Cell microparticles loaded with tumor antigen and resiquimod reprogram tumor-associated macrophages and promote stem-like CD8+ T cells to boost anti-PD-1 therapy

Abstract

The durable response rate to immune checkpoint blockade such as anti-programmed cell death-1 (PD-1) antibody remains relatively low in hepatocellular carcinoma (HCC), mainly depending on an immunosuppressive microenvironment with limited number of CD8⁺ T cells, especially stem-like CD8⁺ T cells, in tumor tissues. Here we develop engineered microparticles (MPs) derived from alpha-fetoprotein (AFP)-overexpressing macrophages to load resiquimod (R848@M2pep-MPs_AFP) for enhanced anti-PD-1 therapy in HCC. R848@M2pep-MPs_AFP target and reprogram immunosuppressive M2-like tumor-associated macrophages (TAMs) into M1-like phenotype. Meanwhile, R848@M2pep-MPs_AFP-reprogrammed TAMs act as antigen-presenting cells, not only presenting AFP antigen to activate CD8⁺ T cell-mediated antitumor immunity, but also providing an intra-tumoral niche to maintain and differentiate stem-like CD8⁺ T cells. Combination immunotherapy with anti-PD-1 antibody generates strong antitumor immune memory and induces abundant stem-like CD8⁺ T cell proliferation and differentiation to terminally exhausted CD8⁺ T cells for long-term immune surveillance in orthotopic and autochthonous HCC preclinical models in male mice. We also show that the R848-loaded engineered MPs derived from macrophages overexpressing a model antigen ovalbumin (OVA) can improve anti-PD-1 therapy in melanoma B16-OVA tumor-bearing mice. Our work presents a facile and generic strategy for personalized cancer immunotherapy to boost anti-PD-1 therapy.

Fig. 1. Schematic of R848@M2pep-MPs_AFP as an efficient therapeutic strategy to potentiate anti-PD-1 antibody therapy in HCC.

a Schematic illustration of the preparation of R848@M2pep-MPs_AFP. R848@M2pep-MPs_AFP were obtained by loading R848 to M2pep-conjugated MPs derived from AFP-overexpressing RAW264.7 cells by lentivirus transduction. b Schematic illustration of R848@M2pep-MPs_AFP to boost anti-PD-1 antibody therapy in HCC. R848@M2pep-MPs_AFP targeted and reprogrammed M2-like TAMs into M1-like phenotype, followed by presenting AFP antigen to activate antigen-specific CD8⁺ T cell for tumor control and promote stem-like CD8⁺ T cell proliferation and differentiation. Combination with anti-PD-1 antibody generated strong antitumor immune memory and induced abundant stem-like CD8⁺ T cell proliferation and differentiation to terminally exhausted CD8⁺ T cells for long-term immune surveillance.

Fig. 2. Relationship between the sensitivity of anti-PD-1 antibody therapy and tumor microenvironment.

a Volcano plots showing differential gene expression in the tumor tissues of anti-PD-1 antibody-responsive and nonresponsive mice. (n = 3 mice per group; two-tailed Student’s t-test for comparison using Cuffdiff in the Cufflinks package). b Top 20 enrichment ratios (E-ratio) in GO term enrichment analysis of differentially expressed genes in tumor tissues of anti-PD-1 antibody-responsive and nonresponsive mice. (n = 3 mice per group; one-sided hypergeometric test). c GO enrichment analysis of the differentially expressed genes associated with macrophages, T cells and antigen presentation pathways in tumor tissues of anti-PD-1 antibody-responsive and nonresponsive mice. (n = 3 mice per group; one-sided hypergeometric test). d–k The numbers of CD80⁺ TAMs (d), CD86⁺ TAMs (e), CD206⁺ TAMs (f), CD8⁺ T (g), CD8⁺IFNγ⁺ T (h), CD8⁺PD-1⁺ T (i), CD8⁺PD-1⁺TCF-1⁺ T (j), and CD8⁺PD-1⁺TCF-1^- T (k) cells in tumor tissues of orthotopic Hepa1-6 tumor-bearing mice after intraperitoneal injection of PBS or anti-PD-1 antibody at the dosage of 5 mg kg⁻¹ every four days for 5 times indicated in Supplementary Fig. 1a. Data are presented as means ± s.d. (n = 5 mice for control group, n = 11 mice for anti-PD-1 nonresponsive group, n = 12 mice for anti-PD-1 responsive group; one-way ANOVA followed by Tukey’s HSD post-hoc test). Source data are provided as a Source Data file.

Fig. 3. Characterization and M2-like TAM targeting of R848@M2pep-MPs_AFP.

a, b Hydrodynamic diameters (a) and zeta potentials (b) of MPs, M2pep-MPs, MPs_AFP, M2pep-MPs_AFP, R848@MPs, R848@M2pep-MPs, R848@MPs_AFP and R848@M2pep-MPs_AFP by DLS analysis. Data are presented as means ± sd (n = 3 independent samples). c Representative AFM height images (left), amplitude images (upper right) and three-dimensional morphology (lower right) of M2pep-MPs_AFP and R848@M2pep-MPs_AFP. Scale bars: 400 nm. Images are representative of three independent samples. d Relative PKH26 mean fluorescence intensity (MFI) in BMDMs (M0 BMDMs), LPS- and IFNγ-stimulated BMDMs (M1-like BMDMs), IL-4-stimulated BMDMs (M2-like BMDMs) and Hepa1-6 cells after treatment with PKH26-labeled MPs, M2pep-MPs, MPs_AFP or M2pep-MPs_AFP at the concentration of 10 µg protein mL⁻¹ for 4 h by flow cytometry. Data are presented as means ± sd (n = 3 biological independent samples; one-way ANOVA followed by Tukey’s HSD post-hoc test). e, f Ex vivo imaging (e) and relative fluorescence intensity (f) of IR780 in major organs and tumors of orthotopic Hepa1-6 tumor-bearing mice at 24 h after intravenous injection of IR780-labeled MPs, M2pep-MPs, MPs_AFP or M2pep-MPs_AFP at the dosage of 15 mg protein kg⁻¹. Data are presented as means ± s.d. for (f). (n = 3 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). g Relative PKH26 MFI in tumor cells (CD45^- cells), M1-like TAMs (CD11b⁺F4/80⁺CD80⁺ cells), M2-like TAMs (CD11b⁺F4/80⁺CD206⁺ cells), T cells (CD45⁺CD3⁺ cells), DCs (CD45⁺F4/80^-CD11c⁺ cells), MDSCs (CD45⁺CD11b⁺Gr1⁺ cells) and Tregs (CD45⁺CD3⁺CD4⁺CD25⁺FoxP3⁺ cells) in tumor tissues of orthotopic Hepa1-6 tumor-bearing mice at 24 h after intravenous injection of PKH26-labeled MPs, M2pep-MPs, MPs_AFP or M2pep-MPs_AFP at the dosage of 15 mg protein kg⁻¹. Data are presented as means ± sd (n = 5 mice per group; two-way ANOVA followed by Bonferroni’s multiple comparisons post-test). h Relative PKH26 MFI in TAMs, liver Kupffer cells, splenic macrophages, pulmonary macrophages and renal macrophages (CD11b⁺F4/80⁺ cells) of orthotopic Hepa1-6 tumor-bearing mice at 24 h after treatment indicated in (g). Data are presented as means ± sd (n = 5 mice per group; two-way ANOVA followed by Bonferroni’s multiple comparisons post-test). Source data are provided as a Source Data file.

Fig. 4. Efficiently reprogramming M2-like TAMs and activating CD8⁺ T cells by R848@M2pep-MPs_AFP in vivo.

a Schematic schedule for reprogramming M2-like TAMs and activating CD8⁺ T cells by R848@M2pep-MPs_AFP in orthotopic Hepa1-6 tumor-bearing mice. b–e The numbers of CD80⁺ (b), CD86⁺ (c), MHC II⁺ (d), and CD206⁺ (e) TAMs in tumor tissues of orthotopic Hepa1-6 tumor-bearing mice after intravenous injection of PBS, MPs, M2pep-MPs, MPs_AFP, M2pep-MPs_AFP, R848, R848@MPs, R848@M2pep-MPs, R848@MPs_AFP or R848@M2pep-MPs_AFP at the R848 dosage of 0.5 mg kg⁻¹ every three days for six times as indicated in (a). Data are presented as means ± s.d. (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). f–i Representative flow plots (f) and percentages of proliferated CD8⁺ T cells (g), IFNγ⁺ (h), and GzmB⁺ (i) cells in CD8⁺ T cells at 5 days after co-culture with TAMs isolated from tumor tissues of orthotopic Hepa1-6 tumor-bearing mice which were intravenously injected with PBS, MPs, M2pep-MPs, MPs_AFP, M2pep-MPs_AFP, R848, R848@MPs, R848@M2pep-MPs, R848@MPs_AFP or R848@M2pep-MPs_AFP at the R848 dosage of 0.5 mg kg⁻¹ every three days for six times as indicated in (a) by flow cytometry. Data are presented as means ± s.d. for (g–i) (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). j, k Representative flow plots (j) and ratios of antigen-specific lysis (k) in splenocytes of orthotopic Hepa1-6 tumor-bearing mice at 24 h after intravenous injection of PBS, R848@M2pep-MPs or R848@M2pep-MPs_AFP at the R848 dosage of 0.5 mg kg⁻¹ every three days for six times, followed by intravenous injection of the mixtures of CFSE^low OVA_257-264-loaded and CFSE^high AFP₂₁₂-loaded splenocytes at the ratio of 1:1 as indicated in (a) by flow cytometry. Data are presented as means ± s.d. for (k). (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). Panels (f, j) show representative results of five independent samples. Source data are provided as a Source Data file.

Fig. 5. Potent antitumor activity and improved antitumor immunity of R848@M2pep-MPs_AFP in orthotopic Hepa1-6 tumor-bearing mice.

a Schematic schedule for the antitumor experiment of R848@M2pep-MPs_AFP in orthotopic Hepa1-6 tumor-bearing mice. b, c Tumor images (b) and tumor weight (c) of orthotopic Hepa1-6 tumor-bearing mice at 16 days after intravenous injection of PBS, MPs, M2pep-MPs, MPs_AFP, M2pep-MPs_AFP, R848, R848@MPs, R848@M2pep-MPs, R848@MPs_AFP or R848@M2pep-MPs_AFP at the R848 dosage of 0.5 mg kg⁻¹ every three days for six times. Data are presented as means ± s.d. for (c). (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). d Kaplan–Meier survival plot of orthotopic Hepa1-6 tumor-bearing mice after treatment indicated in (a). (n = 8 mice per group). e–l The numbers of CD8⁺ T cells (gated as CD45⁺CD3⁺CD8⁺, e), CD8⁺Ki67⁺ T cells (gated as CD45⁺CD3⁺CD8⁺Ki67⁺, f), CD8⁺CD69⁺ T cells (gated as CD45⁺CD3⁺CD8⁺CD69⁺, g), CD8⁺IFNγ⁺ T cells (gated as CD45⁺CD3⁺CD8⁺IFNγ⁺, h), CD8⁺GzmB⁺ T cells (gated as CD45⁺CD3⁺CD8⁺GzmB⁺, i), CD8⁺PD-1⁺TCF-1⁺ T cells (gated as CD45⁺CD3⁺CD8⁺PD-1⁺TCF-1⁺, j), CD8⁺PD-1⁺TCF-1^- T cells (gated as CD45⁺CD3⁺CD8⁺PD-1⁺TCF-1^-, k) and CD8⁺PD-1⁺TCF-1^-GzmB⁺ T cells (gated as CD45⁺CD3⁺CD8⁺PD-1⁺TCF-1^-GzmB⁺, l) in tumor tissues of orthotopic Hepa1-6 tumor-bearing mice at 16 days after treatment indicated in (a). Data are presented as means ± s.d. (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). m Colocalization of M1-like TAMs (F4/80 (red) and CD86 (pink)-positive cells) and TCF-1 (green) in tumor tissues of orthotopic Hepa1-6 tumor-bearing mice at 16 days after treatment indicated in (a) by immunofluorescent staining. Scale bars: 10 μm. Images are representative of three independent samples. Source data are provided as a Source Data file.

Fig. 6. Improved antitumor effects and antitumor immunity of anti-PD-1 antibody by R848@M2pep-MPs_AFP in orthotopic Hepa1-6 tumor-bearing mice.

a Schematic schedule for the antitumor experiment of combination of R848@M2pep-MPs_AFP and anti-PD-1 antibody in orthotopic Hepa1-6 tumor-bearing mice. b, c Tumor images (b) and tumor weight (c) of orthotopic Hepa1-6 tumor-bearing mice after treatment with R848@M2pep-MPs_AFP in the presence or absence of anti-PD-1 antibody at the anti-PD-1 antibody dosage of 5 mg kg⁻¹ and R848 dosage of 0.5 mg kg⁻¹ indicated in (a). Data are presented as means ± s.d. for (c). (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). d Kaplan–Meier survival plot of orthotopic Hepa1-6 tumor-bearing mice after treatment indicated in (a). (n = 8 mice per group). e–l The numbers of CD8⁺ T cells (e), CD8⁺Ki67⁺ T cells (f), CD8⁺CD69⁺ T cells (g), CD8⁺IFNγ⁺ T cells (h), CD8⁺GzmB⁺ T cells (i), CD8⁺PD-1⁺TCF-1⁺ T cells (j), CD8⁺PD-1⁺TCF-1^- T cells (k) and CD8⁺PD-1⁺TCF-1^-GzmB⁺ T cells (l) in tumor tissues of orthotopic Hepa1-6 tumor-bearing mice after treatment indicated in (a). Data are presented as means ± s.d. (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). m Schematic schedule for ELISPOT and T cell cytotoxic assay. n, o Images (n) and numbers (o) of IFNγ immune spots from OVA_257-264- or AFP₂₁₂-restimulated splenocytes isolated from orthotopic Hepa1-6 tumor bearing mice after treatment indicated in (m) by the ELISPOT assay. Data are presented as means ± s.d. (n = 5 biological independent samples; two-way ANOVA followed by Bonferroni’s multiple comparisons post-test). p Cytotoxicity of T cells against Hepa1-6 cells when T cells isolated from OVA_257-264- or AFP₂₁₂-restimulated splenocytes were incubated with Hepa1-6 cells at the effector/target ratio of 10:1 for 6 h as indicated in (m) by LDH assay. Data are presented as means ± s.d. (n = 5 biological independent samples; two-way ANOVA followed by Bonferroni’s multiple comparisons post-test). Source data are provided as a Source Data file.

Fig. 7. Improved antitumor activity and antitumor immunity of anti-PD-1 antibody by R848@M2pep-MPs_AFP in DEN-induced autochthonous HCC mice.

a Schematic schedule for the anticancer experiment of combination of R848@M2pep-MPs_AFP and anti-PD-1 antibody in DEN-induced autochthonous HCC mice. b–e Tumor images (b), tumor weight (c), nodule numbers (d) and H&E staining of liver tissues (e) in DEN-induced autochthonous HCC mice at 30 weeks after treatment with R848@M2pep-MPs_AFP in the presence or absence of anti-PD-1 antibody at the anti-PD-1 antibody dosage of 5 mg kg⁻¹ and R848 dosage of 0.5 mg kg⁻¹ indicated in (a). Data are presented as means ± s.d. for (c, d). (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). Scale bar: 5000 μm for (e). f Kaplan–Meier survival plot of DEN-induced autochthonous HCC mice after treatment indicated in (a). (n = 6 mice per group). g–r The numbers of CD80⁺ TAMs (g), CD86⁺ TAMs (h), MHC II⁺ TAMs (i), CD206⁺ TAMs (j), CD8⁺ T cells (k), CD8⁺Ki67⁺ T cells (l), CD8⁺CD69⁺ T cells (m), CD8⁺IFNγ⁺ T cells (n), CD8⁺GzmB⁺ T cells (o), CD8⁺PD-1⁺TCF-1⁺ T cells (p), CD8⁺PD-1⁺TCF-1^- T cells (q) and CD8⁺PD-1⁺TCF-1^-GzmB⁺ T cells (r) in tumor tissues of DEN-induced autochthonous HCC mice at 30 weeks after treatment indicated in (a). Data are presented as means ± s.d. (n = 5 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). Source data are provided as a Source Data file.

Fig. 8. Improved anticancer activity and antitumor immunity of anti-PD-1 antibody by 848@M2pep-MPs_OVA in B16-OVA tumor-bearing mice.

a–e Percentages of CD80⁺ (a), CD86⁺ (b), MHC II⁺ (c), and CD206⁺ cells (d) and SIINFEKL-H-2Kb⁺ M1-like macrophages (e) in IL-4-stimulated RAW264.7 cells after treatment with the indicated formulations at R848 concentration of 2 nM for 24 h. Data are presented as means ± sd (n = 4 and 3 biological independent samples for (a–d) and (e), respectively; one-way ANOVA followed by Tukey’s HSD post-hoc test). f, g Percentages of SIINFEKL-H-2Kb tetramer⁺ (f) and IFNγ⁺ cells (g) in CD8⁺ T cells at 5 days after co-culture with IL-4-stimulated RAW264.7 cells pretreated indicated in (a–e). Data are presented as means ± sd (n = 4 biological independent samples; one-way ANOVA followed by Tukey’s HSD post-hoc test). h Cytotoxicity of CD8⁺ T cells treated indicated in (f, g) against B16-OVA cells after co-incubation at the effector/target ratio of 20:1 for 6 h. Data are presented as means ± s.d. (n = 4 biological independent samples; one-way ANOVA followed by Tukey’s HSD post-hoc test). i Schematic schedule for anticancer experiment of combination of R848@M2pep-MPs_OVA and anti-PD-1 antibody. j, k Tumor growth curves (j) and Kaplan–Meier survival plots (k) of B16-OVA tumor-bearing mice after treatment with R848@M2pep-MPs_OVA in the presence or absence of anti-PD-1 antibody at anti-PD-1 antibody and R848 dosage of 5 and 0.5 mg kg⁻¹ indicated in (i), respectively. Data are presented as means ± sem for (j). (n = 6 and 8 mice per group for (j) and (k), respectively; one-way ANOVA followed by Tukey’s HSD post-hoc test). l Relative immune cell numbers in tumors of B16-OVA tumor-bearing mice after treatment indicated in (i). (n = 6 mice per group). m–q Representative flow plots of SIINFEKL-H-2Kb tetramer⁺ in CD3⁺CD8⁺T cells (m) and numbers of CD8⁺tetramer⁺ (n), CD8⁺tetramer⁺PD-1⁺TCF-1⁺ (o), CD8⁺tetramer⁺PD-1⁺TCF-1^- (p) and CD8⁺tetramer⁺PD-1⁺TCF-1^-GzmB⁺ T (q) cells in tumors of B16-OVA tumor-bearing mice after treatment indicated in (i). Data are presented as means ± s.d. (n = 6 mice per group; one-way ANOVA followed by Tukey’s HSD post-hoc test). Source data are provided as a Source Data file.