (Fig. 3a and Supplementary Figs. 23 and 24). K-nanoadjuvant strongly supported maturation of DCs (Supplementary Fig. 25) and generated a high local concentration of IL-12(p70) (Fig. 3b) and IFN-γ (Fig. 3c) in the TME. As high IL-12 expression in the TME is related to prolonged survival, according to the survival curves of cutaneous melanoma patients, the high concentration of IL-12 induced by K-nanoadjuvant could be a crucial factor for good clinical outcomes (Supplementary Fig. 26). Compared with a soluble admixture of R848 and poly(I:C), immunization of mice with K-nanoadjuvant induced significantly enhanced T cell infiltration into the TME (Supplementary Fig. 27) and increased the frequency of tumour-infiltrating antigen-specific CD8+ T cells producing cytokines (IFN-γ, Granzyme B and TNF-α) (Fig. 3d and Supplementary Fig. 28). Furthermore, the expression of inhibitory molecules such as PD-1 and LAG-3 on CD8+ T cells in the TME was decreased in K-nanoadjuvant-treated mice (Fig. 3e,f). The frequency of activated NK cells producing IFN-γ, Granzyme B and TNF-α were also significantly increased in the K-nanoadjuvant-treated group (Fig. 3g and Supplementary Fig. 29a,b). We also observed high expression of CD69 on tumour-infiltrating NK and NKT cells (Supplementary Fig. 29c,d). Interestingly, K-nanoadjuvant also decreased the infiltration of immunosuppressive myeloid-derived suppressor cells (MDSCs) (Fig. 3h)44. We also observed that K-nanoadjuvant could modulate immune responses within the TDLNs in favour of T- and NK-cell-based antitumour immunity. K-nanoadjuvant generated mature DCs (CD80+ and CD86+) in the TDLNs (Supplementary Fig. 30). K-nanoadjuvant also elicited higher frequencies of antigen-specific CD8+ T cells producing cytokines (IFN-γ, Granzyme B and TNF-α) and expressing activation markers (4-1BB, CD28 and CD69) (Supplementary Fig. 31), activated NK cells producing IFN-γ and TNF-α, and NK and NKT cells with higher CD69 expression (Supplementary Fig. 32) in the TDLNs than did the admixture of R848 and poly(I:C). Neutralization of IL-12 with anti-IL-12 significantly decreased the antitumour efficacy of K-nanoadjuvant (Fig. 3i,j), suggesting a role of IL-12 as the crucial linker between K-nanoadjuvant-mediated innate immune stimulation and adaptive antitumour immunity45,46. The abrogated effects of K-nanoadjuvant in the anti-IL-12-treated group were related to the decreases in the frequency of antigen-specific CD8+ T cells and activated NK cells in both the TDLNs (Fig. 3k–m and Supplementary Figs. 33 and 34) and the TME (Fig. 3n,p and Supplementary Figs. 35–37) and the increase in PD-1 expression in CD8+ T cells in the TME (Fig. 3o).

Fig. 3: In vivo antitumour immune response induced by K-nanoadjuvant in the B16OVA model and its dependence on IL-12.
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a, Schedule of immune cell analysis in the TME (R848, 79.5 μmol; t-TLR7/8a, 79.5 μmol; poly(I:C), 6.25 μg; SIINFEKL, 15 μg). The flow cytometry gating strategy is shown in Supplementary Figs. 23 and 24. b,c, Concentrations of IL-12(p70) (n = 3 for G1, G2 and G5; n = 4 for G3, G4 and G6) (b) and IFN-γ (n = 5) (c) in the TME. d–h, Representative flow cytometry dot plots and percentage of IFN-γ+ cells (d) in CD8+ T cells (CD3+CD8+) after SIINFEKL restimulation and incubation with GolgiPlug in the presence of IL-2 for 12 h (n = 5); PD-1+ (e) and LAG-3+ (f) cells in CD8+ T cells (CD3+CD8+) after SIINFEKL restimulation in the presence of IL-2 for 12 h (n = 5); IFN-γ+ cells (g) in NK cells (CD3−NK1.1+) after incubation with cell activation cocktail (with brefeldin A), a mixture of optimized concentrations of PMA, ionomycin and brefeldin A for 4 h (n = 5); and myeloid-derived suppressor cells (h) (MDSCs, CD11b+Gr-1+) (n = 5 for G3 and G5; n = 6 for the others). i, Schedule of vaccination, IL-12 depletion and collection of TDLN and tumour tissues (anti-IL-12, 200 μg). j, Average tumour growth curves (n = 6). k–m, Representative flow cytometry dot plots and percentage of IFN-γ+ (k) and GrB+ (l) cells in CD8+ T cells (CD3+CD8+) (n = 5), and IFN-γ+ cells (m) in NK cells (CD3−NK1.1+) (n = 4 for G3; n = 5 for the others). n–p, Representative flow cytometry dot plots and percentage of IFN-γ+ cells (n) in CD8+ T cells (CD3+CD8+) (n = 5), PD-1+ cells (o) in CD8+ T cells (CD3+CD8+) (n = 5), and IFN-γ+ cells (p) in NK cells (CD3−NK1.1+) (n = 5). All data are presented as the mean ± s.d. Statistical significance was evaluated by one-way ANOVA with Tukey’s multiple-comparison test in b–h and k–p and by an unpaired two-tailed t-test in j. P values: NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001).

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To examine whether K-nanoadjuvant is effective against a wide spectrum of cancer types, we also investigated K-nanoadjuvant-enabled modulation of antitumour immune responses within the TME and TDLNs in a TC-1 tumour model (Fig. 4a and Supplementary Figs. 38 and 39). Mice immunized with K-nanoadjuvant exhibited significantly enhanced T-cell and NK-cell infiltration into the TME (Supplementary Fig. 40), and a high local IFN-γ concentration was generated. (Supplementary Fig. 41). In the TME of K-nanoadjuvant-treated mice, the frequency of antigen-specific CD8+ T cells producing cytokines (IFN-γ, Granzyme B and TNF-α) was increased (Fig. 4b,c and Supplementary Fig. 42), and the expression of inhibitory molecules such as PD-1 and TIM-3 on CD8+ T cells was decreased (Fig. 4d,e). We also observed that the frequency of activated NK cells producing IFN-γ, Granzyme B and TNF-α were significantly increased (Fig. 4f,g) and the expression of CD69 on tumour-infiltrating NK and NKT cells was high (Supplementary Fig. 43) in the K-nanoadjuvant-treated group. Compared with the admixture of R848 and poly(I:C), K-nanoadjuvant also enhanced T- and NK-cell-based antitumour immune responses in TDLNs, with higher frequencies of antigen-specific CD8+ T cells producing cytokines (IFN-γ, Granzyme B and TNF-α) and expressing activation markers (4-1BB, CD28 and CD69) (Fig. 4h,i and Supplementary Figs. 44 and 45), higher frequencies of activated NK cells producing IFN-γ, Granzyme B and TNF-α (Fig. 4j,k), higher CD69 expression in NK and NKT cells (Supplementary Fig. 46) and higher local concentrations of IFN-γ (Supplementary Fig. 47).

Fig. 4: In vivo antitumour immune responses by K-nanoadjuvant in a TC-1 model.
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a, Schedule of immune cell analysis in the TME and TDLN in the TC-1 model. Tumour and TDLN tissues were collected 3 days after the last vaccination (R848, 25 μg, 79.5 μmol; t-TLR7/8a, 72.1 μg, 79.5 μmol; poly(I:C), 6.25 μg; E7-long peptide, 10 μg). The flow cytometry gating strategy is shown in Supplementary Figs. 38 and 39. b–g, Immune cell analysis in the TME. Representative flow cytometry dot plots and percentage of IFN-γ+ (b) (n = 5) and GrB+ (c) cells in CD8+ T cells (CD3+CD8+) (n = 5 for G1, G3 and G5; n = 6 for G2; n = 7 for G4 and G6) after E7-long peptide (10 μg ml−1) restimulation and incubation with GolgiPlug in the presence of IL-2 (30 ng ml−1) for 12 h, and PD-1+ (d) and TIM-3+ (e) cells in CD8+ T cells (CD3+CD8+) after E7-long peptide restimulation in the presence of IL-2 for 12 h (n = 4 for G4; n = 5 for the others). Representative flow cytometry dot plots and percentage of IFN-γ+ (f) and GrB+ (g) cells in NK cells (CD3−NK1.1+) after incubating with cell activation cocktail (with brefeldin A), mixture of optimized concentrations of PMA, ionomycin and brefeldin A for 4 h (n = 5). h–k, Immune cell analysis in the TDLN. Representative flow cytometry dot plots and percentage of IFN-γ+ (h) (n = 5) and GrB+ (i) (n = 6 for G6, the rest n = 5) cells in CD8+ T cells (CD3+CD8+) after E7-long peptide restimulation and incubation with GolgiPlug in the presence of IL-2 for 12 h. Representative flow cytometry dot plots and percentage of IFN-γ+ (j) and GrB+ (k) cells in NK cells (CD3−NK1.1+) after incubating with cell activation cocktail (with brefeldin A) for 4 h (n = 4). All data are presented as the mean ± s.d. Statistical significance was evaluated by one-way ANOVA with Tukey’s multiple-comparison test in b–k. P values: NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Antitumour therapeutic efficacy of K-nanoadjuvant

As shown by haematoxylin and eosin (H&E) staining, we did not observe any noticeable signs of tissue damage or toxicity in the liver, lung, spleen or kidney after multiple K-nanoadjuvant treatments, while an increase in the number of inflammatory cells (leading to vessel wall thickening and alveolar damage) in the lungs (red arrow) was observed in the R848 + poly(I:C)-treated group (Fig. 5a and Supplementary Fig. 48). Subcutaneous vaccination with K-nanoadjuvant was effective in suppressing tumour growth in the TC-1 subcutaneous tumour model and enhancing mouse survival (Fig. 5b). When we administered K-nanoadjuvant locally near the primary tumour after inoculation of a secondary tumour contralateral to the primary tumour, the growth of the secondary and primary tumours was drastically inhibited (Fig. 5c). In a spontaneous 4T1 lung metastasis model, we observed that metastatic tumour nodules developed in the lungs in the PBS-treated group, while no noticeable signs of metastasis were detected in the lungs in the K-nanoadjuvant-treated group (Fig. 5d and Supplementary Fig. 49). In a TC-1 lung orthotopic tumour model, tumour cells directly inoculated in the lungs grew aggressively in the non-treated group, while the K-nanoadjuvant-treated group showed near complete tumour regression, as verified by India ink staining and H&E staining of lung tissues (Fig. 5e,f). Taken together, these experimental results suggested that local injection of K-nanoadjuvant generated systemic antitumour immune responses. Many clinical studies have shown that vaccines can enhance the antitumour effects of chemotherapy or immune checkpoint blockade therapy (ICBT)47. Local treatment with K-nanoadjuvant combined with systemically injected liposomes (doxorubicin) led to tumour regression and prolonged survival in the TC-1 cancer model (Fig. 6a). Compared to no treatment, K-nanoadjuvant treatment resulted in the increased activity of cytotoxic lymphocytes and an increased concentration of IFN-γ (as shown in Fig. 4), which led to increased expression of PD-L1 (in CD45− cells) in the TME (Fig. 6b). Upon PD-L1 upregulation after K-nanoadjuvant treatment, combination therapy with K-nanoadjuvant and anti-PD-L1 exhibited enhanced antitumour efficacy, leading to complete tumour regression in three out of eight mice and prolonged survival over the 120-day experimental period (Fig. 6c,d). The strong synergistic effect achieved with K-nanoadjuvant and anti-PD-L1 significantly increased the size of the central and effector memory T cell populations in the TDLNs and spleen (Fig. 6e,f). When we inoculated TC-1 tumour cells contralateral to the primary tumour site at 21 days after primary tumour inoculation and treatment, tumour growth after rechallenging was completely inhibited in K-nanoadjuvant and anti-PD-L1-treated mice, while continuous tumour growth was observed in control mice (Fig. 6g,h).

Fig. 5: Histological analysis and antitumour immunity of K-nanoadjuvant.
figure 5

a, Representative H&E staining images of major organs. Scale bars, 20 μm. The indicated samples were subcutaneously injected four times at 3-day intervals (R848, 159 μmol; t-TLR7/8a, 159 μmol; poly(I:C), 12.5 μg). b–f, The antitumour efficacy of the indicated samples (R848, 79.5 μmol; t-TLR7/8a, 79.5 μmol; poly(I:C), 6.25 μg; E7-long peptide, 10 μg or 4T1 tumour lysate, 10 μg) was assessed in TC-1 subcutaneous (b), TC-1 primary and secondary (c), lung metastasis in subcutaneous 4T1 (d) and lung orthotopic TC-1 (e,f) tumour models. b, Average tumour growth curve and the percentage of mouse survival after vaccination three times at 3-day intervals (n = 6 for K-nanoadjuvant; n = 7 for the others). c, Left: primary and secondary tumour growth curves (n = 9 for PBS, n = 7 for K-nanoadjuvant); right: representative photographs of mice 22 days after primary or secondary tumour inoculation after four vaccinations with the indicated samples. Scale bars, 10 cm. d, Lung metastasis in subcutaneous 4T1 tumour model. Left: representative image of the lungs 28 days after 4T1 tumour inoculation; right: the number of lung metastatic nodules (n = 3). Mice were vaccinated four times with PBS or K-nanoadjuvant with 4T1 tumour lysate. e, Left: schedule and survival curves (n = 7); right: representative image of the lungs 14 or 31 days after TC-1 tumour inoculation. Mice were vaccinated four times with the indicated samples. f, Representative images of H&E-stained lung tissue sections. Scale bars, 20 μm. The red dotted line indicates the tumour. All data are presented as the mean ± s.d. Statistical significance was evaluated by an unpaired two-tailed t-test in b–d. Survival P values were calculated by the log-rank (Mantel–Cox) test in b,e. P values: NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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Fig. 6: Synergy of K-nanoadjuvant with chemotherapy or ICBT.
figure 6

a, Left: schedule and average tumour growth (n = 5 for G3; n = 6 for the others); right: survival curves (n = 6 for G1 and G4; n = 7 for G2 and G4) for K-nanoadjuvant (t-TLR7/8a, 72.1 μg, 79.5 μmol; poly(I:C), 6.25 μg) and liposome (DOX) (DOX, 80 μg) therapy with E7-long peptide (10 μg) in the TC-1 tumour model. b–d, Synergy of K-nanoadjuvant and E7-long peptide (10 μg) with anti-PD-L1 (100 μg): PD-L1 expression in the TME of TC-1 tumour-bearing mice (n = 6) (b); schedule and survival curves (c) and average tumour growth curves (d) for K-nanoadjuvant + anti-PD-L1 (n = 8). e–h, Tumour rechallenge study with K-nanoadjuvant + anti-PD-L1 treatment: schedule for injection (e), representative flow cytometry plots and percentages of central memory (CD44+CD62L+) and effector memory (CD44+ CD62L−) CD8+ T cells (CD3+CD8+) in the TDLN and spleen (n = 5) (f), average tumour growth curves after tumour rechallenge (n = 5 for PBS and n = 4 for K-nanoadjuvant + anti-PD-L1) (g), and survival curves of mice after tumour rechallenge (n = 5 for PBS; n = 4 for K-nanoadjuvant + anti-PD-L1) (h). All data are presented as the mean ± s.d. Statistical significance was evaluated by an unpaired two-tailed t-test. Survival P values were calculated by the log-rank (Mantel–Cox) test in c,h. P values: NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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Conclusions

We report the design and application of K-nanoadjuvant, which can dynamically integrate two waves of innate immune stimuli to achieve effective antitumour immunity while avoiding immune cell exhaustion. The combinatorial code of K-nanoadjuvant for signal integration of t-TLR7/8a, processed by GILT, and TLR3a or TLR4a can be optimized in terms of the order, duration and time window, resulting in the generation of effector/non-exhausted DCs that induce enhanced and durable secretion of IL-12 and high migration capability. We further demonstrate that mice immunized with K-nanoadjuvant exhibit substantially enhanced antigen-specific CD8+ T cells producing cytokines (IFN-γ, Granzyme B and TNF-α) and expressing activation markers (4-1BB, CD28 and CD69), and activated NK cells producing IFN-γ and Granzyme B, in both the TDLNs and TME, while the expression of inhibitory molecules such as PD-1 and TIM-3 is decreased on CD8+ T cells in the TME, compared with mice administered soluble admixture of R848 and poly(I:C). Neutralization of IL-12 substantially abrogates the effects of K-nanoadjuvant on the antitumour activities of T and NK cells in both TDLNs and TME, suggesting an important role of IL-12 as the crucial linker between K-nanoadjuvant-mediated innate immune modulation and adaptive antitumour immunity. We also note that locally injected K-nanoadjuvant induces notable systemic and memory immune responses and synergizes with cancer immunotherapy when combined with anti-PD-L1 or liposomes (doxorubicin).

Our study should be differentiated from several innovative advancements, which have suggested utilizing the potent immunostimulatory properties of TLR7/8a while minimizing systemic toxicity48,49,50,51,52, in that this approach demonstrates kinetic immune modulation of APCs based on the ‘dormancy (benign) at the off-target site and recovery (active) at the target site’ strategy and GILT-induced temporal regulation of multiple TLR stimuli for optimal signal integration. Furthermore, the strategy suggested in this research can be extended as an approach to minimize systemic immune-related side effects of TLR stimuli through a spatiotemporal activation principle (Supplementary Fig. 50). We anticipate the generalizability of our kinetic modulation strategy based on transient chemical inactivation to other TLR7/8a pharmaceutical ingredients53, which could endow them with a dynamic immunomodulatory capability (Supplementary Fig. 51). It is also observed that the GILT enzyme is highly expressed in human moDCs, suggesting that the immunomodulatory properties of K-nanoadjuvant can be applied in humans as a novel clinical protocol for maturation of DCs with a strong chemotactic ability and high IL-12(p70) production (Extended Data Fig. 1f,g). Furthermore, our results also have implications for clinical trials, with broad applicability, in that t-TLR7/8a can be combined with various innate immune stimuli that have been studied in the clinical setting (poly(I:C), stimulator of interferon genes (STING) agonist, monophosphoryl lipid A (MPLA), a TLR2/4 agonist (BCG-cell wall skeleton) and α-galactosylceramide) (Extended Data Fig. 2 and Supplementary Figs. 52–56). For example, when t-TLR7/8a and MPLA (FDA-approved TLR4a) are combined and used as a vaccine with a model tumour antigen protein (OVA), it increases the Th1-related phenotype (T-bet) and the levels of proinflammatory cytokines (IFN-γ, TNF-α, IL-12(p70) and IL-2), while decreasing the regulatory-related phenotype (Foxp3) and the levels of anti-inflammatory cytokine (IL-10) in the lung (Extended Data Fig. 2 and Supplementary Fig. 55). K-nanoadjuvant can be also formulated into vaccines with the spike protein antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the aetiological agent of the coronavirus disease 2019 (COVID-19) pandemic) to improve both humoral and cellular immune responses (Extended Data Fig. 3 and Supplementary Fig. 56). Taken together, such a kinetically activating adjuvant, which optimizes the combinatorial code of t-TLR7/8a with other innate stimuli, can be translated into the development of human vaccines for immunotherapy and future pandemics22.

Methods

Synthesis and characterization of t-TLR7/8a and K-nanoadjuvant

Cholesterol-conjugated TLR7/8a was fabricated through the chemical scheme shown in Supplementary Figs. 1–3. The structures of the synthesized compounds were characterized by 1H NMR (Bruker Avance III, 700 MHz). For the preparation of liposomes (timely activating TLR7/8a, t-TLR7/8a), dimethyl dioctadecyl ammonium bromide (Sigma-Aldrich), 1,2-dioleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipid) and cholesterol-conjugated TLR7/8a (molar ratio, 2:5.3:1) were dissolved in chloroform/methanol (volume ratio, 9:1). The organic solvent was completely removed with a rotary evaporator at r.t. for 30 min. The thin film was hydrated with PBS, and the resulting solution was sonicated for 2 min via tip sonication in ice-bath conditions. Then, the final liposomes were extruded through filter membranes with 0.4 μm and 0.2 μm pore sizes using a Mini Extruder (Avanti Polar Lipid). As controls, blank liposomes and liposomes containing R848 (MedChemExpress) or an admixture of R848 and cholesterol (Sigma-Aldrich) were fabricated by the same method. The loaded amounts of t-TLR7/8a and free R848 were quantified by ultraviolet–visible light spectrometry (UV-1800). The hydrodynamic size and zeta potential of liposome formulations were measured using dynamic light scattering (DLS, ELS-Z electrophoretic light scattering photometer). Morphology was analysed by cryogenic transmission electron microscopy (FEI Tecnai F20 G2, Advanced Analysis Center, Korea Institute of Science and Technology).

To obtain and confirm the optimal conditions for K-nanoadjuvant, poly(I:C) was added so that the mass ratio of t-TLR7/8a to poly(I:C) (Sigma-Aldrich) was approximately 12:1. Then, poly(I:C), t-TLR7/8a and K-nanoadjuvant were run on a 1% agarose gel by electrophoresis in 1× Tris acetate–EDTA buffer (TAE, LPS solution) at 100 V for 40 min. Gel separation was visualized using a BioDoc-It imaging system (UVP).

Animals, cell lines and antibodies

The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University School of Medicine (SKKUIACUC2020-12-13-1), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and abides by the Institute of Laboratory Animal Resources guidelines. C57BL/6 and BALB/C mice (6- to 8-week-old female) were purchased from Orient Bio and DBL (Korea). IFNAR1−/− mice were received from Sang-Jun Ha (Yonsei University, Korea). OT-I mice were received from Yong-Soo Bae (Sungkyunkwan University, Korea). All animals were housed in individually ventilated cage under conditions of 30–70% humidity, 21–26 °C temperature and a 12 h light–dark cycle. RAW 264.7 cells (macrophage cell line, ATCC) and murine B16OVA tumour cells (melanoma, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher). Murine TC-1 tumour cells (cervical cancer, ATCC) and murine 4T1 tumour cells (breast cancer, ATCC) were cultured in RPMI 1640 medium (Thermo Fisher). RAW-Blue cells (InvivoGen) were cultured in DMEM containing Zeocin (200 μg ml−1, InvivoGen). The cell lines examined in this study were used after confirming they were free of mycoplasma contamination and were not listed in the misidentified cell lines. All media were supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher), penicillin (50 IU ml−1) and streptomycin (50 μg ml−1, Thermo Fisher). Detailed information on the antibodies such as fluorescent antibody type, manufacturer, clone and catalogue number used in this study are provided in Supplementary Table 3.

In vitro BMDC culture and cellular uptake assay

BMDCs were generated from the bone marrow of C57BL/6 mice (6- to 8-week-old female). The femurs and tibias were isolated, and the bone marrow was flushed with RPMI 1640 medium (without HEPES, Thermo Fisher Scientific) using a 26-gauge syringe. The red blood cells (RBCs) were removed with RBC lysis buffer (BioLegend). After washing, the cells were resuspended in RPMI medium (24 ml) containing mGM-CSF (20 ng ml−1, CreaGene) and seeded (2.5 × 106 per well) in a 6-well culture plate. On day 2, the medium containing mGM-CSF (20 ng ml−1) was changed after vigorous washing with PBS to remove the non-adherent cells. On day 4, fresh medium containing mGM-CSF (20 ng ml−1) was added. Differentiated immature BMDCs were used on day 6.

For cellular uptake of liposomes, liposomes were prepared with FITC–cholesterol (TopFluor Cholesterol, Avanti Polar Lipid). Immature BMDCs (4 × 104 cells) were seeded in an ibidi μ-slide 8-well microscopy chamber. The cells were preincubated with Dynasore (40 μM, Sigma-Aldrich) for 1 h and incubated with liposome(FITC–cholesterol) at 37 °C for 4 h. After washing with PBS, the cell membrane was stained with wheat germ agglutinin Texas red (Thermo Fisher), and the nuclei were stained with Hoechst 33342 (Invitrogen). Cell imaging was performed using a DeltaVision PD (GE Life Sciences) equipped with a 100× objective and the following filter sets (excitation (nm) /emission (nm)): Cy5 (645/679), FITC (490/525), TRITC (555/605) and DAPI (360/465).

GILT-dependent t-TLR7/8a analysis

GILT-dependent t-TLR7/8a cleavage

t-TLR7/8a (1.27 mM, 50 μl) was prepared in a 5 ml tube. Cysteine (1 μM or 200 nM, 50 μl, Sigma-Aldrich) was dissolved in PBS with or without GILT (2.5 μg, recombinant human IFI30, RayBiotech) and added to the tube. All samples were incubated in a 37 °C shaking incubator. Samples were snap-frozen with liquid nitrogen every hour and stored at −20 °C. After 12 h, all samples were lyophilized at −80 °C in a vacuum freeze dryer (FDU-2100, EYELA) under 10 Pa for 24 h. The lyophilized samples were analysed by liquid chromatography–mass spectrometry (Agilent 1100) to quantify the amount of R848.

GILT gene knockdown analysis

RAW 264.7 cells (2 × 105 cells per well) were seeded in a 6-well culture plate, and after 24 h, the cells were refed with fresh DMEM. Lipofectamine RNAiMAX (Thermo Fisher, 12 μl), IFI30-specific siRNA (Genolotion) and opti-MEM (Thermo Fisher) were mixed in a total volume of 300 μl, and the solution was incubated at r.t. for 5 min. The cells were treated with the solution (40 pmol per well) and incubated for 18 h. After GILT gene knockdown induction, the cells were refed with fresh medium and treated with R848 (1 μg ml−1, 3.18 μM) and t-TLR7/8a (2.9 μg ml−1, 3.18 μM). The cell supernatants were collected 24 h later, and TNF-α secretion was measured by enzyme-linked immunosorbent assay (ELISA).

In vitro Th1/Th2 polarization of CD4+ T cells

BMDCs were pretreated with R848 (1 μg ml−1, 3.18 μM) or t-TLR7/8a (2.9 μg ml−1, 3.18 μM) in the presence of OVA (10 μg ml−1) for 12 h. Spleens were harvested from C57BL/6 mice, and CD4+ T cells were isolated using a naive CD4+ T-cell isolation kit, mouse (Miltenyi Biotec) according to the manufacturer’s protocol. Preincubated DCs were co-cultured with CD4+ T cells (DC:T-cell ratio = 1:10) in a 96-well flat-bottom plate. After 3, 5 or 7 days, the cell culture supernatants were collected, and IL-4 and IFN-γ secretion was measured by ELISA.

Histological analysis

Naive C57BL/6 mice (6-week-old female) were subcutaneously injected with R848 (50 μg, 159 μmol), poly(I:C) (12.5 μg), t-TLR7/8a (144.2 μg, 159 μmol), R848 + poly(I:C) or K-nanoadjuvant four times every 3 days. Three days after the last injection, the liver, lung, spleen and kidney were harvested. The samples were sectioned and stained with H&E. The stained sections were visualized by inverted microscopy (Eclipse Ts2, A-FRONTIER) with a 40× objective.

In vivo flow cytometry analysis

B16OVA or TC-1 tumour cells (5 × 105 cells per mouse) were subcutaneously inoculated into the right flanks of C57BL/6 mice (6-week-old female). After randomly distributing tumour-bearing mice to groups 5–7 days later, R848 (25 μg, 79.5 μmol), poly(I:C) (6.25 μg), t-TLR7/8a (72.1 μg, 79.5 μmol), R848 + poly(I:C), or K-nanoadjuvant with SIINFEKL (15 μg, OVA257-264 peptide antigen, MIMOTOPES) or the E7-long peptide (AGQAEPDRAHYNIVTFCCKCDS) (10 μg, Anygen) was administered. All immunizations were repeated every 3 days for a total of three times. The non-treated group was used as a control.

Preparation of single-cell suspensions

Tumours and TDLNs were mechanically disrupted and resuspended in medium containing collagenase D (1 mg ml−1, Sigma-Aldrich). The solutions were incubated in a shaking incubator for 40 min at 37 °C. Then, the cells were washed twice with PBS after filtration through 70 μm cell strainers. To isolate splenocytes, the spleens were mechanically homogenized and resuspended in RBC lysis buffer (BioLegend) to remove RBCs. The solutions were filtered through a 70 μm cell strainer, and medium was added. Splenocytes were obtained after the suspensions were centrifuged at 488g for 3 min.

DC activation and tumor-infiltrating leukocytes (TIL) population

The single cells from TDLNs and tumours were stained with antibodies specific for activated DCs (anti-mouse CD11c, CD80 and CD86). The single cells from tumours were stained with antibodies specific for MDSCs (anti-mouse CD11b and Gr-1) and T cells and NK cells (anti-mouse CD45, CD3, CD8, NK1.1 and CD69). Detailed information on the antibodies used is provided in Supplementary Table 3.

Peptide restimulation of CD8+ T cells and stimulation of NK cells

For antigen-specific CD8+ T-cell analysis, single cells (5 × 105 per well) were seeded in a round-bottom 96-well plate. Then, the cells were restimulated with the OVA SIINFEKL peptide (10 μg ml−1) or Long-E7 peptide (10 μg ml−1), IL-2 (30 ng ml−1, PeproTech), and GolgiPlug or GolgiStop (protein transport inhibitor, 0.6 μg ml−1, BD Bioscience) for 12 h. For NK-cell stimulation analysis, single cells (5 × 105 per well) were seeded in a round-bottom 96-well plate. The cells were stimulated with a cell activation cocktail (with brefeldin A) (BioLegend, 500X), which is a mixture of optimized concentrations of PMA, ionomycin and a protein transport inhibitor (brefeldin A) for 4 h. After stimulation, the cells were collected and washed twice. Cells were stained with surface marker antibodies for 30 min at 4 °C. Single cells from TDLNs and tumours were stained with antibodies specific for exhausted CD8+ T cells (anti-mouse CD3, CD8, PD-1, LAG-3 and TIM-3) or activated CD8+ T cells (anti-mouse CD3, CD8, CD69, 4-1BB and CD25). Detailed information on the antibodies used is provided in Supplementary Table 3.

For intracellular staining, the cells were washed and then resuspended in fixation/permeabilization solution for 20 min at 4 °C. Fixed cells were washed twice with BD Perm/Wash buffer (BD Bioscience) and stained with antibodies for 30 min at 4 °C. Single cells from TDLNs, tumours and splenocytes were stained with antibodies specific for cytokine-producing CD8+ T cells (anti-mouse CD3, CD8, IFN-γ, TNF-α and Granzyme B) or cytokine-producing NK cells (anti-mouse CD3, NK1.1, IFN-γ, TNF-α and Granzyme B). After staining, the cells were washed twice with BD Perm/Wash buffer and resuspended in staining buffer. Flow cytometry data were analysed using a BD FACSCanto II (at the BIORP of the Korea Basic Science Institute) and quantified using FlowJo v.10. Detailed information on the antibodies and gating strategies used are provided in Supplementary Table 3 and Supplementary Figs. 15, 23, 24, 38 and 39.

In vivo cytokine secretion in tumours and TDLNs

For cytokine secretion analysis in tissues, TDLNs were mechanically disrupted and resuspended in a medium containing collagenase D (1 mg ml−1, Sigma-Aldrich). Single-cell suspensions obtained from the TDLNs were seeded in a 96-well round-bottom culture plate and incubated at 37 °C for 24 h. The cells were harvested, and the supernatants were collected after centrifugation. The tumours were suspended in CellLytic MT cell lysis reagent (100 mg tissue per ml, Sigma-Aldrich) and mechanically disrupted. The solutions were centrifuged at 10,000g for 10 min at 4 °C, and the supernatants were collected. All collected supernatants were analysed with IL-12(p70) or IFN-γ ELISA kits.

In vivo kinetics of IL-12(p70) cytokine secretion and immune cell numbers in LNs

To analyse the kinetics of IL-12(p70) cytokine secretion in LNs, naive C57BL/6 mice received a single injection of poly(I:C) (6.25 μg) + R848 (25 μg, 79.5 μmol) or t-TLR7/8a (72.1 μg, 79.5 μmol) with SIINFEKL (15 μg). The draining inguinal LNs were harvested at serial time points (6, 12, 24, 48 and 72 h). Then, we proceed in the same way as described above. All collected supernatants were analysed with IL-12(p70) or IFN-γ ELISA kits. To analyse the kinetics of immune cell numbers in the LNs, naive C57BL/6 mice received a single injection equal to the above dose without antigen. The draining inguinal LNs were harvested at serial time points (0, 1, 2, 4, 7 and 14 days). Then, we proceeded in the same way as described above to obtain single cells from the lymph node above.

In vivo antitumour study

TC-1 tumour cells (5 × 105 cells per mouse) were subcutaneously inoculated into the right flanks of C57BL/6 mice (6-week-old female). Poly(I:C) (6.25 μg) + R848 (25 μg, 79.5 μmol) or t-TLR7/8a (72.1 μg, 79.5 μmol) with E7-long peptide (10 μg) was injected according to the indicated schedule after random distribution of tumour-bearing mice to the groups. The PBS-treated group was used as a control. Mouse tumour growth and survival were monitored at various time points. The tumour volume was calculated using the following formula: (long-axis diameter) × (short-axis diameter)2/2. The mice were killed when the tumour volume reached the maximum tumour size (1,000 mm3) approved by the IACUC, Sungkyunkwan University School of Medicine.

Combination with immune checkpoint blockade

After 4 days of tumour inoculation, the mice received K-nanoadjuvant (t-TLR7/8a, 72.1 μg; poly(I:C), 6.25 μg) with the E7-long peptide (10 μg), and immunizations were performed every 3 days for a total of six times. Anti-PD-L1 antibodies (clone: 10 F. G2, BioXCell, 100 μg) were administered intraperitoneally every 2 days for a total of eight times.

Combination with chemotherapy

After 4 days of tumour inoculation, mice were injected with K-nanoadjuvant (t-TLR7/8a, 72.1 μg; poly(I:C), 6.25 μg) with the E7-long peptide (10 μg), and immunizations were repeated five times every 3 days. Liposome(doxorubicin) was fabricated by the method described in the Supplementary Methods. Mice were injected with liposome(doxorubicin) (80 μg of doxorubicin) two times at 6-day intervals.

In vivo IL-12 depletion study

B16OVA tumour cells (5 × 105 cells per mouse) were subcutaneously inoculated into the right flanks of mice. The mice received a subcutaneous injection of K-nanoadjuvant (t-TLR7/8a, 72.1 μg; poly(I:C), 6.25 μg) with SIINFEKL (15 μg) three times every 3 days. Beginning 3 days before the first injection of K-nanoadjuvant, anti-mouse IL-12p75 (clone: R2-9A5, BioXcell, 300 μg) was administered intraperitoneally for a total of five times at 3-day intervals.

In vivo distant tumour model study

TC-1 tumour cells (5 × 105 cells per mouse) were subcutaneously inoculated into the right flanks of C57BL/6 mice (6-week-old female) on day 0. At 4 days after primary tumour inoculation, secondary tumour cells (2.5 × 105 cells per mouse) were subcutaneously inoculated in the left flank. K-nanoadjuvant (t-TLR7/8a, 72.1 μg; poly(I:C), 6.25 μg) was injected four times every 3 days beginning 4 days after primary tumour inoculation. Mouse tumour growth was monitored at various time points. On day 22, the tumours were harvested and photographed.

In vivo orthotopic TC-1 model study

C57BL/6 mice (6-week-old female) were anaesthetized via inhaled anaesthesia. The right side of the thorax skin was incised. Then, the mice were inoculated with TC-1 cells (5 × 105 cells per mouse) on the right side of the lung lobe. After 3 days, the K-nanoadjuvant (t-TLR7/8a, 72.1 μg, 79.5 μmol; poly(I:C), 6.25 μg) and R848 (25 μg, 79.5 μmol) + poly(I:C) (6.25 μg) groups were subcutaneously administered, and immunizations were performed every 3 days for a total of four times. On day 14, the lungs were harvested, sectioned, and stained with H&E. The H&E-stained sections were visualized by using a microscope (Zeiss Axiovert 200 M) equipped with a 40× objective. The control group and the R848 + poly(I:C) and K-nanoadjuvant groups were killed on days 14 and 31, respectively, for lung staining.

Lung metastasis analysis

BALB/C mice (6-week-old female) were inoculated in the right flank with 4T1 tumour cells (5 × 105 cells per mouse). After 5 days, the mice were administered K-nanoadjuvant (t-TLR7/8a, 72.1 μg; poly(I:C), 6.25 μg) with tumour lysate (10 μg), and immunization was performed every 3 days for a total of four times. After approximately 1 month, lung metastasis was evaluated using India ink (3 ml in 47 ml of PBS), which was administered by intratracheal injection. The lungs were excised, washed with PBS, and immersed in a fixative solution (70% ethanol (40 ml), 4% formaldehyde (1 ml), and acetic acid (0.5 ml)). Lung metastatic nodules were counted by visual observation.

In vivo tumour rechallenge model study

TC-1 tumour cells (5 × 105 cells per mouse) were subcutaneously inoculated into the right flanks of C57BL/6 mice (6-week-old female) on day 0. After 4 days, mice were injected with K-nanoadjuvant five times every 3 days, and the mice were injected with anti-PD-L1 antibodies (100 μg) five times every 2 days beginning 7 days after tumour inoculation. On day 17, the mice were killed and TDLNs and spleen were collected. Single cells from TDLNs and splenocytes were stained with antibodies specific for memory T cells (anti-mouse CD3, CD8, CD44 and CD62L). Detailed information on the antibodies used is provided in Supplementary Table 3. Flow cytometry was then performed using a BD FACSCanto II to analyse the stained cell suspensions. Twenty-one days after tumour inoculation, naive mice and K-nanoadjuvant-treated tumour-free mice were rechallenged with TC-1 tumour cells (5 × 105 cells per mouse) in the same right flank.

Statistics and reproducibility

All results are indicated as the mean ± s.d. A two-tailed unpaired t-test was used to compare the two groups. One-way analysis of variance (ANOVA) (or two-way ANOVA) with Tukey’s multiple comparisons test (or Sidak’s multiple comparisons test) was used to analyse multiple groups of data. The log-rank (Mantel–Cox) test was used for survival data. All statistical analyses were performed using GraphPad Prism 8 and Microsoft Excel 2016. P values (NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001) were used to indicate statistical significance. Experiments in Figs. 1c,e,f,h and 2j were repeated three times taken from distinct samples. Experiments in Figs. 2i and 5a,f were repeated two times taken from distinct samples.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.