recombinant mouse ifn g  (R&D Systems)

Journal: The Journal of Experimental Medicine

Article Title: Chimeric MHC class I– and II–restricted non-self epitopes broaden antitumor T cell reactions

doi: 10.1084/jem.20250025

Figure Lengend Snippet: Identification of neoantigens of Bpmel. (A) Distribution of exome mutations at the chromosomal level in Bpmel. Colors indicate the number of mutations. This WES analysis identified 14,035 exomic mutations. (B) Workflow for the identification of Bpmel neoantigens. The numbers of peptide candidates screened at each step are shown. (C) Mutation types of Bpmel, as identified through WES analysis. At the RNA level, 328 missense variants were identified in 4,934 genes. (D) The 328 epitopes generated by missense mutation were screened based on their predicted affinities for H2-Kb and H2-Db. The top 1% of the affinity ranking was selected as high-affinity peptides (left and center). Candidates were further selected based on the ratio of affinity before and after mutation and on fragments per kb of transcript per million mapped reads (FPKM) (right). This screening identified 44 candidates (detailed sequences are listed in ). (E) The 44 selected peptides were synthesized for the in vitro T cell screening assay. CD8 + T cells were isolated from the DLNs of Bpmel-bearing mice and stimulated with the indicated peptides (5 µg/ml) for 8 h. The percentage of IFN-γ–releasing CD8 + T cells was evaluated using IFN-γ–catching assays. Four WT and mutant peptide sequences that induced IFN-γ secretion are shown (center). (F) DLN single cells were stimulated with the indicated peptides (5 µg/ml) for 8 h. IFN-γ secretion was detected using the ELISpot assay. OVAI was used as a negative control. Dot numbers are shown. All four candidates except AZIN1 MUT promoted CD8 + T cell release of IFN-γ. (G) Control tetramers (Tet-ctrl), Scap MUT , and ARAF MUT tetramers (the AIFM MUT MHC tetramer could not be constructed) were used to stain CD8 + TILs. Here, 0.17–3.03% of ARAF MUT -specific CD8 + T cells were detected in the CD8 + TILs of all Bpmel-bearing mice ( n = 5 per group). Representative images are shown.

Article Snippet: The ELISpot assay was performed using a Mouse IFN-γ ELISpot (ImmunoSpot) according to the manufacturer’s instructions.

Techniques: Immunopeptidomics, Mutagenesis, Generated, Synthesized, In Vitro, Screening Assay, Isolation, Enzyme-linked Immunospot, Negative Control, Control, Construct, Staining

Journal: The Journal of Experimental Medicine

Article Title: Chimeric MHC class I– and II–restricted non-self epitopes broaden antitumor T cell reactions

doi: 10.1084/jem.20250025

Figure Lengend Snippet: Notch MUT , a long neoantigen, is an endogenous complete T cell antigen of MC38. (A) The DNA sequence of WT Notch2 (left). The red arrow indicates the region deleted by the mutation in MC38. DNA sequences of mutated Notch2 in MC38 cells were obtained through WES (right). (B) The sequence of the MC38 Notch2 cDNA. (C) Tumor volumes in mice treated with Bpmel-EGFP or Bpmel-Notch2 MUT ( n = 5 per group). (D) MC38 tumor volumes in naïve or Bpmel-Notch2 MUT –rejected mice ( n = 5 per group). (E) Percentage of IFN-γ–releasing CD4 + or CD8 + T cells after zsGreen-HisTag or Notch2 MUT -HisTag stimulation for 16 h ( n = 4 mice per group). CD3 + CD4 − T cells were identified as CD8 + T cells. (F) MHC II peptide-binding map of Notch2 MUT . The binding affinity of Notch2 MUT peptides spanning the Notch2 MUT sequence was predicted in silico and expressed as IC50 to I-Ab using hmMHC. The candidate peptide used for verification is marked in yellow. (G) Sorted CD4 + T cells from the DLN of Bpmel-Notch2 MUT tumor-bearing mice were co-cultured with DCs isolated from naïve mice and stimulated with the indicated peptides for 24 h. The left panel shows the number of IFN-γ–producing spot-forming cells (SFC) following peptide stimulation ( n = 3 per group). The right panel displays three representative wells from the IFN-γ ELISPOT assay. All data are representative of three similar experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison.

Article Snippet: The ELISpot assay was performed using a Mouse IFN-γ ELISpot (ImmunoSpot) according to the manufacturer’s instructions.

Techniques: Sequencing, Mutagenesis, Binding Assay, In Silico, Cell Culture, Isolation, Enzyme-linked Immunospot, Comparison

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: Effects of MC1R deletion on T cell subsets in CIA mice. (a-d) Fluorescence-activated cell sorting (FACS) kanalysis of splenic tissue cells from WT and MC1R-KO mice under CIA. Percentages of Th1 (a and c) and Th2 (b and d) cells were measured. (e-h) FACS analysis of splenic tissue cells from WT and MC1R-KO mice under CIA. Percentages of Treg (e and g) and Th17 (f and h) cells were measured. n =3. Data are the mean ± Standard deviation. ✶ P < 0.05, ✶ ✶ P < 0.01, ns: Not significant. MC1R: Melanocortin 1 receptor, CIA: Collagen-induced arthritis, MC1R KO: Melanocortin 1 receptor knockout, Th: T helper, WT: Wild type, IFN-g: Interferon-g; Treg: Regulatory T, Foxp3: Forkhead box protein P3.

Article Snippet: IL-17 (DY421), IFN-g (DY485), IL-10 (DY417), IL-4 (DY404), tumor necrosis factor-a (TNF-a) (DY410), and IL-6 (DY406) kits were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Fluorescence, FACS, Standard Deviation, Knock-Out

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: MC1R-KO promotes the production of inflammatory cytokines in CII-treated mice. (a and b) ELISA measurement of pro-inflammatory cytokines, including IL-17 and IFN-g. (c and d) ELISA measurement of anti-inflammatory cytokines, including IL-10 and IL-4. (e-g) qRT-PCR measurement of cytokines, including IL-6, TNF-a, and IL-1b. (h-m) Western blot analysis to validate changes in immune-related markers by detecting the protein expression levels of p-STAT3, STAT3, T-bet, RORgt, IL-17, and IFN-g. n = 3. Data are the mean ± Standard deviation. ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ns: Not significant. MC1R KO: Melanocortin 1 receptor knockout, ELISA: Enzyme-linked immunosorbent assay, IL: Interleukin, IFN-g: Interferon, TNF-a: Tumor necrosis factor-a, p-STAT3: Phospho-signal transducer and activator of transcription 3, RORgt: Retinoic acid receptor-related orphan receptor g-t, GAPDH: Glyceraldehyde-3-phosphate dehydrogenase, qRT-PCR: Quantitative reverse transcription polymerase chain reaction.

Article Snippet: IL-17 (DY421), IFN-g (DY485), IL-10 (DY417), IL-4 (DY404), tumor necrosis factor-a (TNF-a) (DY410), and IL-6 (DY406) kits were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Western Blot, Expressing, Standard Deviation, Knock-Out, Reverse Transcription, Polymerase Chain Reaction

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: MC1R-KO mouse splenocytes displaying higher CII-specific T cell immune response in vitro . (a-d) Proportions of Th17 cells and Treg cells within the cell population measured by FACS analysis. (e and f) Changes in the levels of pro-inflammatory cytokines IL-17 and IFN-g measured by ELISA. (g and h) Changes in the levels of anti-inflammatory cytokines IL-10 and IL-4 measured by ELISA. n =3. Data are the mean ± Standard deviation. ✶ P < 0.05, ✶ ✶ P < 0.01, ns: Not significant. MC1R KO: Melanocortin 1 receptor knockout, Th17: T helper 17, ELISA: Enzyme-linked immunosorbent assay, IL: Interleukin, IFN-g: Interferon.

Article Snippet: IL-17 (DY421), IFN-g (DY485), IL-10 (DY417), IL-4 (DY404), tumor necrosis factor-a (TNF-a) (DY410), and IL-6 (DY406) kits were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Standard Deviation, Knock-Out

Journal: CytoJournal

Article Title: Melanocortin 1 receptor alleviates collagen-induced arthritis by upregulating T helper 1/T helper 17 cells and downregulating regulatory T cells

doi: 10.25259/Cytojournal_16_2025

Figure Lengend Snippet: Stattic inhibits the expression levels of proinflammatory factors in MC1R-KO mice. (a-e) Changes in the levels of pro-inflammatory cytokines IL-17, IFN-g, IL-6, IL-1b, and TNF-a measured by ELISA. (f and g) Changes in the levels of anti-inflammatory cytokines IL-10 and IL-4 measured by ELISA. (h-m) Western blot analysis to validate changes in immune-related markers by detecting the protein expression levels of p-STAT3, STAT3, T-bet, RORgt, IL-17, and IFN-g. n = 3. Data are the mean ± Standard deviation. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001. MC1R KO: Melanocortin 1 receptor knockout, IL: Interleukin, IFN-g: Interferon, TNF-a: Tumor necrosis factor-a, ELISA: Enzyme-linked immunosorbent assay, p-STAT3: Phospho-signal transducer and activator of transcription 3, RORgt: Retinoic acid receptor-related orphan receptor g-t.

Article Snippet: IL-17 (DY421), IFN-g (DY485), IL-10 (DY417), IL-4 (DY404), tumor necrosis factor-a (TNF-a) (DY410), and IL-6 (DY406) kits were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Standard Deviation, Knock-Out