Fig 2: Light-induced activation of immunological adjuvant effect of LIA in vitro.a–f Flow cytometry analysis of bone marrow-derived dendritic cells (BMDCs) maturation after the cells were incubated with PBS, HAD, rHAD or lipopolysaccharide (LPS). a–c Flow cytometric histograms of CD80 (a), CD86 (b) and CD40 (c) expressions in BMDCs after different treatments (gated on CD11C+ cells). d–f Quantification of CD80 (d), CD86 (e) and CD40 (f) expressions after BMDCs treated with different groups. g, h TNF-a (g) and IL-6 (h) concentrations in BMDCs culture supernatants after 12 h incubation with different formulations (n = 3). i Schematic illustration of the transwell system experiment. 4T1 tumour cells were first placed in the upper chamber and treated with different formulations, then BMDCs were cultured in the lower chamber. j Proportions of CD80+ CD86+ BMDCs after treated with different treatments (gated on CD11C+ cells, n = 3). k, l Transcriptomic analysis of BMDCs after treated with PBS, HAD and rHAD. Three biological replicates are shown. k Heat map of DEGs is indicated for each group and Key DEGs are listed on the right. l KEGG enrichment analysis of the pathways between rHAD treatment and PBS treatment. m Molecular docking of HAD and rHAD head groups with TLR7. Grid score values are calculated from the simulation. Data in d–h are presented as mean ± s.d. (n = 3) and statistically significant differences between groups were identified by one-way ANOVA. Data in j are presented as mean ± s.d. (n = 3) and statistically significant differences between groups were identified by two-tailed unpaired Student’s t-test. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

Fig 3: In vivo immunotherapy of BMS. (a) Quantified signal intensity after different formulation treatment at 16 d. (b) The body weight of GBM bearing mice in the progress of treatment. (c) Representative propotion of CD80 and CD86 cells expression after different formulation (PBS, SAL, BM, BMS) treatment. (d) The quantitative analysis of CD80, CD86 cells in GBM tissues after treatment according to Fig. 6c. (e) Representative propotion of CD163 and CD206 cells expression after different formulation (PBS, SAL, BM, BMS) treatment by flow cytometry. (f) The quantitative analysis of CD163, CD206 cells in GBM tissues after treatment according to Fig. 6e. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: Administration of R848 promotes systemic immune activation. (A) TLR7 expression on DCs, macrophages, MDSCs, NK, CD4+ T and CD8+ T cells was analyzed by flow cytometry (n=3). (B) CD80 and (C) CD86 expression on DCs was detected by flow cytometry (n=3). (D) CD69 expression was assessed in NK, CD4+ T and CD8+ T cells (n=3). (E) Levels of cytokines were evaluated in the serum (n=3). *P<0.05, **P<0.01 and ***P<0.001 compared with PBS. TLR, Toll-like receptor; DCs, dendritic cells; MDSCs, myeloid-derived suppressor cells; NK, natural killer; NS, not significant.

Fig 5: Nck binds to human but not mouse CD28. a, b Human primary CD4+ T cells were stimulated for 5 min with B7-negative (-) or Dap3/B7 (B7) cells and anti-CD28 IP were performed on cellular extracts. Anti-Vav, anti-Nck, anti-p85 PI3K, and anti-CD28 western blottings were performed on anti-CD28 IP or total lysates (TL). Data represent one of three independent experiments. c, d CD4+ T cells isolated from murine spleen (n = 10) were pooled and stimulated for 5 min with with B7-negative (-) or B7.1-expressing RAW (B7) cells and anti-CD28 IP were performed on cellular extracts. Anti-Vav, anti-Nck, anti-p85 PI3K, and anti-CD28 western blottings were performed on anti-CD28 IP or total lysates (TL). The position of immunoprecipitated proteins as well as of immunoglobulin heavy chain (IgH) is indicated. e Equal amounts of total lysates from human or mouse CD4+ T cells were analysed by western blotting for Vav1 (upper panel) and Nck (lower panel) content. The position of molecular weight markers, expressed in kDa, is indicated on the right