pe antimouse cd4 antibody  (Proteintech)

Journal: Advanced Science

Article Title: Ribonuclease 1 Induces T‐Cell Dysfunction and Impairs CD8 + T‐Cell Cytotoxicity to Benefit Tumor Growth through Hijacking STAT1

doi: 10.1002/advs.202404961

Figure Lengend Snippet: The ribonucleolytic activity‐independent function of RNase1 induces the dysfunction of CD4 + and CD8 + T cells, and reduces CD8 + T cell cytotoxicity in vitro. A) Membrane PD‐1, LAG‐3, and TIM‐3 expression by flow cytometric analysis in CD4 + T cells isolated from activated PBMC‐derived T cells treated with or without 1 µg mL −1 recombinant RNase1 for 24 h. Quantitative data of flow cytometric analysis from three independent experiments. B) Membrane PD‐1, LAG‐3, and TIM‐3 expression by flow cytometric analysis in CD8 + T cells isolated from activated PBMC‐derived T cells treated with or without 1 µg mL −1 recombinant RNase1 for 24 h. Quantitative data of flow cytometric analysis from three independent experiments. C) RNase1 levels detected by flow cytometric analysis in CD4 + and CD8 + T cells isolated from activated PBMC‐derived T cells treated with 1 µg mL −1 recombinant RNase1 for 24 h. Quantitative data of flow cytometric analysis from three independent experiments. D) Membrane PD‐1, LAG‐3, and TIM‐3 expression by flow cytometric analysis in CD4 + T cells isolated from activated PBMC‐derived T cells treated with CM collected from RNase1‐expressing (R1), enzyme‐dead RNase1‐expressing (R1‐H12A), or control (pCDH) MDA‐MB‐231 cells for 24 h. Quantitative data of flow cytometric analysis from three independent experiments. E) Membrane PD‐1, LAG‐3, and TIM‐3 expression by flow cytometric analysis in CD8 + T cells isolated from activated PBMC‐derived T cells treated with CM collected from R1, R1‐H12A, or pCDH MDA‐MB‐231 cells for 24 h. Quantitative data of flow cytometric analysis from three independent experiments. F) Quantitative RT‐PCR analysis of IL‐2 , INF‐γ , PD‐1 , LAG‐3 , and TIM‐3 mRNA expression in CD4 + T cells isolated from activated PBMC‐derived T cells treated with CM collected from R1, R1‐H12A, or pCDH MDA‐MB‐231 cells for 24 h. Representative data from three independent experiments (each experiment contains three technical replicates). G) Quantitative RT‐PCR analysis of IL‐2 , INF‐γ , PD‐1 , LAG‐3 , and TIM‐3 mRNA expression in CD8 + T cells isolated from activated PBMC‐derived T cells treated with CM collected from R1, R1‐H12A, or pCDH MDA‐MB‐231 cells for 24 h. Representative data from three independent experiments (each experiment contains three technical replicates). H) Representative images and quantitative results of T cell‐mediated cancer cell killing assay. Left: RNase1‐knockdown (sh‐R1#1 and #2) and control (sh‐Ctrl) FaDu cells (6 × 10 4 cells) cocultured with or without activated PBMC‐derived T cells for 72 h were subjected to crystal violet staining to evaluate T‐cell cytotoxicity. FaDu to T‐cell ratio, 1:4. Right: RNase1‐expressing (R1), and control (pCDH) SAS cells (5 × 10 4 cells) cocultured with activated PBMC‐derived T cells for 48 h were subjected to crystal violet staining to evaluate T‐cell cytotoxicity. SAS to T‐cell ratio, 1:6. Three independent experiments with three technical replicates were carried out. I) Representative images and quantitative results of T cell‐mediated cancer cell killing assay. Left: RNase1‐knockout (KO‐R1) and control (KO‐Ctrl) KPL4 cells (5 × 10 4 cells) cocultured with or without activated PBMC‐derived T cells for 24 h were subjected to crystal violet staining to evaluate T‐cell cytotoxicity. KPL4 to T‐cell ratio, 1:4. Right: R1, R1‐H12A, and pCDH MDA‐MB‐231 cells (3 × 10 4 cells) cocultured with or without activated PBMC‐derived T cells for 48 h were subjected to crystal violet staining to evaluate T‐cell cytotoxicity. MDA‐MB‐231 to T‐cell ratio, 1:4. Three independent experiments with three technical replicates were carried out. J) Time‐course quantitative results of T cell‐meditated cancer cell killing assay of dead cells. R1, R1‐H12A, and pCDH MDA‐MB‐231 cells (2000 cells) labeled with Incucyte Nuclight Rapid Red Dye were cocultured with CD8 + T cells isolated from activated PBMC‐derived T cells for 24 h. The caspase‐3/7 activity of dead cells was normalized to that at the zero‐time point. MDA‐MB‐231 to T‐cell ratio, 1:5. Two independent experiments with three technical replicates were carried out. Data are presented as mean ± SD, ** p , 0.001–0.01, *** p < 0.001, and ns, not significant by A–I) two‐sided unpaired Student's t ‐test or J) an ANOVA test.

Article Snippet: [ ] Mice bearing tumors were treated with 200 μg mouse IgG1 isotype control (#BE0083; BioXCell), antimouse CD4 (#BE0003‐1; BioXCell), or antimouse CD8α (#BE0061; BioXCell) antibodies on day 3, 6, 9, 12, and 15 through intraperitoneal injections.

Techniques: Activity Assay, In Vitro, Membrane, Expressing, Isolation, Derivative Assay, Recombinant, Control, Quantitative RT-PCR, Knockdown, Staining, Knock-Out, Labeling

Journal: Advanced Science

Article Title: Ribonuclease 1 Induces T‐Cell Dysfunction and Impairs CD8 + T‐Cell Cytotoxicity to Benefit Tumor Growth through Hijacking STAT1

doi: 10.1002/advs.202404961

Figure Lengend Snippet: RNase1 impairs T cell cytotoxicity against cancer cells and promotes T‐cell dysfunction. A) Mouse RNase1‐knockdown (sh‐mR1#1 and #2) and control (Ctrl) MOC‐L1 cells (5 × 10 6 ) were subcutaneously inoculated into C57BL/6 mice ( n = 8 mice per group). The tumor volume was measured. B) 4T1‐sh‐Ctrl or sh‐R1#1 cells (5 × 10 4 ) were orthotopically injected into NOD SCID mice ( n = 10 mice per group). The tumor volume was measured. C) Mouse RNase1‐knockdown (sh‐mR1#1) and control (sh‐Ctrl) 4T1 cells (5 × 10 4 ) were orthotopically injected into BALB/c mice ( n = 8 mice per group). The tumor volume was measured. D) The percentage of CD3 + CD8 + T cells‐expressing granzyme B (GB) or IFNγ in 4T1‐sh‐Ctrl and sh‐R1#1 tumor tissues from mice according to flow cytometry analysis ( n = 8 independent tissue samples). E) The percentage of CD3 + CD8 + T cells‐expressing Pd‐1, Lag‐3, or Tim‐3 in 4T1‐sh‐Ctrl and sh‐R1#1 tumor tissues from mice according to flow cytometry analysis ( n = 8 independent tissue samples). F) sh‐mR1#1 and sh‐Ctrl 4T1 cells (1 × 10 5 ) were orthotopically injected into BALB/c mice treated with isotype, anti‐CD4, or anti‐CD8 antibodies every 3 d ( n = 6 mice per group). The tumor volume was measured. G) Mouse RNase1‐expressing (mR1), enzyme‐dead mouse RNase1‐expressing (mR1‐H12A), and control (pCDH) E0771 cells (2 × 10 5 ) were orthotopically injected into C57BL/6 mice ( n = 10 mice per group). The tumor volume is measured. H) The percentage of CD3 + CD8 + T cells‐expressing GB or IFNγ in E0771‐Ctrl, mR1, and mR1‐H12A tumor tissues from mice according to flow cytometry analysis ( n = 7 independent tissue samples). I) The percentage of CD3 + CD8 + T cells‐expressing Pd‐1, Lag‐3, or Tim‐3 in E0771‐Ctrl, mR1, and mR1‐H12A tumor tissues from mice according to flow cytometry analysis ( n = 7 independent tissue samples). J) The analysis of T‐cell cytotoxicity against E0771‐pCDH, mR1, or mR1‐H12A cells expressing MHC class I specific epitope of OVA (E0771‐OVA) by OT‐1 T cells. Three independent experiments with three technical replicates were carried out. K) The analysis of T‐cell cytotoxicity against B16F10‐pCDH, mR1, or mR1‐H12A cells expressing MHC class I specific epitope of OVA (B16F10‐OVA) by OT‐1 T cells. Three independent experiments with three technical replicates were carried out. L) OT‐1 TCR transgenic mice were orthotopically injected with 1 × 10 6 cells of E0771‐pCDH or mR1 cells expressing MHC class I specific epitope of OVA, n = 6 for pCDH group and n = 5 for mR1 group. Data are presented as mean ± SD, ** p , 0.001–0.01, *** p < 0.001, and ns, not significant by D,E,H–K) two‐sided unpaired Student's t ‐test or A–C,F,G,L) an ANOVA test.

Article Snippet: [ ] Mice bearing tumors were treated with 200 μg mouse IgG1 isotype control (#BE0083; BioXCell), antimouse CD4 (#BE0003‐1; BioXCell), or antimouse CD8α (#BE0061; BioXCell) antibodies on day 3, 6, 9, 12, and 15 through intraperitoneal injections.

Techniques: Knockdown, Control, Injection, Expressing, Flow Cytometry, Transgenic Assay