Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti-T. Gondii Immunity and Tumor Immunotherapy.

doi: 10.1002/advs.202400952

Figure Lengend Snippet: Figure 4. GRA4 recruits Tripartite Motif protein 27 (TRIM27) to catalyze TBK1 K48-ubiquitination at Lys251 and Lys372 sites. A) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding FLAG-TBK1, HA-Ub, and MYC-GRA4, followed by treatment with 3-MA (10 mm), and then IP with anti-FLAG beads. B) Immunoblotting analysis of 293T cells transfected with FLAG-TBK1, HA-WT-, -K11-, -K27-, -K33-, -K48- or -K63- linked Ub, together with MYC-EV or -GRA4, followed by treatment with 3-MA (10 mm), and then IP with anti-FLAG beads. C) Immunoblotting analysis of extracts of BMDMs infected with ME49wt or ME49∆gra4 (MOI = 5) for the indicated time points, followed by IP with anti-TBK1. D) Luciferase activity in 293T cells transfected with a luciferase reporter for IFN-𝛽-luc, Scr shRNA or E3 ligases specific shRNAs for 24 h, followed by transfected with FLAG-TBK1, together with HA-EV or -GRA4 for 24 h. E) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding HA-TBK1, FLAG-TRIM27, and MYC-GRA4, is shown. F) Immunoblotting analysis of 293T cells transfected with HA- TBK1, FLAG-TRIM27, and MYC-GRA4, followed by treatment with 3-MA (10 mM), and then IP with anti-FLAG beads. G) Immunoblotting analysis of 293T cells transfected with Scramble shRNA or TRIM27 specific shRNA for 24 h, then transfected with various combinations of plasmid encoding FLAG-TBK1, HA-K48-Ub, and MYC-GRA4 or EV,

Article Snippet: In Vivo siRNA Knockdown of Tbk1: Tbk1 siRNA (Santa Cruz, Cat#sc39059) or Scr siRNAs (Santa Cruz, Cat#sc-37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δompdc/gra4 vaccination.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, shRNA

Journal: Advanced Science

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti‐ T. Gondii Immunity and Tumor Immunotherapy

doi: 10.1002/advs.202400952

Figure Lengend Snippet: GRA4 specifically interacts with activated TANK Binding Kinase 1 (TBK1). A) Luciferase activity in 293T cells transfected with a luciferase reporter for IFN‐β luc, and FLAG tagged ‐RIG‐I, ‐MDA5, ‐MAVS, ‐cGAS plus STING, ‐TBK1, ‐IKKi, ‐IRF3(5D), together with or without GRA4. Results are expressed relative to renilla luciferase activity. B) Immunoblotting analysis of 293T cells transfected with FLAG‐GRA4 or ‐EV, and treated with ME49 RNA for 8 h, followed by IP with anti‐TBK1. C) Representative confocal images of 293T cells overexpressing MYC‐GRA4 and FLAG‐TBK1. Cells are treated with parasitic RNA for 8 h. Nuclei are stained with DAPI. Scale bars, 20 µm. The intensity analysis is next to the image. D) Schematic of two‐step IP in 293T cells. Protein extracted from 293T cells are immunoprecipitated through anti‐p‐TBK1 and protein A/G as first IP, using anti‐TBK1 and protein A/G antibodies as second IP. E) Immunoblotting analysis of 293T cells transfected with FLAG‐GRA4, and treated with ME49 RNA for indicated time, followed by IP with anti‐p‐TBK1 and second IP with anti‐TBK1. F,G) Immunoblotting analysis of 293T cells transfected with FLAG‐GRA4 and truncations of HA‐ or MYC‐TBK1, followed by IP with anti‐FLAG or anti‐MYC beads. H) Protein structure and molecular docking maps of p‐TBK1 and GRA4. I) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding FLAG‐WT TBK1, ‐A104G mutant, ‐K251R mutant or ‐Y329A mutant of TBK1 and MYC‐GRA4, followed by IP with anti‐FLAG beads. J) Luciferase activity in 293T cells, transfected with a luciferase reporter for IFN‐β luc, WT TBK1 and Y329A mutant of FLAG‐TBK1, together with or without MYC‐GRA4. Results are expressed relative to renilla luciferase activity. luc: luciferase, IP: immunoprecipitation, WCL, whole cell lysis, IB: immunoblotting. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, and ns (not significant) as determined by unpaired Student's t test.

Article Snippet: Tbk1 siRNA (Santa Cruz, Cat#sc‐39059) or Scr siRNAs (Santa Cruz, Cat#sc‐37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δ ompdc / gra4 vaccination.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Western Blot, Staining, Immunoprecipitation, Plasmid Preparation, Mutagenesis, Lysis

Journal: Advanced Science

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti‐ T. Gondii Immunity and Tumor Immunotherapy

doi: 10.1002/advs.202400952

Figure Lengend Snippet: GRA4 induces the autophagic degradation of TBK1 in a Sequestosome 1 (SQSTM1)/p62 dependent manner. A,B) Immunoblotting and qPCR analysis of TBK1 protein and mRNA level extracts of 293T cells transfected with FLAG‐TBK1, HA‐EV or increasing amounts of HA‐GRA4. C) Immunoblotting analysis of total and phosphorylated TBK1 in 293T cells transfected with MYC‐EV or ‐GRA4, followed by treatment with ME49 RNA at indicated time points. D) Immunoblotting analysis of total TBK1 in 293T cells transfected with FLAG‐EV or ‐GRA4, pre‐stimulated by ME49 RNA for 8 h, and then treated with cycloheximide (CHX) (100 µg/mL) for indicated time points. E) Immunoblotting analysis of total and phosphorylated TBK1 in BMDMs followed by ME49wt or ME49 ∆gra4 (MOI = 5) infection at indicated time points. F) Immunoblotting analysis of 293 T cells transfected with FLAG‐TBK1, together with HA‐EV or ‐GRA4, followed by treatments of 3‐MA (10 m m ), bafilomycin A1 (Baf A1) (0.2 µ m ), MG132 (10 µ m ), and Z‐VAD (50 µ m ) for 6 h respectively. G) Immunoblotting analysis of total TBK1 in 293T cells transfected with FLAG‐EV or ‐GRA4, pre‐stimulated by ME49 RNA for 8 h and then treated with rapamycin (250 nM) for indicated time points. H) Representative confocal images of 293T cells overexpressing FLAG‐TBK1, GFP‐LC3B, together with MYC‐EV or GRA4. Cells are treated with parasitic RNA for 8 h. Nuclei are stained with DAPI. The intensity analysis is next to it. Scale bars, 20 µm. I) Immunoblotting analysis of WT, BECN1 KO, and ATG5 KO 293T cells transfected with FLAG‐TBK1, together with HA‐EV or HA‐GRA4. J) Immunoblotting analysis of 293T cells transfected with FLAG‐EV, ‐p62, ‐NDP52, ‐OPTN, ‐NIX, ‐TOLLIP, or ‐NBR1, and HA‐GRA4, followed by IP with anti‐FLAG beads. K) Luciferase activity in WT, p62 KO, OPTN KO or TOLLIP KO 293T cells transfected with a luciferase reporter for IFN‐β‐luc, FLAG‐TBK1, HA‐EV or increasing amounts of HA‐GRA4, is shown. L) Immunoblotting analysis of 293T cells transfected with HA‐TBK1, FLAG‐p62 and MYC‐ GRA4, followed by treatment with 3‐MA (10 m m ), and then IP with anti‐FLAG beads. M) Representative confocal images of 293T cells overexpressing FLAG‐TBK1, HA‐p62, together with MYC‐GRA4. Scale bars, 20 µm. N) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding HA‐TBK1, MYC‐GRA4, and Flag‐p62, is shown. O) Immunoblotting analysis of p62 KO 293T cells transfected with HA‐TBK1, FLAG‐p62 or ‐p62ΔUBA, and MYC‐GRA4 or ‐EV. luc: luciferase, IP: immunoprecipitation, WCL, whole cell lysis, IB: immunoblotting. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. *** p < 0.001 and ns (not significant) as determined by unpaired Student's t test.

Article Snippet: Tbk1 siRNA (Santa Cruz, Cat#sc‐39059) or Scr siRNAs (Santa Cruz, Cat#sc‐37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δ ompdc / gra4 vaccination.

Techniques: Western Blot, Transfection, Infection, Staining, Luciferase, Activity Assay, Plasmid Preparation, Immunoprecipitation, Lysis

Journal: Advanced Science

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti‐ T. Gondii Immunity and Tumor Immunotherapy

doi: 10.1002/advs.202400952

Figure Lengend Snippet: GRA4 recruits Tripartite Motif protein 27 (TRIM27) to catalyze TBK1 K48‐ubiquitination at Lys251 and Lys372 sites. A) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding FLAG‐TBK1, HA‐Ub, and MYC‐GRA4, followed by treatment with 3‐MA (10 m m ), and then IP with anti‐FLAG beads. B) Immunoblotting analysis of 293T cells transfected with FLAG‐TBK1, HA‐WT‐, ‐K11‐, ‐K27‐, ‐K33‐, ‐K48‐ or ‐K63‐linked Ub, together with MYC‐EV or ‐GRA4, followed by treatment with 3‐MA (10 m m ), and then IP with anti‐FLAG beads. C) Immunoblotting analysis of extracts of BMDMs infected with ME49wt or ME49 ∆gra4 (MOI = 5) for the indicated time points, followed by IP with anti‐TBK1. D) Luciferase activity in 293T cells transfected with a luciferase reporter for IFN‐β‐luc, Scr shRNA or E3 ligases specific shRNAs for 24 h, followed by transfected with FLAG‐TBK1, together with HA‐EV or ‐GRA4 for 24 h. E) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding HA‐TBK1, FLAG‐TRIM27, and MYC‐GRA4, is shown. F) Immunoblotting analysis of 293T cells transfected with HA‐ TBK1, FLAG‐TRIM27, and MYC‐GRA4, followed by treatment with 3‐MA (10 mM), and then IP with anti‐FLAG beads. G) Immunoblotting analysis of 293T cells transfected with Scramble shRNA or TRIM27 specific shRNA for 24 h, then transfected with various combinations of plasmid encoding FLAG‐TBK1, HA‐K48‐Ub, and MYC‐GRA4 or EV, followed by treatment of 3‐MA for 6 h, and then IP with anti‐FLAG beads. H,I) Immunoblotting analysis of 293T cells transfected with MYC‐Full Length or truncations of TBK1, HA‐K48‐Ub, together with or without FLAG‐GRA4, followed by treatment with 3‐MA (10 m m ), and then IP with anti‐MYC beads. J) Luciferase activity in 293T cells transfected with a luciferase reporter for IFN‐β‐luc, FLAG‐WT or K251R/K372R mutant of TBK1, together with HA‐EV or ‐GRA4, is shown. K) Immunoblotting analysis of 293T cells transfected with FLAG‐WT or K251R/K372R mutant of TBK1, together with HA‐EV or ‐GRA4, is shown. L) qPCR analysis of IFNB and ISG56 in TBK1 KO 293T cells transfected with FLAG‐WT or ‐K251R/K372R mutant of TBK1, together with HA‐EV or GRA4, is shown. M) Immunoblotting analysis of 293T cells transfected with FLAG‐WT or ‐K251R/K372R mutant of TBK1, HA‐K48‐Ub, together with or without HA‐GRA4, followed by treatment with 3‐MA (10 m m ), and then IP with anti‐FLAG beads. luc: luciferase, IP: immunoprecipitation, WCL, whole cell lysis, IB: immunoblotting. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. *** p < 0.001 and ns (not significant) as determined by unpaired Student's t test.

Article Snippet: Tbk1 siRNA (Santa Cruz, Cat#sc‐39059) or Scr siRNAs (Santa Cruz, Cat#sc‐37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δ ompdc / gra4 vaccination.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, shRNA, Mutagenesis, Immunoprecipitation, Lysis

Journal: Advanced Science

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti‐ T. Gondii Immunity and Tumor Immunotherapy

doi: 10.1002/advs.202400952

Figure Lengend Snippet: GRAPH ABSTRACT: The dual character of GRA4 in regulating anti‐ T. gondii and anti‐tumor immunity via suppressing IFN‐I signaling. Toxoplasma effector protein GRA4 induces TRIM27‐p62‐dependent selective autophagic degradation of TBK1 to inhibit host IFN‐I responses. On one hand, GRA4 helps host to prevent severe toxoplasmosis. On the other hand, GRA4 limits anti‐tumor efficiency induced by attenuated T. gondii , and vaccination with the modified ME49Δ ompdc/gra4 activates stronger IFN‐I production to promote the proliferation of CD64 + MAR‐1 + CD11b + DCs and drive enhanced T cell responses, which confers host complete resistance to the tumor.

Article Snippet: Tbk1 siRNA (Santa Cruz, Cat#sc‐39059) or Scr siRNAs (Santa Cruz, Cat#sc‐37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δ ompdc / gra4 vaccination.

Techniques: Modification

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti-T. Gondii Immunity and Tumor Immunotherapy.

doi: 10.1002/advs.202400952

Figure Lengend Snippet: Figure 2. GRA4 specifically interacts with activated TANK Binding Kinase 1 (TBK1). A) Luciferase activity in 293T cells transfected with a luciferase reporter for IFN-𝛽luc, and FLAG tagged -RIG-I, -MDA5, -MAVS, -cGAS plus STING, -TBK1, -IKKi, -IRF3(5D), together with or without GRA4. Results are expressed relative to renilla luciferase activity. B) Immunoblotting analysis of 293T cells transfected with FLAG-GRA4 or -EV, and treated with ME49 RNA for 8 h, followed by IP with anti-TBK1. C) Representative confocal images of 293T cells overexpressing MYC-GRA4 and FLAG-TBK1. Cells are treated with parasitic RNA for 8 h. Nuclei are stained with DAPI. Scale bars, 20 μm. The intensity analysis is next to the image. D) Schematic of two-step IP in 293T cells. Protein extracted from 293T cells are immunoprecipitated through anti-p-TBK1 and protein A/G as first IP, using anti-TBK1 and protein A/G antibodies as second IP. E) Immunoblotting analysis of 293T cells transfected with FLAG-GRA4, and treated with ME49 RNA for indicated time, followed by IP with anti-p-TBK1 and second IP with anti-TBK1. F,G) Immunoblotting analysis of 293T cells transfected with FLAG-GRA4 and truncations of HA- or MYC-TBK1, followed by IP with anti-FLAG or anti-MYC beads. H) Protein structure and molecular docking maps of p-TBK1 and GRA4. I) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding FLAG-WT TBK1, -A104G mutant, -K251R mutant or -Y329A mutant of TBK1 and MYC-GRA4, followed by IP with anti-FLAG beads. J) Luciferase activity in 293T cells, transfected with a luciferase reporter for IFN-𝛽luc, WT TBK1 and Y329A mutant of FLAG-TBK1, together with or without MYC-GRA4. Results are expressed relative to renilla luciferase activity. luc: luciferase, IP: immunoprecipitation, WCL, whole cell lysis, IB: immunoblotting. Data with error bars are represented as means ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, and ns (not significant) as determined by unpaired Student’s t test.

Article Snippet: In Vivo siRNA Knockdown of Tbk1: Tbk1 siRNA (Santa Cruz, Cat#sc39059) or Scr siRNAs (Santa Cruz, Cat#sc-37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δompdc/gra4 vaccination.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Western Blot, Staining, Immunoprecipitation, Plasmid Preparation, Mutagenesis, Lysis

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti-T. Gondii Immunity and Tumor Immunotherapy.

doi: 10.1002/advs.202400952

Figure Lengend Snippet: Figure 3. GRA4 induces the autophagic degradation of TBK1 in a Sequestosome 1 (SQSTM1)/p62 dependent manner. A,B) Immunoblotting and qPCR analysis of TBK1 protein and mRNA level extracts of 293T cells transfected with FLAG-TBK1, HA-EV or increasing amounts of HA-GRA4. C) Immunoblot- ting analysis of total and phosphorylated TBK1 in 293T cells transfected with MYC-EV or -GRA4, followed by treatment with ME49 RNA at indicated time points. D) Immunoblotting analysis of total TBK1 in 293T cells transfected with FLAG-EV or -GRA4, pre-stimulated by ME49 RNA for 8 h, and then treated with cycloheximide (CHX) (100 μg/mL) for indicated time points. E) Immunoblotting analysis of total and phosphorylated TBK1 in BMDMs followed by ME49wt or ME49∆gra4 (MOI = 5) infection at indicated time points. F) Immunoblotting analysis of 293 T cells transfected with FLAG-TBK1, together with HA-EV or -GRA4, followed by treatments of 3-MA (10 mm), bafilomycin A1 (Baf A1) (0.2 μm), MG132 (10 μm), and Z-VAD (50 μm) for 6 h respec- tively. G) Immunoblotting analysis of total TBK1 in 293T cells transfected with FLAG-EV or -GRA4, pre-stimulated by ME49 RNA for 8 h and then treated with rapamycin (250 nM) for indicated time points. H) Representative confocal images of 293T cells overexpressing FLAG-TBK1, GFP-LC3B, together with MYC-EV or GRA4. Cells are treated with parasitic RNA for 8 h. Nuclei are stained with DAPI. The intensity analysis is next to it. Scale bars, 20 μm. I) Immunoblotting analysis of WT, BECN1 KO, and ATG5 KO 293T cells transfected with FLAG-TBK1, together with HA-EV or HA-GRA4. J) Immunoblotting analysis of 293T cells transfected with FLAG-EV, -p62, -NDP52, -OPTN, -NIX, -TOLLIP, or -NBR1, and HA-GRA4, followed by IP with anti-FLAG beads. K) Luciferase activity in WT, p62 KO, OPTN KO or TOLLIP KO 293T cells transfected with a luciferase reporter for IFN-𝛽-luc, FLAG-TBK1, HA-EV or

Article Snippet: In Vivo siRNA Knockdown of Tbk1: Tbk1 siRNA (Santa Cruz, Cat#sc39059) or Scr siRNAs (Santa Cruz, Cat#sc-37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δompdc/gra4 vaccination.

Techniques: Western Blot, Transfection, Infection, Staining, Luciferase, Activity Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti-T. Gondii Immunity and Tumor Immunotherapy.

doi: 10.1002/advs.202400952

Figure Lengend Snippet: Figure 4. GRA4 recruits Tripartite Motif protein 27 (TRIM27) to catalyze TBK1 K48-ubiquitination at Lys251 and Lys372 sites. A) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding FLAG-TBK1, HA-Ub, and MYC-GRA4, followed by treatment with 3-MA (10 mm), and then IP with anti-FLAG beads. B) Immunoblotting analysis of 293T cells transfected with FLAG-TBK1, HA-WT-, -K11-, -K27-, -K33-, -K48- or -K63- linked Ub, together with MYC-EV or -GRA4, followed by treatment with 3-MA (10 mm), and then IP with anti-FLAG beads. C) Immunoblotting analysis of extracts of BMDMs infected with ME49wt or ME49∆gra4 (MOI = 5) for the indicated time points, followed by IP with anti-TBK1. D) Luciferase activity in 293T cells transfected with a luciferase reporter for IFN-𝛽-luc, Scr shRNA or E3 ligases specific shRNAs for 24 h, followed by transfected with FLAG-TBK1, together with HA-EV or -GRA4 for 24 h. E) Immunoblotting analysis of 293T cells transfected with various combinations of plasmid encoding HA-TBK1, FLAG-TRIM27, and MYC-GRA4, is shown. F) Immunoblotting analysis of 293T cells transfected with HA- TBK1, FLAG-TRIM27, and MYC-GRA4, followed by treatment with 3-MA (10 mM), and then IP with anti-FLAG beads. G) Immunoblotting analysis of 293T cells transfected with Scramble shRNA or TRIM27 specific shRNA for 24 h, then transfected with various combinations of plasmid encoding FLAG-TBK1, HA-K48-Ub, and MYC-GRA4 or EV,

Article Snippet: In Vivo siRNA Knockdown of Tbk1: Tbk1 siRNA (Santa Cruz, Cat#sc39059) or Scr siRNAs (Santa Cruz, Cat#sc-37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δompdc/gra4 vaccination.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Plasmid Preparation, Infection, Luciferase, Activity Assay, shRNA

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: The Toxoplasma Effector GRA4 Hijacks Host TBK1 to Oppositely Regulate Anti-T. Gondii Immunity and Tumor Immunotherapy.

doi: 10.1002/advs.202400952

Figure Lengend Snippet: Figure 7. GRAPH ABSTRACT: The dual character of GRA4 in regulating anti-T. gondii and anti-tumor immunity via suppressing IFN-I signaling. Toxo- plasma effector protein GRA4 induces TRIM27-p62-dependent selective autophagic degradation of TBK1 to inhibit host IFN-I responses. On one hand, GRA4 helps host to prevent severe toxoplasmosis. On the other hand, GRA4 limits anti-tumor efficiency induced by attenuated T. gondii, and vaccina- tion with the modified ME49Δompdc/gra4 activates stronger IFN-I production to promote the proliferation of CD64+MAR-1+CD11b+ DCs and drive enhanced T cell responses, which confers host complete resistance to the tumor.

Article Snippet: In Vivo siRNA Knockdown of Tbk1: Tbk1 siRNA (Santa Cruz, Cat#sc39059) or Scr siRNAs (Santa Cruz, Cat#sc-37007) were intravenously injected in mice at indicated time points: i) in the T. gondii infection model, siRNAs were injected at Day 0, 4, 8 post T. gondii infection; ii) in the tumor model, siRNAs were injected at Day −7, −1 post ME49Δompdc/gra4 vaccination.

Techniques: Clinical Proteomics