Simultaneous enhancement of cellular and humoral immunity by the lymph node-targeted cholesterolized TLR7 agonist liposomes

Abstract

Toll-like receptor (TLR) agonists, as promising adjuvants and immunotherapeutic agents, have the potential to enhance immune responses and modulate antigen-dependent T-cell immune memory through activation of distinct signaling pathways. However, their clinical application is hindered by uncontrolled systemic inflammatory reactions. Therefore, it is imperative to create a vaccine adjuvant for TLR receptors that ensures both safety and efficacy. In this study, we designed lymph node-targeted cholesterolized TLR7 agonist cationic liposomes (1V209-Cho-Lip⁺) to mitigate undesired side effects. Co-delivery of the model antigen OVA and cholesterolized TLR7 agonist facilitated DC maturation through TLR activation while ensuring optimal presentation of the antigen to CD8⁺ T cells. The main aim of the present study is to evaluate the adjuvant effectiveness of 1V209-Cho-Lip⁺ in tumor vaccines. Following immunization with 1V209-Cho-Lip⁺+OVA, we observed a pronounced "depot effect" and enhanced trafficking to secondary lymphoid organs. Prophylactic vaccination with 1V209-Cho-Lip⁺+OVA significantly delays tumor development, prolongs mouse survival, and establishes durable immunity against tumor recurrence. Additionally, 1V209-Cho-Lip⁺+OVA, while used therapeutic tumor vaccine, has demonstrated its efficacy in inhibiting tumor progression, and when combined with anti-PD-1, it further enhances antitumor effects. Therefore, the co-delivery of antigen and lymph node-targeted cholesterolized TLR7 agonist shows great promise as a cancer vaccine.

Keywords: Cancer nanovaccines; Cationic liposomes; Cellular immunity; Cross-presentation; Humoral immunity; Immunotherapy; Lymph node; TLR7 agonist.

Graphical abstract

Scheme 1

1V209-Cho-Lip⁺+OVA effectively co-delivers antigen and TLR7 agonist, thereby augmenting both humoral and cellular immunity to exert a superior anti-tumor effect.

Figure 1

(A) DLS and TEM image of 1V209-Cho-Lip⁺, scale bar = 200 nm. (B) DLS and TEM image of 1V209-Cho-Lip⁺+OVA, scale bar = 200 nm. (C) The cellular uptake of BMDCs were measured by FACS analysis. (D–F) The expression levels of (D) CD40, (E) CD80, and (F) CD86 on DCs. (G and H) Representative flow cytometry pictures and quantification of CFSE-OT-I CD8 T cells after coculture with DC activated by PBS, OVA, Blank-Lip⁺+OVA, and 1V209-Cho-Lip⁺+OVA for 72 h. (I and J) Representative flow cytometry pictures and quantification of CD69 expression on OT-I CD8 T cells after coculture with DC activated by PBS, OVA, Blank-Lip⁺+OVA, and 1V209-Cho-Lip⁺+OVA for 72 h. (K) In vivo fluorescent imaging of mice. (L) Ex vivo imaging and semiquantification of in inguinal and popliteal lymph node fluorescence at 24 h post-injection. Data are presented as mean ± SEM (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2

(A) Scheme of immunizations. (B–E) Individual tumor growth curves are shown. CR, complete regression. (F) Percentage survival rate of mice following tumor challenge. (G) Percentage of mice remaining tumor-free after tumor challenge. (H) Quantification of active CD4⁺ T cells in inguinal LNs. (I) Representative flow cytometry plots and quantification of effector memory CD4⁺ T cells (EM), central memory CD4⁺ T cells (CM), and nai$\ddot{\mathrm{v}}$e CD4⁺ T cells (Naive) in inguinal LNs. (J) Quantification of active CD8⁺ T cells in drain inguinal LNs. (K) Representative flow cytometry plots and quantification of effector memory CD8⁺ T cells (EM), central memory CD8⁺ T cells (CM), and nai$\ddot{\mathrm{v}}$e CD8⁺ T cells (Naive) in inguinal LNs. Data are presented as mean ± SEM (n = 5). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s., not significant.

Figure 3

1V209-Cho-Lip⁺+OVA elicited robust cellular immune responses in mice. (A) Quantification of stem-like CD8⁺ T cells in inguinal LNs. (B–D) Quantification of (B) CD4⁺ T cells, (C) activated CD4⁺ T and (D) CD8⁺ T cells in spleens. (E) Representative flow cytometry pictures and frequency of GC B cells (GL7⁺ CD95⁺ CD19⁺) in inguinal LNs B cell population. (F) Representative flow cytometry pictures and frequency of T_FH cells (CXCR5⁺ PD-1⁺ CD4⁺) in the CD4 T cell population from inguinal LNs. (G) Representative flow cytometry plots and frequency OVA-specific CD8 T cells bearing a T-cell receptor specific for OVA_257-264-H2Kb tetramer. (H and I) Splenocytes from immunized mice were restimulated OVA_257-264ex vivo, and the percentages of (H) IFN-γ⁺ and (I) Granzyme B⁺ among CD8α⁺ splenocytes are shown. (J) Cytokine production including IFN-γ, Granzyme B, TNF-α, IL-6, and IL-10 after ex vivo stimulation of splenocytes for 72 h is presented. (K) Secretion levels of IFN-γ, TNF-α, and IL-6 were measured in the serum samples from immunized mice. (L) The OVA-specific killing ability of CD8⁺ T cells on tumors. Data are presented as mean ± SEM (n = 3–4). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. n.s., not significant.

Figure 4

(A) Scheme of the immunization procedures and experimental design. (B–E) Curves depicting the growth of tumors in individual mice that were injected with B16F10-OVA tumor cells. CR, complete regression. (F) Percentage of mice remaining tumor-free after tumor challenge. (G) Quantification of BMEM in bone marrow. (H and I) ELISPOT was used to determine the abundance of IFN-γ⁺ splenocytes (H) and cells in the blood (I). Representative images of ELISPOT wells are shown. (J) Schematic representation depicting therapeutic immunization against established B16F10-OVA tumors. (K–P) Curves depicting the growth of tumors in individual mice. CR, complete regression. (Q) Percentage survival rate of mice following tumor challenge. Data are presented as mean ± SEM (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 5

In vivo prophylactic and therapeutic efficacy of the 1V209-Cho-Lip⁺+OVA vaccine in E.G7-OVA syngeneic allograft tumor mode. (A) Experimental design for tumor challenge in the immune-prophylaxis group was illustrated. (B–E) Curves depicting the growth of tumors in individual mice. CR, complete regression. (F) Percentage survival rate of mice following tumor challenge. (G) Schematic of therapeutic immunization against established E.G7-OVA tumors. (H–M) Curves depicting the growth of tumors in individual mice. CR, complete regression. (N) Percentage survival rate of mice following tumor challenge.

Figure 6

Tolerability of 1V209-Cho-Lip⁺+OVA was evaluated in C57BL/6J mice through intramuscular immunization with 1V209-Cho-Lip⁺+OVA on Days 0, 14, and 21. On Day 28, blood samples were collected to assess complete blood count parameters (A) and biochemical indexes (B). (C) Vital organs were subjected to hematoxylin and eosin staining for histopathological examination. Scale bar = 200 μm. Data are presented as mean ± SEM (n = 5).