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human pd 1 fc fusion protein (Sino Biological)

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Structured Review

Sino Biological human pd 1 fc fusion protein
Human Pd 1 Fc Fusion Protein, supplied by Sino Biological, used in various techniques. Bioz Stars score: 94/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+pd+1+fc+fusion+protein/Human+PD1+%2F+PDCD1+%2F+CD279+Protein/pmc12914813-85-7-14
Average 94 stars, based on 28 article reviews

human pd 1 fc fusion protein - by Bioz Stars, 2026-11

94/100 stars

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Protein Binding:

Article Title: Targeting the ANXA2-CD63-exosomal PD-L1 axis in hepatocellular carcinoma: A novel mechanism of immune evasion and its implications for precision immunotherapy
Article Snippet: For human HCC cell–derived exosomes, exosomes were captured using CD63 exosome capture beads (Abcam; ab239686) under the same conditions and stained with PE-conjugated anti-human PD-L1 antibody (BD; 557924; 1:10) for 40 min at 4°C, with PE-conjugated IgG isotype control (BD; 556650) as the negative control. .. High protein-binding 96-well plates were coated with human PD-1-Fc fusion protein (5 μg/mL, 10377-H02H, SinoBiological, China) and incubated at 37°C for 1 hour. .. High protein-binding 96-well plates were coated with human PD-1-Fc fusion protein (5 μg/mL, 10377-H02H, SinoBiological, China) and incubated at 37°C for 1 hour.

Article Title: LSD1 deletion decreases exosomal PD-L1 and restores T-cell response in gastric cancer
Article Snippet: .. Human PD-1-Fc fusion protein (5 μg/ml) (10377-H02H, SinoBiological, China) was coated on high protein-binding 96-well plates for 1 h at 37°C and BSA (5 μg/ml) (A8020, Solarbio, China) was used as a control. ..

Incubation:

Concentration Assay:

Article Title: Anti-PD-1 antibodies and compositions
Article Snippet: Materials and Methods Investigation of PD-L1 or PD-L2 ligand blocking activity was performed by Bio-Layer lnterferometry (BLI) analysis using an Octet QK384 instrument (Fortebio, USA). .. Commercially available human PD-1 Fc fusion protein (Sino Biological) at 5 μg/ml concentration was captured on anti-human Fc sensor chips (Fortebio, USA) and residual anti-Fc sites blocked with Herceptin® negative control antibody. ..

Article Title: Anti-PD-1 antibodies and compositions
Article Snippet: Materials and Methods Investigation of PD-L1 or PD-L2 ligand blocking activity was performed by Bio-Layer Interferometry (BLI) analysis using an Octet QK384 instrument (Fortebio, USA). .. Commercially available human PD-1 Fc fusion protein (Sino Biological) at 5 μg/ml concentration was captured on anti-human Fc sensor chips (Fortebio, USA) and residual anti-Fc sites blocked with Herceptin® negative control antibody. ..

Negative Control:

Article Title: Anti-PD-1 antibodies and compositions
Article Snippet: Materials and Methods Investigation of PD-L1 or PD-L2 ligand blocking activity was performed by Bio-Layer Interferometry (BLI) analysis using an Octet QK384 instrument (Fortebio, USA). .. Commercially available human PD-1 Fc fusion protein (Sino Biological) at 5 μg/ml concentration was captured on anti-human Fc sensor chips (Fortebio, USA) and residual anti-Fc sites blocked with Herceptin® negative control antibody. ..

Control:

Image Search Results

a γ c level is negatively correlated with PD-L1 level in human NSCLC tumor biopsies. Representative images from IHC staining of γ c and PD-L1 in human NSCLC tumor biopsies are shown (left panels). Quantification of PD-L1 and γ c staining intensities was performed by semi-quantitative scoring (right panel). n = 127 independent samples, R = –0.2966. Correlation coefficients were calculated using the Pearson test. Two-sided P -value was given. Scale bars, 100 μm. b PD-1 blockade increases γ c level in tumor tissues. B16F10 cells (5 × 10 5 ) were subcutaneously injected into C57BL/6J mice. CT26 cells (5 × 10 5 ) were subcutaneously injected into Balb/c mice. Mice were intraperitoneally injected with control or anti-PD-1 antibody (100 μg per mouse) every three days (four times in total) five days after inoculation of cells. After 17 days, tumor-bearing mice were euthanized and tumor tissues were analyzed. Representative images from IHC staining of γ c in tumor sections are shown. Scale bars, 100 μm. c PD-1 blockade increases γ c level in tumor-infiltrating CD8 + T cells. TILs were isolated from B16F10 tumor tissues (left panels) or CT26 tumor tissues (right panels), stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 8 independent samples. Data were analyzed using Student’s unpaired t -test with GraphPad Prism 8. MFI, median fluorescence intensities. d PD-1 ligation down-regulates γ c level. Human CD8 + T cells or PD-1-expressing Jurkat (Jurkat-PD-1) cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days. The cells were stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 3 technical repeats. Data were analyzed using two-way ANOVA with GraphPad Prism 8. e The level of γ c is down-regulated after PD-1 ligation. Human CD8 + T cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before immunoblotting analysis with the indicated antibodies. f PD-1 ligation down-regulates γ c level. Left panels: Jurkat cells transduced with empty vector (Jurkat-EV) or Jurkat-PD-1 cells were analyzed by immunoblots with the indicated antibodies. Middle panels: Jurkat-EV and Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) as indicated for 3 days before immunoblotting analysis with the indicated antibodies. Right panels: Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. g Effects of PD-1 ligation on transcription of γ c . Human CD8 + T cells or Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before qPCR analysis of mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. The experiments in c – g were repeated for at least two times with similar results.

Journal: Cell Research

Article Title: PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity

doi: 10.1038/s41422-023-00890-4

Figure Lengend Snippet: a γ c level is negatively correlated with PD-L1 level in human NSCLC tumor biopsies. Representative images from IHC staining of γ c and PD-L1 in human NSCLC tumor biopsies are shown (left panels). Quantification of PD-L1 and γ c staining intensities was performed by semi-quantitative scoring (right panel). n = 127 independent samples, R = –0.2966. Correlation coefficients were calculated using the Pearson test. Two-sided P -value was given. Scale bars, 100 μm. b PD-1 blockade increases γ c level in tumor tissues. B16F10 cells (5 × 10 5 ) were subcutaneously injected into C57BL/6J mice. CT26 cells (5 × 10 5 ) were subcutaneously injected into Balb/c mice. Mice were intraperitoneally injected with control or anti-PD-1 antibody (100 μg per mouse) every three days (four times in total) five days after inoculation of cells. After 17 days, tumor-bearing mice were euthanized and tumor tissues were analyzed. Representative images from IHC staining of γ c in tumor sections are shown. Scale bars, 100 μm. c PD-1 blockade increases γ c level in tumor-infiltrating CD8 + T cells. TILs were isolated from B16F10 tumor tissues (left panels) or CT26 tumor tissues (right panels), stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 8 independent samples. Data were analyzed using Student’s unpaired t -test with GraphPad Prism 8. MFI, median fluorescence intensities. d PD-1 ligation down-regulates γ c level. Human CD8 + T cells or PD-1-expressing Jurkat (Jurkat-PD-1) cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days. The cells were stained with the indicated antibodies and analyzed by flow cytometry. Graph shows mean ± SEM, n = 3 technical repeats. Data were analyzed using two-way ANOVA with GraphPad Prism 8. e The level of γ c is down-regulated after PD-1 ligation. Human CD8 + T cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before immunoblotting analysis with the indicated antibodies. f PD-1 ligation down-regulates γ c level. Left panels: Jurkat cells transduced with empty vector (Jurkat-EV) or Jurkat-PD-1 cells were analyzed by immunoblots with the indicated antibodies. Middle panels: Jurkat-EV and Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) as indicated for 3 days before immunoblotting analysis with the indicated antibodies. Right panels: Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. g Effects of PD-1 ligation on transcription of γ c . Human CD8 + T cells or Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before qPCR analysis of mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. The experiments in c – g were repeated for at least two times with similar results.

Article Snippet: Reagents and antibodies used in this study were purchased from the indicated companies: recombinant human PD-L1-Fc fusion protein (BPS Bioscience, Catalog #71104), recombinant mouse PD-L1-Fc fusion protein (Sino, Catalog #50010-M02H), anti-human CD3ε (clone OKT3, Biolegend, Catalog #317326), anti-human CD28 (clone CD28.2, Biolegend, Catalog #302934), anti-mouse CD3ε (clone 145-2C11, Biolegend, Catalog #100340), anti-mouse CD28 (clone 37.51, Biolegend, Catalog #102116), PHA (Sigma, Catalog #L8954), polybrene (Millipore, Catalog #3924803), SYBR (Bio-Rad, Catalog #1725125), cycloheximide (Sigma, Catalog #239763), MG132 (Sigma, Catalog #M8699), NH 4 Cl (Sigma, Catalog #254134), 3-MA (Sigma, Catalog #189490), Pitavastatin calcium (Aladdin, Catalog #P129617), Rosuvastatin calcium (Aladdin, Catalog #R129220), Simvastatin (Aladdin, Catalog #S129538), Lovastatin (Aladdin, Catalog #L107709), Fluvastatin Sodium (Aladdin, Catalog #F129852), human IL-2 (SL Pharm, Catalog #S19991010), human IL-7 (Peprotech, Catalog #200-07), mouse IL-7 (Peprotech, Catalog #217-17) and human IL-9 (Peprotech, Catalog #200-09).

Techniques: Immunohistochemistry, Staining, Injection, Control, Isolation, Flow Cytometry, Fluorescence, Ligation, Expressing, Western Blot, Transduction, Plasmid Preparation

a PD-1 ligation promotes γ c degradation. Jurkat-PD-1 cells were pre-stimulated with PHA (50 ng/mL) for 36 h and then treated with CHX (0.1 mM) for the indicated times before immunoblotting analysis with the indicated antibodies. The γ c band intensities relative to the corresponding β-actin bands were shown in the histograph. b NH 4 Cl inhibits γ c degradation. Jurkat cells were pre-treated with MG132 (100 μM), NH 4 Cl (25 mM) or 3-MA (500 ng/mL) for 4 h, and then treated with CHX (0.1 mM) for 2 h before immunoblotting analysis with the indicated antibodies. c NH 4 Cl inhibits PD-1 ligation-induced degradation of γ c . Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 36 h, and then treated with MG132 (100 μM), NH 4 Cl (25 mM) or 3-MA (500 ng/mL) for 12 h before immunoblotting analysis with the indicated antibodies. d PD-1 ligation induces polyubiquitination of γ c . Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. e MARCH5 promotes degradation of γ c in a dose-dependent manner. HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis with the indicated antibodies. f Association of γ c with MARCH5. Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. g MARCH5-deficiency up-regulates the level of γ c but not IL2Rβ. Control or MARCH5-deficient Jurkat cells were collected for immunoblotting analysis with the indicated antibodies. h Effects of MARCH5-deficiency on transcription of γ c . Control or MARCH5-deficient Jurkat cells were analyzed by qPCR for mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. i MARCH5-deficiency impairs PD-1 ligation-induced degradation of γ c . Control or MARCH5-deficient Jurkat-PD-1 cell were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before immunoblotting analysis with the indicated antibodies. j PD-1 ligation-induced degradation of γ c is impaired in MARCH5-deficient cells. Mouse CD8 + T cells from sex- and age-matched March5 f/f or March5 f/f:CD4-Cre mice were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound mPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before immunoblotting analysis with the indicated antibodies. k MARCH5 promotes polyubiquitination of γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. l MARCH5-deficiency impairs PD-L1-induced K27-linked polyubiquitination of γ c . Control or MARCH5-deficient PD-1-expressing Jurkat cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. m MARCH5 increases K27-linked polyubiquitination of wild-type γ c and γ c K294R but not γ c K315R . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. n PD-1 induces down-regulation of wild-type γ c but not γ c K315R . Jurkat-PD-1 cells were expressed with Flag-tagged wild-type γ c or γ c K315R mutant and then stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Journal: Cell Research

Article Title: PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity

doi: 10.1038/s41422-023-00890-4

Figure Lengend Snippet: a PD-1 ligation promotes γ c degradation. Jurkat-PD-1 cells were pre-stimulated with PHA (50 ng/mL) for 36 h and then treated with CHX (0.1 mM) for the indicated times before immunoblotting analysis with the indicated antibodies. The γ c band intensities relative to the corresponding β-actin bands were shown in the histograph. b NH 4 Cl inhibits γ c degradation. Jurkat cells were pre-treated with MG132 (100 μM), NH 4 Cl (25 mM) or 3-MA (500 ng/mL) for 4 h, and then treated with CHX (0.1 mM) for 2 h before immunoblotting analysis with the indicated antibodies. c NH 4 Cl inhibits PD-1 ligation-induced degradation of γ c . Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 36 h, and then treated with MG132 (100 μM), NH 4 Cl (25 mM) or 3-MA (500 ng/mL) for 12 h before immunoblotting analysis with the indicated antibodies. d PD-1 ligation induces polyubiquitination of γ c . Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. e MARCH5 promotes degradation of γ c in a dose-dependent manner. HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblot analysis with the indicated antibodies. f Association of γ c with MARCH5. Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. g MARCH5-deficiency up-regulates the level of γ c but not IL2Rβ. Control or MARCH5-deficient Jurkat cells were collected for immunoblotting analysis with the indicated antibodies. h Effects of MARCH5-deficiency on transcription of γ c . Control or MARCH5-deficient Jurkat cells were analyzed by qPCR for mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. i MARCH5-deficiency impairs PD-1 ligation-induced degradation of γ c . Control or MARCH5-deficient Jurkat-PD-1 cell were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before immunoblotting analysis with the indicated antibodies. j PD-1 ligation-induced degradation of γ c is impaired in MARCH5-deficient cells. Mouse CD8 + T cells from sex- and age-matched March5 f/f or March5 f/f:CD4-Cre mice were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound mPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before immunoblotting analysis with the indicated antibodies. k MARCH5 promotes polyubiquitination of γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. l MARCH5-deficiency impairs PD-L1-induced K27-linked polyubiquitination of γ c . Control or MARCH5-deficient PD-1-expressing Jurkat cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. m MARCH5 increases K27-linked polyubiquitination of wild-type γ c and γ c K294R but not γ c K315R . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. n PD-1 induces down-regulation of wild-type γ c but not γ c K315R . Jurkat-PD-1 cells were expressed with Flag-tagged wild-type γ c or γ c K315R mutant and then stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Techniques: Ligation, Western Blot, Control, Immunoprecipitation, Transfection, Expressing, Mutagenesis

a USP5 removes K27-linked polyubiquitin moieties from γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblotting analysis with the indicated antibodies. b Association of γ c with USP5. Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c USP5 but not its enzymatic inactive mutant USP5 C335A removes K27-linked polyubiquitin moieties from γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. d USP5 removes K27-linked polyubiquitin moieties of γ c catalyzed by MARCH5. HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblotting analysis with the indicated antibodies. e USP5-deficiency enhances PD-1 ligation-induced K27-linked polyubiquitination of γ c . Control or USP5-deficient Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Journal: Cell Research

Article Title: PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity

doi: 10.1038/s41422-023-00890-4

Figure Lengend Snippet: a USP5 removes K27-linked polyubiquitin moieties from γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblotting analysis with the indicated antibodies. b Association of γ c with USP5. Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c USP5 but not its enzymatic inactive mutant USP5 C335A removes K27-linked polyubiquitin moieties from γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. d USP5 removes K27-linked polyubiquitin moieties of γ c catalyzed by MARCH5. HEK293 cells were transfected with the indicated plasmids for 24 h before immunoblotting analysis with the indicated antibodies. e USP5-deficiency enhances PD-1 ligation-induced K27-linked polyubiquitination of γ c . Control or USP5-deficient Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Techniques: Transfection, Western Blot, Immunoprecipitation, Mutagenesis, Ligation, Control

a MARCH5 is up-regulated after PD-L1 but not PHA stimulation. Human CD8 + T cells or Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before immunoblotting analysis with the indicated antibodies (left panels). Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (right panels). b Effects of PD-1 ligation on MARCH5 degradation. Jurkat-PD-1 cells were pre-stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 36 h and then treated with CHX (0.1 mM) for the indicated times before immunoblotting analysis with the indicated antibodies. The MARCH5 band intensities relative to the corresponding β-actin bands were shown in the histograph. c MARCH5-deficiency impairs PD-1 ligation-induced degradation of γ c . Control or MARCH5-deficient Jurkat-PD-1 cells were reconstituted with wild-type MARCH5 or MARCH5 H43W mutant and then stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 2 days before immunoblotting analysis with the indicated antibodies. d Effects of PD-1 ligation on MARCH5 mRNA level. Human CD8 + T cells or Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before qPCR analysis of mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. e BATF binds to the promoter region of MARCH5 gene. Jurkat-PD-1 cells were analyzed by ChIP with the indicated antibodies, and then de-crosslinked DNA was subjected to qPCR analysis using specific primers. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using a Student’s unpaired t -test with GraphPad Prism 8. f BATF-deficiency impairs PD-1 ligation-induced transcription of MARCH5. Control or BATF-deficient Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 2 days before qPCR analysis of mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. g BATF-deficiency impairs PD-1 ligation-induced degradation of γ c . Control or BATF-deficient Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 2 days before immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Journal: Cell Research

Article Title: PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity

doi: 10.1038/s41422-023-00890-4

Figure Lengend Snippet: a MARCH5 is up-regulated after PD-L1 but not PHA stimulation. Human CD8 + T cells or Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before immunoblotting analysis with the indicated antibodies (left panels). Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (right panels). b Effects of PD-1 ligation on MARCH5 degradation. Jurkat-PD-1 cells were pre-stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 36 h and then treated with CHX (0.1 mM) for the indicated times before immunoblotting analysis with the indicated antibodies. The MARCH5 band intensities relative to the corresponding β-actin bands were shown in the histograph. c MARCH5-deficiency impairs PD-1 ligation-induced degradation of γ c . Control or MARCH5-deficient Jurkat-PD-1 cells were reconstituted with wild-type MARCH5 or MARCH5 H43W mutant and then stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 2 days before immunoblotting analysis with the indicated antibodies. d Effects of PD-1 ligation on MARCH5 mRNA level. Human CD8 + T cells or Jurkat-PD-1 cells were stimulated with plate-bound anti-CD3 (1 μg/mL) and solubilized anti-CD28 (5 μg/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 3 days before qPCR analysis of mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. e BATF binds to the promoter region of MARCH5 gene. Jurkat-PD-1 cells were analyzed by ChIP with the indicated antibodies, and then de-crosslinked DNA was subjected to qPCR analysis using specific primers. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using a Student’s unpaired t -test with GraphPad Prism 8. f BATF-deficiency impairs PD-1 ligation-induced transcription of MARCH5. Control or BATF-deficient Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 2 days before qPCR analysis of mRNA levels of the indicated genes. Graph shows mean ± SEM, n = 3 independent samples from one representative experiment. Data were analyzed using two-way ANOVA with GraphPad Prism 8. g BATF-deficiency impairs PD-1 ligation-induced degradation of γ c . Control or BATF-deficient Jurkat-PD-1 cells were stimulated with PHA (50 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for 2 days before immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Techniques: Control, Western Blot, Ligation, Mutagenesis

a Association of γ c with SHP2. Jurkat-PD-1 cells were stimulated with PHA (150 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. b Effects of SHP2 on dephosphorylation of γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c JAK3 mediates phosphorylation of γ c Y357 . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. d IL-7 treatment induces γ c Y357 phosphorylation. Control or γ c -deficient HPB-ALL cells were stimulated with IL-7 (100 ng/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. e SHP2-deficiency impairs PD-L1-induced γ c Y357 dephosphorylation. Control or SHP2-deficient Jurkat-PD-1 cells were stimulated with PHA (150 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. f SHP2-deficiency enhances IL-7-induced γ c Y357 phosphorylation. Control or SHP2-deficient HPB-ALL cells were stimulated with IL-7 (100 ng/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. g SHP2-deficiency promotes γ c family cytokine-induced phosphorylation of STAT5 Y694/Y699 . Control or SHP2-deficient HPB-ALL cells were stimulated with IL-9 (100 ng/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (left panels). Control or SHP2-deficient CTLL2 cells were stimulated with IL-2 (400 IU/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (right panels). h Effects of γ c Y357F mutant on the γ c family cytokine-induced phosphorylation of STAT5 Y694/Y699 . γ c -deficient HPB-ALL cells were reconstituted with wild-type γ c or γ c Y357F mutant and then stimulated with IL-7 (100 ng/mL) or IL-9 (100 ng/mL) for the indicated time lengths before immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Journal: Cell Research

Article Title: PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity

doi: 10.1038/s41422-023-00890-4

Figure Lengend Snippet: a Association of γ c with SHP2. Jurkat-PD-1 cells were stimulated with PHA (150 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein or control hIgG1 (2 μg/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. b Effects of SHP2 on dephosphorylation of γ c . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. c JAK3 mediates phosphorylation of γ c Y357 . HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. d IL-7 treatment induces γ c Y357 phosphorylation. Control or γ c -deficient HPB-ALL cells were stimulated with IL-7 (100 ng/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. e SHP2-deficiency impairs PD-L1-induced γ c Y357 dephosphorylation. Control or SHP2-deficient Jurkat-PD-1 cells were stimulated with PHA (150 ng/mL) in the presence of plate-bound hPD-L1-Fc fusion protein for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. f SHP2-deficiency enhances IL-7-induced γ c Y357 phosphorylation. Control or SHP2-deficient HPB-ALL cells were stimulated with IL-7 (100 ng/mL) for the indicated times before co-immunoprecipitation and immunoblotting analysis with the indicated antibodies. g SHP2-deficiency promotes γ c family cytokine-induced phosphorylation of STAT5 Y694/Y699 . Control or SHP2-deficient HPB-ALL cells were stimulated with IL-9 (100 ng/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (left panels). Control or SHP2-deficient CTLL2 cells were stimulated with IL-2 (400 IU/mL) for the indicated times before immunoblotting analysis with the indicated antibodies (right panels). h Effects of γ c Y357F mutant on the γ c family cytokine-induced phosphorylation of STAT5 Y694/Y699 . γ c -deficient HPB-ALL cells were reconstituted with wild-type γ c or γ c Y357F mutant and then stimulated with IL-7 (100 ng/mL) or IL-9 (100 ng/mL) for the indicated time lengths before immunoblotting analysis with the indicated antibodies. All the experiments were repeated for at least two times with similar results.

Techniques: Control, Immunoprecipitation, Western Blot, De-Phosphorylation Assay, Transfection, Mutagenesis

In tumor microenvironment, PD-L1/PD-1 signaling results in inhibition of the γ c family cytokine-triggered signaling and immune activation by two mechanisms. Immediately after PD-1 ligation, SHP2 is recruited to PD-1 and activated, which in turn dephosphorylates γ c at Y357, leading to its inactivation and unresponsiveness to γ c family cytokines. More later after PD-1 ligation, the transcription factor BATF is induced, which up-regulates the expression of the membrane-associated E3 ubiquitin ligase MARCH5. MARCH5 is recruited to γ c and mediates its K27-linked polyubiquitination at K315 and lysosomal degradation. Targeting of components involved in these regulatory mechanisms, such as by a combination of PD-1 blocking antibody (1), IL-2 (2) and MARCH5 inhibitor (3) leads to potent anti-tumor immunity.

Journal: Cell Research

Article Title: PD-1 signaling negatively regulates the common cytokine receptor γ chain via MARCH5-mediated ubiquitination and degradation to suppress anti-tumor immunity

doi: 10.1038/s41422-023-00890-4

Figure Lengend Snippet: In tumor microenvironment, PD-L1/PD-1 signaling results in inhibition of the γ c family cytokine-triggered signaling and immune activation by two mechanisms. Immediately after PD-1 ligation, SHP2 is recruited to PD-1 and activated, which in turn dephosphorylates γ c at Y357, leading to its inactivation and unresponsiveness to γ c family cytokines. More later after PD-1 ligation, the transcription factor BATF is induced, which up-regulates the expression of the membrane-associated E3 ubiquitin ligase MARCH5. MARCH5 is recruited to γ c and mediates its K27-linked polyubiquitination at K315 and lysosomal degradation. Targeting of components involved in these regulatory mechanisms, such as by a combination of PD-1 blocking antibody (1), IL-2 (2) and MARCH5 inhibitor (3) leads to potent anti-tumor immunity.

Techniques: Inhibition, Activation Assay, Ligation, Expressing, Membrane, Blocking Assay

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