Journal: Science Advances

Article Title: TLR9-activating cholesterol azetidine derivative–assisted therapeutic vaccines for cancer immunotherapy

doi: 10.1126/sciadv.aeb2465

Figure Lengend Snippet: ( A ) Zeta potential and quantification of OVA protein adsorption on Aze-Chol NP@OVA nanovaccines. ( B ) STORM image of Aze-Chol NP@OVA. Green (rhodamine B), Aze-Chol NP; red (Cy5), OVA. ( C ) Representative confocal fluorescence images of DC2.4 cells incubated with Cy5-OVA or Aze-Chol NP@Cy5-OVA (Aze-Chol NP at 20 μg/ml). Red, Cy5-OVA; blue, nuclei. Scale bars, 10 μm. ( D and E ) Flow cytometry analysis of OVA uptake by BMDCs incubated with free OVA or Aze-Chol NP@OVA (Aze-Chol NP at 20 μg/ml) for 24 hours. (D) Representative histograms; (E) MFI quantification. FITC, fluorescein isothiocyanate. ( F ) Heatmap showing MFI of CD80, CD86, CD40, and MHCII expression on BMDCs after incubation with OVA, Aze-Chol NP, or Aze-Chol NP@OVA for 24 hours. ( G and H ) Biodistribution kinetics of Aze-Chol NP@Cy5-OVA (Aze-Chol NP at 20 mg/kg) in ILNs of C57BL/6 mice following subcutaneous administration. (G) Ex vivo fluorescence imaging and (H) semiquantitative analysis at indicated time points postinjection with free Cy5-OVA or Aze-Chol NP@Cy5-OVA. ( I and J ) Flow cytometry analysis of Cy5-OVA uptake by (I) resident CD8α + CD11c + MHCII + cDC1s and (J) migratory CD103 + CD11c + MHCII + cDC1s in ILNs following subcutaneous injection with free Cy5-OVA or Aze-Chol NP@Cy5-OVA. ( K ) SIINFEKL-MHCI complex levels on BMDCs after incubation with OVA (10 μg/ml) or Aze-Chol NP@OVA (Aze-Chol NP at 20 μg/ml) for 24 hours. ( L and M ) OT-I CD8 + T cell proliferation and activation after 72-hour coculture with OVA- or Aze-Chol NP@OVA–treated BMDCs. Left shows representative flow cytometry plots; right shows quantification of (L) proliferation (by CFSE dilution) and (M) activation (by CD69 expression). ( N ) In vivo evaluation of OVA-specific CTL responses. Representative flow cytometry plots and quantification of OVA peptide–specific target cell lysis percentages are shown. ** P < 0.01 and **** P < 0.0001.

Article Snippet: OVA peptide (SIINFEKL) and E7 peptide (AGQAEPDRAHYNIVTFCCKCDS) were synthesized by Sangon Biotech (Shanghai, China), which were purified by reversed-phase high-performance liquid chromatography to a purity exceeding 95%.

Techniques: Zeta Potential Analyzer, Adsorption, Fluorescence, Incubation, Flow Cytometry, Expressing, Ex Vivo, Imaging, Injection, Activation Assay, In Vivo, Lysis

Journal: Science Advances

Article Title: TLR9-activating cholesterol azetidine derivative–assisted therapeutic vaccines for cancer immunotherapy

doi: 10.1126/sciadv.aeb2465

Figure Lengend Snippet: ( A ) Schematic showing the immunization schedule of C57BL/6 mice subcutaneously (sc) injected with PBS (G1; Group 1), OVA alone (G2; Group 2), CpG + OVA (G3; Group 3), or Aze-Chol NP@OVA nanovaccine (G4; Group 4; Aze-Chol NP at 20 mg/kg) once weekly for 3 weeks. ( B ) Flow cytometry quantification of cDCs presenting SIINFEKL peptide on H-2K b in dLNs of immunized mice. ( C ) Representative dot plots (left) and quantification (right) of OVA-specific (SIINFEKL-MHCI tetramer-positive) CD8 + T cells in peripheral blood after immunization. ( D and E ) Representative dot plots (D) and quantification (E) of IFN-γ–positive CD8 + T cells in peripheral blood postimmunization. ( F ) Serum IFN-γ concentrations quantified by ELISA at day 21 postimmunization. ( G ) Experimental timeline for assessing antigen-specific immune responses after immunization with OVA, CpG + OVA, or Aze-Chol NP@OVA. ( H ) Representative ELISPOT images of IFN-γ–secreting splenocytes stimulated ex vivo with SIINFEKL peptide for 48 hours. ( I and J ) Quantification of (I) IFN-γ spot-forming units (SFU) and (J) sum of spot volume (SSV) from splenocytes stimulated as described in (G). ( K ) Levels of cytokines (IFN-γ, TNF-α, IL-2, and granzyme B) associated with T cell activation and effector function, measured by ELISA after ex vivo stimulation of splenocytes with SIINFEKL peptide for 72 hours. ( L ) Percentages of specific tumor cell lysis measured by Hoechst 33342 staining following 72-hour coculturing of splenocytes from immunized mice with OVA-expressing B16F10 cells. ( M ) Schemes showing the tumor challenge experiment design. D 0, D 5, D 10 and D15 indicate Day 0, Day 5, Day 10 and Day 15, respectively. ( N ) Average (left) and individual (right) tumor growth curves of C57BL/6 mice bearing B16F10-OVA tumors, treated as indicated. ( O ) Survival curves of tumor-bearing mice after treatment with PBS, OVA, CpG + OVA, or Aze-Chol NP@OVA. ( P ) Representative immunofluorescence staining showing CD8 + T cell infiltration (red) and nuclei (blue) in tumor sections from indicated treatment groups. Scale bars, 100 μm. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: OVA peptide (SIINFEKL) and E7 peptide (AGQAEPDRAHYNIVTFCCKCDS) were synthesized by Sangon Biotech (Shanghai, China), which were purified by reversed-phase high-performance liquid chromatography to a purity exceeding 95%.

Techniques: Injection, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Ex Vivo, Activation Assay, Lysis, Staining, Expressing, Immunofluorescence

Journal: Science Advances

Article Title: TLR9-activating cholesterol azetidine derivative–assisted therapeutic vaccines for cancer immunotherapy

doi: 10.1126/sciadv.aeb2465

Figure Lengend Snippet: ( A ) Therapeutic regimen of Aze-Chol NP-OVAp vaccine in the B16F10-OVA melanoma model. ( B ) (Left) Average and (right) individual tumor growth curves of C57BL/6 mice treated with OVA peptide, CpG + OVA peptide, and Aze-Chol NP-OVAp (Aze-Chol NP at 20 mg/kg). ( C ) Frequency of OVA antigen–specific effector memory CD8 + T cells in various tissues, analyzed by flow cytometry. ( D ) Effector function of tumor-infiltrating CD8 + T cells. Percentages of TNF-α, IFN-γ, or granzyme B–secreted CD8 + T cells within tumor after OVA peptide restimulation are shown. ( E ) Proportion of multifunctional CD8 + T cells capable of simultaneously secreting one, two, and three effector cytokines (TNF-α, IFN-γ, and/or granzyme B) in tumor was displayed as a 10 by 10 dot plot. ( F ) Schematic of therapeutic regimen and tumor rechallenge study of Aze-Chol NP-E7p in combination with αPD-L1 antibody in TC-1 cervical cancer model. ip, intraperitoneal. ( G ) Average TC-1 tumor growth curves of C57BL/6 mice treated with αPD-L1 antibody, Aze-Chol NP-E7p, and Aze-Chol NP-E7p in combination with αPD-L1 antibody (Aze-Chol NP at 20 mg/kg). ( H and I ) (H) Survival curves of C57BL/6 mice following indicated treatments were obtained using the Kaplan-Meier method, (I) along with pie charts representing the percentage of cured mice quantified by tumor-free survival (>60 days). ( J and K ) Long-term immune effect induced by Aze-Chol NP-E7p against TC-1 rechallenge. (J) Average tumor growth curves and (K) survival curves of cured mice from indicated groups after TC-1 tumor rechallenge on day 45. Age-matched naive C57BL/6 mice served as controls. ( L and M ) Representative flow cytometry plots and frequencies of central memory (CD44 + CD62L + ) CD8 + T cells (top) and CD4 + T cells (down) in the (L) spleen and (M) bone marrow. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: OVA peptide (SIINFEKL) and E7 peptide (AGQAEPDRAHYNIVTFCCKCDS) were synthesized by Sangon Biotech (Shanghai, China), which were purified by reversed-phase high-performance liquid chromatography to a purity exceeding 95%.

Techniques: Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Condensate nanovaccine adjuvants augment CD8 + T-Cell-dependent antitumor immunity through mtDNA leakage-triggered cGAS-STING axis activation

doi: 10.1038/s41392-025-02447-w

Figure Lengend Snippet: Evaluation of the in vivo antitumor efficacy and immunological mechanisms of OVA PCD. a Schematic timeline of vaccine and B16-OVA tumor cell inoculation in the C57BL/6 mouse model. The mice were immunized three times via subcutaneous injection at the tail base with OVA PCD, free OVA, or saline. On the seventh day after the final immunization, 5 × 10 5 B16-OVA tumor cells were subcutaneously inoculated into the right thighs of the mice. b Tumor growth curves within 18 days after the subcutaneous inoculation of B16-OVA tumor cells. Compared with that in the free OVA or saline group, tumor growth in the OVA PCD group was significantly inhibited ( n = 5). c Body weight change curves after tumor inoculation. The OVA PCD group showed no significant change in body weight, indicating that there was no apparent systemic toxicity ( n = 4–5). d Individual tumor growth curves for each group after subcutaneous inoculation of B16-OVA cells. e Tumor images from day 18 posttumor inoculation. “x” indicates mice that died on day 18 posttumor inoculation (experimental endpoint). The tumor volume data of this deceased mouse from the statistical analysis for day 18 are included in Fig. 4b. The data in b , c are presented as mean ± s.d. from two independent experiments. Groups were compared via one-way ANOVA with Tukey’s post hoc test. Flow cytometry analysis of the DC maturation rate (CD80 + CD86 + CD11c + ) ( f ) and the percentage of IFN-γ + CD8 + T cells ( g ) in the spleens of mice subjected to different treatments ( n = 3). Representative flow cytometry results ( h ) and quantification ( i ) of OVA antigen peptide-specific CD8 + T cells after ex vivo restimulation with the SIINFEKL peptide (8 μg/mL) ( n = 3). Representative images ( j ) and statistical analysis ( k ) of IFN-γ spot-forming cells in splenocytes after ex vivo restimulation with the SIINFEKL peptide (8 μg/mL) via the ELISPOT assay ( n = 3). l Evaluation of the cytotoxic effect of effector splenocytes (E) on target B16-OVA-GFP cells (T) after coincubation for 24 h at specified ratios, assessing the in vitro tumor cell-targeting cytotoxicity of effector T cells ( n = 3). Statistical analysis data in ( f–l ) are presented as mean ± s.d. from three independent experiments. Groups were compared via one-way ANOVA with Tukey’s post hoc test. Significance levels are indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns (not significant)

Article Snippet: The SIINFEKL (OVA 257-264 ) peptide was synthesized by Shanghai Bioengineering Co., Ltd. CpG ODN and the Alhydrogel® adjuvant were purchased from InvivoGen.

Techniques: In Vivo, Injection, Saline, Flow Cytometry, Ex Vivo, Enzyme-linked Immunospot, In Vitro

Journal: Signal Transduction and Targeted Therapy

Article Title: Condensate nanovaccine adjuvants augment CD8 + T-Cell-dependent antitumor immunity through mtDNA leakage-triggered cGAS-STING axis activation

doi: 10.1038/s41392-025-02447-w

Figure Lengend Snippet: Mechanistic research. Representative flow cytometry results ( a ) and quantitative analysis ( b , n = 3) of BMDC maturation. BMDCs were incubated with OVA PCD (10 μg/mL) for 48 h, followed by flow cytometry analysis of DC maturation (CD11c + CD80 + CD86 + ). Representative flow cytometry plots ( c ) and quantitative analysis ( d , n = 3) of MHC II molecule upregulation in BMDCs. BMDCs were incubated with OVA PCD (10 μg/mL) for 48 h, followed by flow cytometry detection of MHC II expression. Quantitative analysis of DC maturation ( e , n = 3) and OVA epitope presentation ( f , n = 3) in lymph nodes. C57BL/6 mice were immunized with two doses (containing an equivalent of 2.5 mg/kg OVA and 0.5 mg/kg CpG ODN). Lymph nodes were harvested 48 h after the final immunization, and single-cell suspensions were prepared for flow cytometry analysis of DC maturation and SIINFEKL-H-2Kb complex presentation on DC surfaces. g Cytokine levels (IL-12 and IFN-β) in supernatants measured by ELISA after BMDCs were incubated with OVA PCD (20 μg/mL) for 24 h ( n = 5). h Representative fluorescence images of mitochondrial colocalization with OVA PCD. Red: mitochondria. Green: FITC-OVA PCD. Scale bar: 15 μm. After 72 h of incubation of OVA PCD with DC2.4 cells, the mitochondria were labeled with a red tracker and observed via confocal microscopy. The white arrows indicate yellow fluorescent signals from the colocalization of FITC-OVA PCD (green) with mitochondria (red). i qPCR analysis of mtDNA leakage in the cytosol after BMDCs were incubated with OVA PCD (20 μg/mL) for 24 h ( n = 5). j cGAMP levels in BMDCs. After treatment with different concentrations of OVA PCD, ELISA was used to detect changes in the cGAMP content in BMDCs ( n = 5). k cGAMP levels in the lymph nodes. C57BL/6 mice ( n = 5) were immunized once a week 2 times. Lymph nodes were harvested 48 h after the final immunization, and single-cell suspensions were prepared for ELISA detection of cGAMP content in lymph node tissues. l Relative mRNA expression levels of type I interferons analyzed by RT‒qPCR after BMDCs were pretreated with different concentrations of the STING-IN-2 inhibitor followed by incubation with OVA PCD (20 μg/mL) for 24 h ( n = 4). All data are expressed as mean ± s.d. from three independent experiments. Unpaired two-tailed Student’s t tests were used for ( g ), and all other statistical analyses were performed via one-way ANOVA with Tukey’s post hoc test. Significance levels are indicated as ** p < 0.01, *** p < 0.001, **** p < 0.0001, and ns (not significant)

Article Snippet: The SIINFEKL (OVA 257-264 ) peptide was synthesized by Shanghai Bioengineering Co., Ltd. CpG ODN and the Alhydrogel® adjuvant were purchased from InvivoGen.

Techniques: Flow Cytometry, Incubation, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Labeling, Confocal Microscopy, Two Tailed Test

Journal: Mucosal immunology

Article Title: Triggering mouth-resident antiviral CD8 + T cells potentiates experimental periodontitis

doi: 10.1016/j.mucimm.2025.02.003

Figure Lengend Snippet: ( A ) VPEP model and experimental design. ( B ) Enumeration of gingival OT-I cells, CD4 + T cells, and PMNs seven days post-LIP in mice orally swabbed with gp33 or SIIN peptide. Data representative of two independent experiments with four-six mice per group per experiment. ( C ) Quantification of total bone within predefined coordinates surrounding the non-ligated second molar (Ref.; Left) and ligated second molar (Target; Middle), and their difference (Change; Right). ( D ) Representative maxilla iso -surfaces from LIP mice exposed to gp33 or SIIN peptide. Shaded regions highlight superficial differences between groups. ( E ) Alveolar bone loss, normalized to gp33/LIP mice from each of two independent experiments with two-four mice per group per experiment. Normal. = Normalized. ( F ) Experimental strategy to access the impact of oral CD8 + T RM reactivation on cLN cellularity and gingival inflammatory infiltrate. ( G ) Abundance of Ki67 + OT-I T cells within cLNs of day three LIP mice exposed to either gp33 or SIIN peptide. White circles represent % Ki67 + OT-I T cells within cLNs of non-ligated VPEP mice (No LIP). ( H ) Enumeration of total LN cellularity and individual DC subsets within cLNs of day three LIP mice exposed to gp33 (grey) or SIIN (blue) peptide. White bars represent DCs of the indicated subset within cLNs of non-ligated VPEP mice. Data in G and H representative of two independent experiments with four-five mice per group per experiment. ( I ) Ce3D imaging of day three LIP gingiva from mice of the indicated treatment group, stained with antibodies against E-Cadherin (teal) or pan-CD45 (red). ‘1′ indicates location of first molar. ‘B’ and ‘P’ denote buccal and palatal orientation, respectively. Insets magnify regions of interest. Data representative of three-four mice per group. Scale bar represents 1 mm. ( J ) Immunofluorescence (IF) microscopy of interstitial gingiva separating the second and third molar of non-ligated VPEP mice (Left), gp33/LIP mice (Middle), and SIIN/LIP mice (Right) seven days post ligation. White and yellow arrowheads indicate OT-I T cells in gingiva and adjacent molar dental pulp, respectively. Representative images are from at least three decalcified maxillae sections, per mouse, of three or more individual mice per group. I.P. = interproximal; 2nd = second molar; 3rd = third molar; NS = non-specific. Scale bar represents 50 μm. Error bars in all graphs represent mean ± SEM. Dots in C, E, and G represent individual mice. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 as determined by an unpaired Student’s t test between the relevant comparisons.

Article Snippet: For in vitro peptide stimulations, cells were isolated from the oral mucosa or cLNs and cultured for 5 h at 37 ◦ C in RPMI 1640 supplemented with 10 % FBS containing 1X Golgi-plug (BD) ± SIIN peptide (MedChemExpress) at a final concentration of 10 μg/ml.

Techniques: Imaging, Staining, Immunofluorescence, Microscopy, Ligation

Journal: Mucosal immunology

Article Title: Triggering mouth-resident antiviral CD8 + T cells potentiates experimental periodontitis

doi: 10.1016/j.mucimm.2025.02.003

Figure Lengend Snippet: ( A ) Experimental design to assess the contribution of oral CD103 + CD8 + T RM from CD103 neg recirculating CD8 + T cells on aggravated bone loss following LIP. Experiments were performed in CD103 KO host mice. ( B ) Fold change in the number of CD103 + OT-I T cells, CD4 + T cells, and PMNs isolated from LIP gingiva from mice of the indicated treatment group, normalized to their abundance in IgG/gp33 treated mice. Data representative of two independent experiments with three-four mice per group per experiment. ( C ) Quantification of total bone within predefined coordinates surrounding the non-ligated second molar (Ref.; Left) and ligated second molar (Target; Middle), and their differences (Change; Right). ( D ) Representative maxilla iso -surfaces from LIP mice exposed to SIIN peptide with an intact (Left; blue) or depleted (Right; green) oral CD103 + CD8 + T RM compartment. Shaded regions highlight superficial differences between groups. ( E ) Alveolar bone loss, normalized to IgG/gp33 mice, from each of two independent experiments with three-five mice per group per experiment. Error bars in all graphs represent mean ± SEM. Dots in C and E represent individual mice. **, P < 0.01; ***, P < 0.001 as determined by an unpaired Student’s t test between the relevant comparisons.

Article Snippet: For in vitro peptide stimulations, cells were isolated from the oral mucosa or cLNs and cultured for 5 h at 37 ◦ C in RPMI 1640 supplemented with 10 % FBS containing 1X Golgi-plug (BD) ± SIIN peptide (MedChemExpress) at a final concentration of 10 μg/ml.

Techniques: Isolation