siinfekl ova peptide  (InvivoGen)

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: iScience

Article Title: Simulating CD8 T cell exhaustion: A comprehensive approach

doi: 10.1016/j.isci.2025.112897

Figure Lengend Snippet: Characterization of the development of in vitro exhausted CD8 T cells Freshly isolated cells (Naive), cells activated with SIINFEKL for 24h (Activated), and chronically stimulated cells (Exhausted) were phenotypically characterized. (A) In vitro experimental design of chronic stimulation to produce exhausted T cells. (B) Representative gating strategy and percentages of T cells per cell-cycle stage obtained by EdU and Hoescht staining. (C) tSNE using IRs with representative histograms for indicated markers on naive, activated, and in vitro exhausted T cells. (D) Quantification of geometric mean fluorescence intensity (MFI) of individual markers. (E) Intracellular expression of Granzyme B with representative histograms. (F) Background unstimulated corrected percentage of double-positive populations of CD107a+ IFNγ+, CD107a+ TNFα+, and IFNγ+ TNFα+ in stimulated cells with representative contour plot of stimulated samples. Representative data from three independent experiments. Mean ± SEM shown with symbols representing individual mice. Statistical analysis performed by two-way ANOVA with a Tukey’s multiple-comparisons test (B and D) or Mann–Whitney tests (E and F). Only significant p values indicated in the figure.

Article Snippet: The OVA peptide SIINFEKL (AnaSpec) was added at 3 μg/mL with 100 U/mL human IL-2 (Peprotech).

Techniques: In Vitro, Isolation, Staining, Fluorescence, Expressing, MANN-WHITNEY

Journal: iScience

Article Title: Simulating CD8 T cell exhaustion: A comprehensive approach

doi: 10.1016/j.isci.2025.112897

Figure Lengend Snippet: Metabolic alterations of in vitro exhausted T cells Freshly isolated cells (Naive), cells activated with SIINFEKL for 24h (Activated), and chronically stimulated cells (Exhausted) characterized for metabolic changes. (A) Quantification of the spare respiratory capacity. (B) Glucose uptake quantification using 2-NBDG, representative histograms are shown. (C) Determination of cellular ROS content with representative histograms shown. (D) Measurement of mitochondrial mass with MitoTracker Deep Red, with representative histograms shown. (E) Confocal visualization of mitochondria, 3D view with MitoTracker in red and DAPI for nucleus in blue. Scale bar = 5 μm. (F) Mitochondrial membrane potential assessed using TMRE, representative histograms with gates of potential. (G) Transmission electron microscopy (TEM) of naive, activated, and exhausted T cells with inset zoom on mitochondria, scale bars are as follows for cells and mitochondria images; naive 4 μm and 500 nm; activated 4 μm and 1 μm and exhausted 4 μm and 1 μm. (H) Number of mitochondria per cell. (I–L) Area and circularity of mitochondria and cells respectively. Representative data from three (A-E) and two (F) independent experiments. For TEM at least three sample preparations per group were imaged and at least 27 cells per sample were quantified. Mean and ±SEM shown with symbols representing individual mice (A-F), individual cells (H, K, and L) or individual mitochondria (I and J). Statistical analyses were performed by Mann–Whitney tests (A-D and F) and Kruskal-Wallis ANOVA with Dunn’s multiple comparisons test (H-L). Only significant p values indicated in the figure.

Article Snippet: The OVA peptide SIINFEKL (AnaSpec) was added at 3 μg/mL with 100 U/mL human IL-2 (Peprotech).

Techniques: In Vitro, Isolation, Membrane, Transmission Assay, Electron Microscopy, MANN-WHITNEY