hif-2 alpha/epas1 antibody pack  (Bio-Techne corporation)

Journal: Frontiers in Immunology

Article Title: Aging impairs CD8 T cell responses in adoptive T-cell therapy against solid tumors

doi: 10.3389/fimmu.2025.1484303

Figure Lengend Snippet: Epas1 expression is reduced in intra-tumoral aged CD8 TCR-T cells. (A) GSEA was performed using scRNA-seq data obtained in Figure 4 . Red and blue bars represent upregulated and downregulated gene sets in aged OT-I TCR-T cells compared to young OT-I TCR-T cells. (B) Analysis of DEGs (adjusted p values <0.05) in scRNA-seq data obtained in Figure 4 revealed significantly lower Epas1 expression in aged cells in clusters 0 to 4. The violin plot shows Epas1 expression in young and aged OT-I TCR-T cells. (C) CD8 T cells isolated from spleens of young and aged C57BL/6 mice were activated with anti-CD3 and anti-CD28 antibodies and IL-2. Cells were subjected to immunoblot analysis using an antibody against Epas1 at indicated time points. As a loading control, the blot was stained with Ponceau-S. Data are representative of 2 independent experiments.

Article Snippet: Anti-Epas1 antibody (NOVUS Bio, NB100-122SS) was incubated on membranes at 4°C overnight, followed by HRP-tagged secondary antibody incubation at room temperature for 2 h. Chemiluminescent signals were developed with Clarity ECL substrate (Bio-Rad, 170-5060) and detected using an iBrightTM CL1500 Imaging System (Thermo).

Techniques: Expressing, Isolation, Western Blot, Control, Staining

Journal: Frontiers in Immunology

Article Title: Aging impairs CD8 T cell responses in adoptive T-cell therapy against solid tumors

doi: 10.3389/fimmu.2025.1484303

Figure Lengend Snippet: Epas1 is dispensable for activation and cytolytic functions of CD8 T cells in vitro. CD8 T cells were isolated from spleens of young OT-I mice (OT-I T cells) and were nucleofected with Crispr ribonucleoproteins targeting Epas1 (Epas1-Crispr) or control ribonucleoproteins (control-Crispr) on day 0, before being cultured overnight. On day 1, cells were activated with anti-CD3 and anti-CD28 antibodies and IL-2 for 24 h. From day 2 to day 8, cells were cultured without TCR stimulation in the presence of IL-7 and IL-15. (A, B) Cell viability (A) and fold increase in cell numbers (B) were assessed using a Muse cell analyzer every other day between day 2 and day 8. (C) Flow cytometry analysis of expression of CD62L and CD44 in OT-I T cells (gating strategy is shown in Supplementary Figure S4C ) was conducted every other day between day 2 and day 8. Graph shows the percentage of cells with a CD62L + CD44 hi (central memory: CM) phenotype. (D-F) On day 8, OT-I T cells were co-cultured with B16-OVA cells at a ratio of 1 to 3. (D) Flow cytometry analysis of Zombie NIR staining in B16-OVA cells (gated on CD8−: gating strategy is shown in Supplementary Figure S4F ) co-cultured with OT-I T cells for 3 days. B16-OVA cells cultured alone for 3 days were also analyzed for comparison. (E) Flow cytometry analysis of expression of granzyme B, perforin, IFN-γ, and TNF-α in OT-I T cells (gated on CD8+: gating strategy is shown in Supplementary Figure S4G ) co-cultured with B16-OVA for 24 h. (F) Flow cytometry analysis of CFSE dilution. OT-I T cells were stained with CFSE, and CFSE dilution was analyzed before and after 2 and 3 days of co-culture. Graphs show mean fluorescence intensity (MFI) and percentages of cells positive for each protein. (A-F) n = 3 per group. Error bars represent SD. Statistical analysis was performed with RM two-way ANOVA with Fisher’s LSD test (A-C) or unpaired t-tests (E) . ns, not significant, *p <0.05. Data are representative of 2 independent experiments.

Article Snippet: Anti-Epas1 antibody (NOVUS Bio, NB100-122SS) was incubated on membranes at 4°C overnight, followed by HRP-tagged secondary antibody incubation at room temperature for 2 h. Chemiluminescent signals were developed with Clarity ECL substrate (Bio-Rad, 170-5060) and detected using an iBrightTM CL1500 Imaging System (Thermo).

Techniques: Activation Assay, In Vitro, Isolation, CRISPR, Control, Cell Culture, Flow Cytometry, Expressing, Staining, Comparison, Co-Culture Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Aging impairs CD8 T cell responses in adoptive T-cell therapy against solid tumors

doi: 10.3389/fimmu.2025.1484303

Figure Lengend Snippet: Epas1 is essential for the anti-tumor CD8 T cell response. Young OT-I T cells (CD45.1 + CD45.2 + ) were nucleofected with Crispr ribonucleoproteins targeting Epas1 (Epas1-Crispr) or control ribonucleoproteins (control-Crispr), and then activated with anti-CD3 and anti-CD28 antibodies and IL-2 for 24 h. Cells were cultured in the presence of IL-7 and IL-15 for 6 days. Subsequently, 10 6 of these OT-I T cells were adoptively transferred into young congenic recipient mice (CD45.1−CD45.2 + ) that had been subcutaneously injected with 2 x 10 5 of B16-OVA cells 8 days earlier (day 0). Mice injected with PBS instead of adoptive cell transfer were also analyzed for comparison. (A) Left, schematic diagrams show the experimental design (created with BioRender.com ). Right, graphs show results of tumor volume measurements. Mice that received OT-I T cells with Epas1-Crispr (n = 23) or control-Crispr (n = 22) were analyzed. Data are pooled from four independent experiments. Results are shown as mean tumor volume ± SEM. Statistical analysis was performed using RM two-way ANOVA with uncorrected Fisher’s LSD test. *p <0.05, ****p < 0.0001. (B) Photos of tumors isolated from mice analyzed on day 20. n = 3 per group. (C-E) Cells isolated from tumor on day 20 were subjected to flow cytometry analysis. (C, D) The abundance of tumor-infiltrating OT-I T cells (CD8 + CD45.1 + CD45.2 + ) were analyzed. (C) Graph shows percentages of OT-I T cells. n = 6 (control-Crispr), n = 7 (Epas1-Crispr). (D) Graph shows absolute numbers of OT-I T cells per tumor volume (mm 3 ). n = 7 per group. (E) Tim3 expression was analyzed. Graph shows MFI for Tim3 expression. n = 3 per group. Error bars indicate SD. Statistical analysis was performed using unpaired t-tests. *p <0.05, **p < 0.01. (C-E) Data are representative of 2 independent experiments. (F) On day 20 after tumor injection, OT-I T cells (CD8 + CD45.1 + CD45.2 + ) were FACS-sorted from tumors and subjected to bulk RNA-seq analysis. The volcano plot shows DEGs between OT-I T cells with Epas1-Crispr and control-Crispr (log2 fold change > 0.5 or < -0.5 and adjusted p < 0.05). Red and blue colored dots represent upregulated and downregulated genes in cells with Epas1-Crispr, respectively. n = 3 per group.

Article Snippet: Anti-Epas1 antibody (NOVUS Bio, NB100-122SS) was incubated on membranes at 4°C overnight, followed by HRP-tagged secondary antibody incubation at room temperature for 2 h. Chemiluminescent signals were developed with Clarity ECL substrate (Bio-Rad, 170-5060) and detected using an iBrightTM CL1500 Imaging System (Thermo).

Techniques: CRISPR, Control, Cell Culture, Injection, Comparison, Isolation, Flow Cytometry, Expressing, RNA Sequencing

Journal: Frontiers in Immunology

Article Title: Aging impairs CD8 T cell responses in adoptive T-cell therapy against solid tumors

doi: 10.3389/fimmu.2025.1484303

Figure Lengend Snippet: Retroviral expression of Epas1 enhances anti-tumor activity of CD8 TCR-T cells (A, B) CD8 T cells isolated from aged mice were transduced with a retroviral vector expressing Epas1 and Thy1.1 marker (MSCV-Epas1-Thy1.1) or control vector (empty MSCV-Thy1.1), followed by MIGR1-OT-I, generating aged OT-I TCR-T + MSCV-Epas1 cells and aged OT-I TCR-T + MSCV-control cells, respectively. Cells were cultured in the presence of IL-7 and IL-15 for 6 days. (A) Flow cytometry analysis of the frequency of cells transduced with MSCV-Epas1-Thy1.1 (Thy1.1 + ) in cells transduced with MIGR1-OT-I (GFP + ). Representative plots are shown. (B) Cells including 10 6 GFP + cells were adoptively transferred into young mice that had been subcutaneously injected with 2 x 10 5 of B16-OVA cells 8 days earlier (day 0). Left schematic diagrams show the experimental design (created with BioRender.com ). Right graph shows results of tumor volume measurements. n=3 per group. Results are shown as mean tumor volume ± SEM. Statistical analysis was performed using two-way RM ANOVA with uncorrected Fisher’s LSD test. ****p<0.0001. (A, B) Data are representative of 2 independent experiments.

Article Snippet: Anti-Epas1 antibody (NOVUS Bio, NB100-122SS) was incubated on membranes at 4°C overnight, followed by HRP-tagged secondary antibody incubation at room temperature for 2 h. Chemiluminescent signals were developed with Clarity ECL substrate (Bio-Rad, 170-5060) and detected using an iBrightTM CL1500 Imaging System (Thermo).

Techniques: Retroviral, Expressing, Activity Assay, Isolation, Transduction, Plasmid Preparation, Marker, Control, Cell Culture, Flow Cytometry, Injection