mouse monoclonal anti bag1  (Cell Signaling Technology Inc)

Journal: The American Journal of Pathology

Article Title: Penetration of CD8 + Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii , Leads to Its Elimination

doi: 10.1016/j.ajpath.2019.04.018

Figure Lengend Snippet: CD8+ immune T cells eliminate pre-existing cysts of Toxoplasma gondii through perforin-dependent cytotoxic activity. Severe combined immunodeficiency (SCID) mice were infected orally with 10 cysts of the ME49 strain of T. gondii and treated with sulfadiazine beginning at 10 days after infection to establish a chronic infection by forming cysts in their brains. A and B: CD8+ immune T cells (2.1 × 106 cells) purified from the spleens of chronically infected wild-type (WT) or Prf1−/− mice were injected intravenously from a tail vein, and 7 days later, amounts of mRNA for bradyzoite (cyst)-specific BAG1 and CST1 and tachyzoite-specific SAG1 (A) and CD3, a T-cell surface marker (B), were measured by real-time RT-PCR in the brains of the recipient animals. C: Amounts of mRNA for interferon (IFN)-γ and effector molecules [inducible nitric oxide synthase 2 (NOS2), immunity-related GTPases M3 (Irgm3), and guanylate-binding protein 1 (Gbp1)] of the IFN-γ–mediated protective immunity to prevent tachyzoite proliferation were also measured by real-time RT-PCR. P values were obtained using t-test, and corrected P values shown in the figure were calculated by multiplying the P values by the number of comparisons performed among three groups. D and E: Immunohistochemical detection of T cells in the parenchyma (D) and a perivascular area (E) of the brains of infected nude mice at 2 to 3 days after a systemic transfer of Prf1−/− CD8+ immune T cells (4.2 × 106 cells). The T cells (positive for CD3) were stained in red. Arrows indicate the representatives of the T cells. Data are expressed as means ± SEM in each group (A–C). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Original magnification, ×200 (D and E).

Article Snippet: 20 Staining for bradyzoite-specific BAG1 and CD3 was performed in the same manner, with a modification of the use of mouse anti-BAG1 monoclonal antibody after blocking with F(ab) 2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight.

Techniques: Activity Assay, Infection, Purification, Injection, Marker, Quantitative RT-PCR, Binding Assay, Immunohistochemical staining, Staining

Journal: The American Journal of Pathology

Article Title: Penetration of CD8 + Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii , Leads to Its Elimination

doi: 10.1016/j.ajpath.2019.04.018

Figure Lengend Snippet: Invasion of T cells into Toxoplasma gondii cysts in the brains of infected mice. A–J: CBA/J mice were infected orally with 10 cysts of the ME49 strain of T. gondii; and 2 months later, their brains were applied for immunohistologic staining for T. gondii (A, B, D, E, G, and I) or bradyzoite-specific BAG1 (C, F, H, and J) in brown in combination with the staining for CD3, the T-cell marker, in red. The entire fields of four or five sagittal sections from each of the brains of four mice were analyzed. A–C: T cells that attached on the surface of cyst-containing cells. A: An arrow indicates the cyst wall. B: The arrow indicates the T cells in a spindle shape. C: The arrow indicates the bent of cyst wall inward at the site of T-cell attachment, and the arrowhead indicates a projection of the cyst wall outward nearby the site of the T-cell attachment. D–F: T cells located halfway through the cyst wall (arrows). G and H: T cells completely penetrated into the cysts were detected (arrows). I and J: Totally destroyed cysts associated with an accumulation of inflammatory cells, including T cells. K: Comparison of the diameters of cysts with and without T-cell association. Original magnification: ×400 (A–H); ×200 (I and J).

Article Snippet: 20 Staining for bradyzoite-specific BAG1 and CD3 was performed in the same manner, with a modification of the use of mouse anti-BAG1 monoclonal antibody after blocking with F(ab) 2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight.

Techniques: Infection, Staining, Marker, Cell Attachment Assay, Comparison

Journal: The American Journal of Pathology

Article Title: Penetration of CD8 + Cytotoxic T Cells into Large Target, Tissue Cysts of Toxoplasma gondii , Leads to Its Elimination

doi: 10.1016/j.ajpath.2019.04.018

Figure Lengend Snippet: CD8+ immune T cells attach to and invade into Toxoplasma gondii cysts in the brains of infected mice. Athymic nude mice were infected orally with 20 cysts of the ME49 strain of T. gondii and treated with sulfadiazine beginning at 11 days after infection to establish a chronic infection by forming cysts in their brains. A–F: CD8+ T cells (3.5 × 106 cells) purified from the spleens of infected BALB/c mice were injected intravenously from a tail vein; and 2 to 3 days later, their brains were applied for immunohistochemical staining for T. gondii (brown; A, C, E, and F) or bradyzoite-specific BAG1 (brown; B and D) and CD3 (red), the T-cell marker. A and B: T cells attached on the surface of cyst-containing cells. C–F: T cells that had migrated halfway through the cyst wall (arrows). F: T cells that completely penetrated into the cysts were detected (arrowheads). G–K: Confocal microscopy with staining for T. gondii (green) and CD3 (red) was also performed on their brains. G: The bent of the cyst wall inward at the site of CD8+ T-cell attachment (arrow), shown at higher magnification in H. H: Leakage of T. gondii materials at the site of CD8+ T-cell attachment (arrows). I–K: Confocal images of the T cell that was halfway in the invasion into a cyst. Another group of infected and sulfadiazine-treated nude mice received CD8+ normal T cells from uninfected BALB/c mice, and the immunohistochemical studies were performed on their brains in the same manner. L: The frequencies of T. gondii cysts associated with the T cells were calculated for each of these two groups of mice that had received the normal or immune CD8+ T cells. Data are expressed as means ± SEM in each group (L). ∗P < 0.05 (U-test). Original magnification: ×400 (A–F); ×1000 (G–K). SCID, severe combined immunodeficiency; WT, wild type.

Article Snippet: 20 Staining for bradyzoite-specific BAG1 and CD3 was performed in the same manner, with a modification of the use of mouse anti-BAG1 monoclonal antibody after blocking with F(ab) 2 fragments of goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) overnight.

Techniques: Infection, Purification, Injection, Immunohistochemical staining, Staining, Marker, Confocal Microscopy, Cell Attachment Assay