pe cyanine7 anti mouse cd4  (Elabscience Biotechnology)

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: IL22 upregulates PD-L1 expression in gastric cancer cells through the UPR IRE1α–XBP1 axis. A, mIF images show the alterations of PD-L1 + tumor cells (purple), CD4 + (green), and CD8 + (red) T cells in orthotopic gastric cancer tumors from control and Abhd16a -knockdown mice following IL22 treatment. Scale bar, 50 μm. B, KEGG pathway enrichment analysis of RNA-seq data of gastric cancer tissues with or without IL22 treatment. C, RT-PCR was used to assess the mRNA expression of key downstream molecules of the UPR branches ( XBP1 , ATF4 , ATF6 ) in control and IL22RA1 -knockdown gastric cancer cells. D, Western blotting analysis of PD-L1 and XBP1s levels in control and IL22RA1 -knockdown MGC-803 cells treated with IL22 (100 μg/L). E, Western blotting detection of PD-L1 and XBP1s levels in XBP1- knockdown MGC-803 cells treated with IL22 and MGC-803 cells treated with IL22 or XBP1s inhibitor (STF083010, 30 μmol/L) in combination with IL22. F, The binding sequence of XBP1 on the CD274 promoter. G and H, ChIP ( G ) and luciferase reporter assay ( H ) showing the transcriptional regulation of CD274 by XBP1s under IL22 stimulation. I, Orthotopic gastric cancer mouse models ( n = 5 per group) were injected with anti-IL22 (200 μg per mouse), anti-CD90.2 antibody (150 μg per mouse), anti-CD90.2 antibody in combination with IL22 (500 ng per mouse), or anti-CD90.2 antibody in combination with XBP1s inhibitors (STF083010, 30 mg/kg) and IL22 for 2 weeks. IHC analysis was used to show IL22, XBP1s, and PD-L1 levels in gastric cancer tissues. Scale bar, 200 μm. J, Tumor volume of orthotopic gastric cancer models under treatments the same as in I . *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Expressing, Control, Knockdown, RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Binding Assay, Sequencing, Luciferase, Reporter Assay, Injection

Journal: Cancer Research

Article Title: Nerves Stimulate Cross-talk Between Gastric Cancer and Group 3 Innate Lymphoid Cells to Enhance Immunosuppression

doi: 10.1158/0008-5472.CAN-25-3092

Figure Lengend Snippet: Combination therapy enhances the anti–PD-L1 immunotherapeutic effect in gastric cancer. A and B, After tumor formation, the orthotopic gastric cancer mice ( n = 5 per group) were treated with anti–PD-L1 (100 μg per mouse), GPR34 inhibitor (20 mg/kg), or XBP1s inhibitor (30 mg/kg) every 3 days or ACh inhibitor (2.5 mg/kg) daily. Combinations of anti–PD-L1 with each inhibitor followed the every 3-day dosing schedule for a total duration of 2 weeks via i.p. injection. Living images were used to monitor tumor progression at 5-day intervals from the time of drug administration ( A ); IHC and mIF were performed to detect PD-L1 and XBP1s levels and proportions of CD4 + (green) and CD8 + (red) T cells in gastric cancer tissues at the end of treatments ( B ). Scale bars, 1.000e+5 –∼ 5.000e + 5 p/s/cm 2 /sr for living images; 200 μm for IHC; 50 μm for immunofluorescence. C and D, Representative images ( C ) and tumor volume ( D ) of subcutaneous tumors. The administration protocol for the mice was consistent with the description provided in A and B . **, P < 0.01; ***, P < 0.001.

Article Snippet: The antibodies used for flow cytometry: Brilliant Violet 605 anti-mouse CD127 (BioLegend, cat. #135025, RRID: AB_2562114, 5 μL/1 × 10 6 cells), FITC anti-mouse CD3 (BioLegend, cat. #100203, RRID: AB_312660, 2 μL/1 × 10 6 cells), APC anti-mouse CD3 (Elabscience, cat. #E-AB-F1013E, RRID: AB_3675272, 5 μL/1×10 6 cells), PE/Cyanine7 anti-mouse CD4 (Elabscience, cat. #E-AB-F1097H, 5 μL/1 × 10 6 cells), FITC Anti-Mouse CD8a (Elabscience, cat. #E-AB-F1104UC, 5 μL/1 × 10 6 cells), FITC anti-mouse CD19 (BioLegend, cat. #152403, RRID: AB_2629812, 0.25 μL/1 × 10 6 cells), FITC anti-mouse CD11c (BioLegend, cat. #117305, RRID: AB_313774, 0.5 μL/1 × 10 6 cells), FITC anti-mouse NK1.1 (BioLegend, cat. #108705, RRID: AB_313392, 0.5 μL/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD45 (BioLegend, cat. #103133, RRID: AB_10899570, 1 μL/1 × 10 6 cells), PE anti-mouse RORγt (BD Biosciences, cat. #562607, RRID: AB_11153137, 2 μL/1 × 10 6 cells), PerCP/Cyanine5.5 anti-mouse IL22 (BioLegend, cat. #516411, RRID: AB_2563373, 5 μL/1 × 10 6 cells), AF647 anti-STAT3 phospho (BioLegend, cat. #651007, RRID: AB_2572085, 5 μL/1 × 10 6 cells), PE anti-mouse CD45 (BioLegend, cat. #157604, RRID: AB_2876536, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD8b (BioLegend, cat. #126613, RRID: AB_2562774, 0.625 μL/1 × 10 6 cells), APC anti-mouse CD4 (BioLegend, cat. #100411, RRID: AB_312696, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD206 (BioLegend, cat. #141707, RRID: AB_10896057, 2.5 μL/1 × 10 6 cells), FITC anti-mouse F4/80 (BioLegend, cat. #157309, RRID: AB_2876535, 2 μL/1 × 10 6 cells), FITC anti-mouse CD25 (BioLegend, cat. #101907, RRID: AB_961210, 2 μL/1 × 10 6 cells), AF700 anti-mouse FOXP3 (BioLegend, cat. #126421, RRID: AB_2750492, 0.12 μL/1 × 10 6 cells), PE anti-mouse Ly6G (BioLegend, cat. #127607, RRID: AB_1186104, 1.25 μL/1 × 10 6 cells), APC anti-mouse CD274 (Elabscience, cat. #E-AB-F1132E, 5 μL/1 × 10 6 cells), PerCP-Cyanine5.5 anti–T-bet (eBioscience, cat. #45-5825-80, RRID: AB_953658, 0.25 μg/1 × 10 6 cells), PE/Dazzle 594 anti-mouse CD273 (BioLegend, cat. #107215, RRID: AB_2728124, 0.25 μg/1 × 10 6 cells), Brilliant Violet 421 anti-mouse CD274 (BioLegend, cat. #124315, RRID: AB_10897097, 5 μL/1 × 10 6 cells), and PE anti-mouse MHC-I (H-2Kk; BioLegend, cat. #114907, RRID: AB_313614, 0.25 μg/1 × 10 6 cells).

Techniques: Injection, Immunofluorescence