Journal: Stem Cell Research & Therapy

Article Title: Antioxidant preconditioning improves therapeutic outcomes of adipose tissue-derived mesenchymal stem cells through enhancing intrahepatic engraftment efficiency in a mouse liver fibrosis model

doi: 10.1186/s13287-020-01763-y

Figure Lengend Snippet: Antioxidant preconditioning promotes the cell survival of ADSCs in vitro. a Cell viability of ADSCs with or without antioxidant pretreatment in H 2 O 2 -induced cell injury model ( n = 5 per group; ** p < 0.01; *** p < 0.001). b Representative images of cell morphology of ADSCs treated with H 2 O 2 (scale bar, 50 μm). c Cell apoptosis was evaluated by flow cytometry. d Western blotting of Bcl-2, Bax, and Cyclin-D1 in ADSCs treated with H 2 O 2 . e Relative expression of Bcl-2, Bax, and Cyclin-D1 in ADSCs treated with H 2 O 2 ( n = 3 per group; * p < 0.05; ** p < 0.01). ADSCs, adipose tissue-derived mesenchymal stem cells; G-ADSCs, ADSCs pretreated with reduced glutathione; M-ADSCs, ADSCs pretreated with melatonin; H 2 O 2 , hydrogen peroxide

Article Snippet: To determine the protective effect of antioxidant preconditioning on ADSC cell apoptosis, antioxidant pretreated ADSCs (passage 3) were cultured in a 6-well plate at a density of 1 × 10 5 cells/well for 24 h, followed by the incubation with 300 μM H 2 O 2 in fresh complete medium for another 24 h, and finally analyzed using an Annexin V-FITC apoptosis assay kit (Dojindo Molecular Technologies, Tokyo, Japan) in flow cytometry (Becton Dickinson, USA).

Techniques: In Vitro, Flow Cytometry, Western Blot, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Two Contrasting Classes of Nucleolus-Associated Domains in Mouse Fibroblast Heterochromatin

doi: 10.1101/484568

Figure Lengend Snippet: A schematic workflow of nucleolar isolation. Details that differ between the crosslinked and non-crosslinked versions of the preparation are illustrated in . Comparison of NADs to heterochromatin features, and analysis of NAD-seq by multiple bioinformatic packages, including NADfinder . All data shown are from MEF cells. In this panel, the strongly nucleolar-associated chromosome 19 is shown in its entirety. Tracks shown from the top are DNA Replication timing data (early replicating regions have positive values) and LADs (red, ). Below those are raw sequence data from crosslinked cells (blue, experiment #26): read counts for total genomic DNA (Genomic), nucleolar-associated DNA (Nucleolar), and the Nucleolar/Genomic ratio. Next are shown comparisons of peaks called by the bioinformatic packages MACS (broad peaks setting), EDD (50K bins), Hidden Domains (50K bins), and normr (50K bins, v1<0.01, q<10e-03). At the bottom are peaks called by NADfinder (default cutoff q<0.05). All software tested except NADfinder called almost the entire chromosome as peaks. NADs called by NADfinder generally correlate with LADs and late replication timing. As in , showing chromosome 5. Note the gradual decrease in the ratio going from left to right (away from the centromere on the left of this acrocentric chromosome). Unlike MACs and EDD, NADfinder was able to call peaks on regions distal from the centromere.

Article Snippet: An additional plate grown in parallel was reserved for total genomic DNA extraction (Quick-DNA Universal Kit (Zymo Research, CA)).

Techniques: Isolation, Sequencing, Software

Journal: bioRxiv

Article Title: Two Contrasting Classes of Nucleolus-Associated Domains in Mouse Fibroblast Heterochromatin

doi: 10.1101/484568

Figure Lengend Snippet: Schematic representation of crosslinked and non-crosslinked methods used for isolation of nucleoli. The abbreviations of the fractions analyzed by immunoblotting in panel D are indicated. Phase microscopy images of nucleoli purified via crosslinked (experiment #24, see Supplemental Table S1) and non-crosslinked (experiment #29) methods (20x magnification, scale bar 10 μm). qPCR analyses of rDNA enrichment in purified nucleoli compared to genomic DNA. We observed 30-50-fold rDNA enrichment in purified non-crosslinked nucleoli (experiment #33), depending on the primer set used. An 8-13-fold enrichment was observed in crosslinked samples (mean values for experiments #24, 26, 28). Immunoblot analyses of crosslinked and non-crosslinked preparations. Fibrillarin was used as a nucleolar (nus) marker. Nuclear periphery proteins (porin Nup62 and LaminA/C) were present in total (tot) and crude nuclear (nuc, S1) extracts, but depleted from nucleolar (nus) fractions. Cytoskeletal protein actin was enriched in total and crude cytosolic (cyt, S1) fractions, but not in nucleoplasmic (np) or nucleolar (nus) fractions. Analysis of RNAs from fractions obtained during a non-crosslinked preparation. The nucleolar-specific small RNA U3 as well as the ribosomal 5S species are illustrated.

Article Snippet: An additional plate grown in parallel was reserved for total genomic DNA extraction (Quick-DNA Universal Kit (Zymo Research, CA)).

Techniques: Isolation, Western Blot, Microscopy, Purification, Marker

Journal: Nature Medicine

Article Title: Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

doi: 10.1038/s41591-024-02894-y

Figure Lengend Snippet: a , Vaccine-induced responses assessed by IFNγ ELISpot assays without cytokine stimulation ( n = 22). Cumulative magnitudes were collected from positive epitopes before and after treatment. The postvaccination response is the ‘best’ (highest magnitude) response for each patient across time points. SFU, spot-forming units. b , Total neoantigens (gray bars) and positive neoantigens before (black bars) and after (red bars) vaccination in each patient’s PTCV assessed by IFNγ ELISpot. c , Percentage of positive responding epitopes by groups. The definition of a neoantigen-specific ELISpot response can be found in . d , Representative density plots (patient 22) of the T cell markers CD69, Ki67, CD107a, IFNγ and TNF upon stimulation with patient-specific PTCV epitope pools. e , f , Polyfunctionality assessed by Boolean gating of CD4 + or CD8 + cytokine-producing populations. T cell activation (CD69 and CD107a; e ) and proliferation (Ki67; f ) were assessed together with the double-positive expression of GZMA and perforin 1 (PRF1) to evaluate the cytolytic potential of neoantigen-reactive T cells. Four patients (patients 7, 11, 18 and 22) were analyzed in d – f . g , T cell clones expanded in the periphery and the new or expanded clones enriched in the matched tumor sample for each patient ( n = 14). Total PBMC and tumor-associated T cell expansion were calculated by comparing posttreatment to pretreatment PBMC or tumor samples (differential abundance statistical analysis). h , Cumulative frequencies of peripherally expanded TCR rearrangements in tumor biopsy samples. i , Expanded clone numbers in tumor biopsy samples. j , k , TCR clonality ( j ) and repertoire richness ( k ) in tumor biopsy samples ( n = 14). PD (red), SD (gray), and CR/PR (blue). Error bars correspond to the upper s.e.m. of each group. Simpson clonality reports the distribution of TCR rearrangements in a sample, in which 0 indicates an even distribution of frequencies and 1 indicates an asymmetric distribution. TCR repertoire richness reports the mean number of unique rearrangements. Lower numbers indicate focused TCR diversity. Filled symbols in c , e and f and open circles in h and i represent individual patients; the box extends from the 25th to the 75th percentile; the line inside the box is the median; and the whiskers extend from the minimum to the maximum value. Significance between groups was evaluated by a two-tailed Mann–Whitney test ( c ); significance within groups was evaluated by a two-tailed Wilcoxon rank test ( a , h – k ).

Article Snippet: The ELISpot assay was performed (FlowMetric) using the standard ELISpot protocol and the human IFNγ single-color ELISpot kit (all reagents and plates included; stock keeping unit no. hIFNgp-1M, Cellular Technology Limited).

Techniques: Enzyme-linked Immunospot, Immunopeptidomics, Activation Assay, Expressing, Clone Assay, Two Tailed Test, MANN-WHITNEY

Journal: Nature Medicine

Article Title: Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

doi: 10.1038/s41591-024-02894-y

Figure Lengend Snippet: a , A two-tailed, Spearman correlation between positive epitopes versus the total number of neoantigens included in each patient’s PTCV. b , Interquartile survival analysis by magnitude (best response post-PTCV; IFNγ SFU) of evaluable patients. Six patients per group; Log-rank (Mantel-Cox) test. c , T cell reactivity evaluated by IFNγ-ELISpot stratified by responder or non-responder groups. Symbols represent individual patients (CR/PR, n = 6; SD/PD, n = 15 per group), the box extends from the 25th to the 75th percentile, the line inside the box is the median, and the whiskers extend from the minimum to maximum values. Significance was tested by a two-tailed, Mann-Whitney statistical analysis. d , Cumulative number of targetable neoantigens encoded in the vaccines of the 22 pts whose samples were evaluated by IFNγ ELISpot assay. The neoantigens are stratified by the predicted MHC Class I binding affinity as high (< 500 nM), medium (500–1000nM), and low (1000–2000 nM). While the majority of the neoantigens encoded in the PTCVs were categorized as high-affinity antigens, the medium and low-affinity antigens also yielded positive responses in a similar proportion.

Article Snippet: The ELISpot assay was performed (FlowMetric) using the standard ELISpot protocol and the human IFNγ single-color ELISpot kit (all reagents and plates included; stock keeping unit no. hIFNgp-1M, Cellular Technology Limited).

Techniques: Two Tailed Test, Immunopeptidomics, Enzyme-linked Immunospot, MANN-WHITNEY, Vaccines, Binding Assay

Journal: Nature Medicine

Article Title: Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

doi: 10.1038/s41591-024-02894-y

Figure Lengend Snippet: a , Most frequent TCRs identified by TCRseq and RNAseq in a patient (before vaccination versus week 9 after vaccination, pairwise scatterplots). Different superscript letters show selected high-frequency new T cell clones detected in PBMCs after vaccination and their abundance in the tumor. Orange, green, and gray circles represent expanded, contracted and not significantly changed T cell clones, respectively. b , CDR3 sequences of the three TCRs (from patient 8; TCR 1, TCR 2 and TCR 3) selected for cloning and their frequency (freq.) in the tumor before (pre-Vax) and after (post-Vax) vaccination. Selected cloned TCRs were present in high frequency only in the peripheral blood and tracked into the tumor after treatment. c , UMAP (Uniform Manifold Approximation and Projection) and stacked barplot indicating the single-cell cluster identities and number of cells for each of the three TCRs selected for cloning. d , Patient-specific clonal TCR sequences were gene optimized and inserted into the pMXs-IRES-GFP retroviral plasmid vector containing the viral packaging signal, transcriptional and processing elements, and the GFP reporter gene. MuLV, murine leukemia virus; Mo-MuLV, Moloney MuLV; LTR, long terminal repeat; Amp R , ampicillin resistance. e , TCR-engineered T cells (GFP + ) from unvaccinated patient-derived PBMCs were stimulated for 6 h with epitope pools or the nonspecific epitope CTA1 (10 µg ml −1 ), and CD69 expression was evaluated by flow cytometry. Peptide pool 1 included the most reactive epitopes measured by ELISpot, whereas pool 2 (consisting of peptides corresponding to epitopes 21–40) served as an internal negative control.

Article Snippet: The ELISpot assay was performed (FlowMetric) using the standard ELISpot protocol and the human IFNγ single-color ELISpot kit (all reagents and plates included; stock keeping unit no. hIFNgp-1M, Cellular Technology Limited).

Techniques: Clone Assay, Cloning, Retroviral, Plasmid Preparation, Virus, Derivative Assay, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Negative Control

Journal: Nature Medicine

Article Title: Personalized neoantigen vaccine and pembrolizumab in advanced hepatocellular carcinoma: a phase 1/2 trial

doi: 10.1038/s41591-024-02894-y

Figure Lengend Snippet: a , Change in target lesion from baseline (%) evaluated by RECIST 1.1 and tumor imaging scans at various treatment time points. Liver lesion (red arrows); Adrenal lesion (yellow arrows). b , TCR frequencies (%) of 25 expanded T cell clones at pre- or post-treatment in the periphery or in the target lesion (liver). c , PBMCs (3 × 10^5/per well) were stimulated with vaccine-encoded epitopes at the concentration of 10 μg/mL for 18–24 hours. Cells were evaluated for the presence of vaccine-induced neoantigen-specific responses prior to and post-personalized GNOS-PV02 vaccination using an interferon IFNγ ELISPOT assay without cytokine stimulation. The bar indicates the mean SFU of n = 3 individual technical replicates ± SD per group. d , Neoantigen-specific T cell activation was evaluated by stimulating patient-derived PBMCs (week 9) with DMSO, PROS1, or OBSCN peptides ex vivo by intracellular cytokine staining. e , Venn diagram of neoantigens identified in the liver (day 0) and adrenal lesions (week 54) by RNA/DNA sequencing. f , Monitoring of the dynamic expression of liver- or adrenal-specific targets collected over 50 weeks by ctDNA analysis. g , T cell infiltration/activation biomarkers are evaluated at pre-treatment and post-treatment (liver lesion), and from the adrenal lesion (week 54). The T cell suppressor, IDO1, is separated from the other markers with a dashed line. The significance between evaluated stacked gene expression groups was tested by a two-tailed, Mann-Whitney statistical analysis.

Article Snippet: The ELISpot assay was performed (FlowMetric) using the standard ELISpot protocol and the human IFNγ single-color ELISpot kit (all reagents and plates included; stock keeping unit no. hIFNgp-1M, Cellular Technology Limited).

Techniques: Imaging, Clone Assay, Concentration Assay, Enzyme-linked Immunospot, Activation Assay, Derivative Assay, Ex Vivo, Staining, Immunopeptidomics, DNA Sequencing, Expressing, Gene Expression, Two Tailed Test, MANN-WHITNEY

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Ad‐E7P therapeutic vaccine induces antigen‐specific immune responses and potent antitumor protection in TC‐1 tumor model. (A) Schematic of the adenoviral‐based therapeutic vaccine. (B) Experimental schema for evaluating the antitumor efficacy of Ad‐E7P in the TC‐1 tumor model. C57BL/6 mice were subcutaneously inoculated with 1 × 10 6 TC‐1 tumor cells in the right flank, followed by weekly intramuscular (i.m.) immunizations with 10 9 viral particles (VP) of Ad‐E7P or the empty vector (Adv) for a total of two doses when tumor volumes reached approximately 50 mm 3 . Mice in the negative control group received PBS. On Day 17, lymph nodes and spleens were harvested for immune cell analysis. (C) Tumor growth curves of mice treated with Ad‐E7P, Adv, or PBS ( n = 5 mice per group). (D and E) Flow cytometric analysis of DCs, total T cells, and CD8+ T cells in the lymph nodes (D), and CD8+ T cells in the spleen (E) following immunization ( n = 5 mice per group). (F) On Day 17, antigen‐specific T cell activation in splenocytes was assessed by ELISpot assay following in vitro stimulation with the E7 49‐57 peptide. SFU, spot‐forming unit ( n = 5 mice per group). (G) Treatment schedule for Ad‐E7P vaccination and correlative immune kinetics analysis. (H and I) Flow cytometric analysis of the percentages of CD8+ T cells (H) and ELISpot analysis of IFN‐γ–producing cells (I) in the spleen at different time points following Ad‐E7P vaccination ( n = 3 per group). Data are presented as the means ± SD. One‐way analysis of variance (ANOVA) with Tukey's multiple comparisons test was performed for all comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Plasmid Preparation, Negative Control, Cell Analysis, Activation Assay, Enzyme-linked Immunospot, In Vitro

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: The therapeutic vaccine Ad‐E7P induces intratumoral immune activation and reduces tumor recurrence. (A) Representative flow cytometry plots of CD8+ TILs. Numbers indicate the percentage of CD8+ TILs within the gated population. (B) Quantification of CD8+ TILs is shown ( n = 5 per group). (C) Representative hematoxylin and eosin (H&E)–stained images of paraffin‐embedded TC‐1 tumor sections from different treatment groups. TLSs were observed adjacent to tumors in Ad‐E7P‐vaccinated mice and confirmed by both H&E and CD3 immunohistochemical staining. Scale bars: 200 µm (overview) and 50 µm (zoomed‐in view). (D and E) Experimental scheme. TC‐1 tumor‐regressing mice, cured by Ad‐E7P vaccination, were re‐inoculated with the same number of tumor cells in the left flank on day 60, and survival was monitored (D). Untreated mice served as controls. Tumor growth curves (E, left) and corresponding survival kinetics of mice after tumor rechallenge (E, right) (Control, n = 5 mice; Ad‐E7P, n = 10 mice). (F and G) Flow cytometric analysis of splenic CD8+ T cells after tumor rechallenge. Representative plots from Day 7, including a CD8 fluorescence‐minus‐one (FMO) control to identify the positive population (F), and the percentages of CD8+ T cells on Days 7, 14, 21, 30, and 60 (G) ( n = 3 per group). (H) Summary statistics for ELISpot assays on Day 7 and 14 after tumor rechallenge ( n = 5 per group). Data are presented as mean ± SD. r. Statistical analyses were conducted by one‐way ANOVA with Tukey's correction for multiple comparisons in (B), by two‐way ANOVA in (H), and by log‐rank (Mantel‐Cox) test in (E). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Activation Assay, Flow Cytometry, Staining, Immunohistochemical staining, Control, Fluorescence, Enzyme-linked Immunospot

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Ad‐E7P vaccine combined with the oncolytic virus SKV‐012 inhibits tumor progression and induces a potent antitumor immune response in the TC‐1 tumor‐bearing mouse model. (A) Experimental scheme. C57BL/6 mice were subcutaneously inoculated with 1 × 10 6 TC‐1 tumor cells in the right flank. When tumor volumes reached approximately 50 mm 3 , mice were assigned to receive Ad‐E7P, SKV‐012, a combination of Ad‐E7P and SKV‐012, or PBS as a control. For Ad‐E7P group, mice received two doses of 10 9 VP Ad‐E7P administered once a week. For SKV‐012 group, mice received three intratumoral injections of 10 6 PFU SKV‐012 every 3 days. For combination treatment group, mice received two doses of 10 9 VP Ad‐E7P once a week and three doses of 10 6 PFU SKV‐012 intratumorally every three days. (B) Tumor growth curves ( n = 5 per group). (C and D) Representative IFN‐γ ELISPOT images (C) and summary of ELISPOT results (D) from splenocytes stimulated in vitro with the E7 49‐57 peptide ( n = 5 per group). (E) Proportions of various immune cell populations in lymph nodes and spleens ( n = 3 per group). (F and G) Representative flow cytometric analysis of CD86 expression on dendritic cells in the lymph nodes (F), and quantification of mean fluorescence intensity (MFI) of CD86 expression (G) ( n = 3 per group). (H and I) Representative flow cytometric analysis of IFN‐γ+ CD8+ T cells in both lymph nodes and spleen (H), and statistical analysis of the results (I). ( n = 3 per group). Data are presented as mean ± SD. One‐way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Virus, Control, Enzyme-linked Immunospot, In Vitro, Expressing, Fluorescence

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Combination of Ad‐E7P vaccine and SKV‐012 provides long‐term protection in the TC‐1 tumor model and induces robust antitumor immunity in the mEERL tumor model. (A) Experimental design. Mice cured by the SKV‐012+Ad‐E7P combination treatment were re‐inoculated with 1 × 10 6 TC‐1 tumor cells in the left flank on Day 60, and survival was monitored. Untreated mice served as controls. (B) Survival kinetics of mice after tumor rechallenge (Control, n = 5 mice; SKV‐012+Ad‐E7P, n = 10 mice). (C) On Days 7, 14, 21, 30, 60, and 90 post‐tumor challenge, the proportion of CD8+ T cells in the spleen was assessed by flow cytometry ( n = 3 per group). (D) On Days 7 and 14 post‐tumor challenge, spleens were harvested, and the frequency of IFN‐γ‐producing T cells was assessed using an ELISPOT assay following in vitro stimulation with the E7 peptide ( n = 5 per group). (E and F) Differential expression of KLRG1 and CD127 on spleen T cells. Representative contour plots (E, Day 7) and quantification (F) of KLRG1 and CD127 expression are shown ( n = 5 per group). (G) Experimental Scheme. C57BL/6 mice were subcutaneously implanted with 2 × 10 6 mEERL tumor cells. Mice were treated with Ad‐E7P, SKV‐012, Ad‐E7P + SKV‐012, or PBS (control) on Day 12, when tumor volumes reached approximately 50 mm 3 . For Ad‐E7P group, mice received two doses of 10 9 Ad‐E7P administered once a week. For SKV‐012 group, mice received three intratumoral injections of 10 6 PFU SKV‐012 every three days. For combination treatment group, mice received two doses of 10 9 VP Ad‐E7P once a week and three doses of 10 6 PFU SKV‐012 intratumorally every 3 days. Tumor volume was monitored. (H) Tumor growth curves in mEERL model ( n = 5 per group). (I) Survival kinetics in mEERL model (Control, n = 5 mice; Treatment group, n = 7 mice). Mice from independent experimental cohorts. (J and K) Representative images of IHC staining for CD3 in mEERL tumor tissue sections on Day 20 (K), and quantification of CD3+ cells per tumor area (J) (cells/mm 2 ) ( n = 3 mice per group; N = 3 images/field of view per mouse).

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunospot, In Vitro, Quantitative Proteomics, Expressing, Immunohistochemistry

Journal: MedComm

Article Title: Combination of Vaccine With IL‐12‐Armed Oncolytic Virus SKV‐012 Synergistically Potentiates Immune Responses in HPV‐Associated Malignancies

doi: 10.1002/mco2.70737

Figure Lengend Snippet: Ad‐MP triggers antigen‐specific T cell response and enhances antitumor efficacy with SKV‐012 in vitro. (A) Experimental design for assessing the SKV‐012 + Ad‐E7P immune response in vitro. The figure panel was created in BioRender. (B) Representative flow cytometry plots of CD80 and CD86 expression in DCs. DCs were isolated and induced from the peripheral blood of HPV‐related tumor patients and loaded with the Ad‐MP vaccine, and were tracked at Day7. (C and D) Representative flow cytometry plots of activated CD69 expression in T cells (C) and quantification of CD69+CD3+ T cells are shown (D). The Ad‐MP‐loaded DCs were co‐cultured with autologous PBMCs at a ratio of 1:100 for 24 h ( n = 3 per group). Empty‐loaded DC were prepared as a control. (E) The number of IFN‐γSFUin PBMCs was assessed after 24 h of stimulation with single peptides ( n = 3 per group). Before performing the ELISPOT assay, antigen‐loaded DCs were pre‐co‐cultured with autologous PBMCs from HPV‐related tumor patients for 24 h ( n = 3 per group). (F) The concentrations of IL‐12, IFN‐γ, IL‐2, and TNF‐α in the supernatants were measured by ELISA after 48 h of co‐culture of primary tumor cells with autologous PBMCs and DCs. Prior to co‐culturing with activated autologous PBMCs, tumor cells were infected with SKV‐012 at an MOI of 0.01 for 24 h to assess responses ( n = 3 per group). Data are presented as mean ± SD. One‐way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). ns , not significant.

Article Snippet: The frequency of IFN‐γ–producing T cells in human PBMCs was assessed by ELISpot following co‐culture with mature DCs at a DC‐to‐lymphocyte ratio of 1:100 for 24 h. Subsequently, PBMCs were seeded at 2 × 10 5 cells per well in ELISpot plates (human IFN‐γ ELISpot kit, R&D Systems, Cat#EL285) and stimulated with single peptide 1–4 at a concentration of 10 μg/mL for 24 h at 37°C.

Techniques: In Vitro, Flow Cytometry, Expressing, Isolation, Cell Culture, Control, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Infection