human ifn γ elispot kit  (R&D Systems)

Journal: Human Mutation

Article Title: Whole‐Exome Sequencing to Screen Personal Neoantigens With High Immunogenicity in Patients With Microsatellite Stability (MSS)–Advanced Colorectal Cancer

doi: 10.1155/humu/3876230

Figure Lengend Snippet: Evaluating the immunogenicity of neoantigens from patients with CRC. Autologous PBMCs were stimulated with candidate mutated peptides every 3 days in the presence of IL‐2. On Day 10, T‐cell responses to each antigen were measured by an IFN‐ γ ELISpot assay. The PBMCs were obtained from P1 with CRC. No peptide (medium only) or VSV‐NP 43-69 (STKVALNDLRAYVYQGIKSGNPSILHI) stimulation was used as a control. Data are presented as mean ± SD of three independent experiments. ∗ p < 0.05 and ∗∗ p < 0.01 compared with IFN‐ γ production by PBMCs stimulated without peptide or with VSV‐NP 43-69 .

Article Snippet: After 10 days, the IFN‐ γ response from the prestimulated T cells against neoantigens was assessed using ELISpot assays with a Human IFN‐ γ ELISpot kit (Mabtech).

Techniques: Immunopeptidomics, Enzyme-linked Immunospot, Control

Journal: Human Mutation

Article Title: Whole‐Exome Sequencing to Screen Personal Neoantigens With High Immunogenicity in Patients With Microsatellite Stability (MSS)–Advanced Colorectal Cancer

doi: 10.1155/humu/3876230

Figure Lengend Snippet: Cytotoxicity of NRTs raised by in vitro stimulation of PBLs of P1. NRTs were induced with autologous ZNF169‐A275S– and CDH4‐V456M–pulsed DCs derived from the PBLs of P1. On Day 7, after the third stimulation, the NRTs were harvested for analysis. (A) IFN‐ γ secretion by NRT lines in response to mutated and WT peptides. IFN‐ γ ‐positive SFCs/10 5 NRTs were detected by cytokine‐specific ELISpot. (B–F) Cytotoxicity at the indicated E:T ratios measured by a CCK‐8 kit. Peptide‐specific targets were mutated protein–pulsed T2 cells and minimally nucleated SW480 cells, whereas VSV‐NP 43-69 –pulsed T2 cells, T2 cells alone, and SW480 cells alone were used as controls. Data are expressed as mean ± SEM ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001).

Article Snippet: After 10 days, the IFN‐ γ response from the prestimulated T cells against neoantigens was assessed using ELISpot assays with a Human IFN‐ γ ELISpot kit (Mabtech).

Techniques: In Vitro, Derivative Assay, Enzyme-linked Immunospot, CCK-8 Assay

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