rat monoclonal antibody against cd8a  (Thermo Fisher)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer

doi: 10.1016/j.omtm.2019.06.011

Figure Lengend Snippet: MnTBAP Induces Dramatic Loss of CD4 on the Surface of Murine and Human T Lymphocytes and Inhibits Transduction of Human T Lymphocytes by Lentiviral Vectors Pseudotyped with gp120 HXB2 -env (A and B) MnTBAP effect on murine (A) and human (B) T lymphocyte receptor level. Murine splenic (A) and human peripheral blood (B) cells were treated for 2 h with or without MnTBAP and then analyzed by flow cytometry for cell-surface CD3, CD4, and CD8 expression on live cells. The results of geometric means of fluorescence for each receptor are expressed relative to the corresponding values of the control non-treated cells (None, arbitrarily set at 100%); n = 3–4 independent experiments. For each receptor, statistical comparisons between untreated and treated cells were made using the Mann-Whitney test. (C) CD3 + T cells from human peripheral blood were transduced in the absence or presence of 400 μM MnTBAP on retronectin-coated plates, with gp120 HXB2 -env pseudotyped lentiviral vectors expressing GFP reporter gene. Transduction efficiency was assessed by flow cytometry via GFP expression 72 h post-transduction. Representative CD4/GFP dot plots are shown, with inset numbers indicating the percentage of cells in each quadrant. (D) Normalized percentages of GFP + cells obtained 72 h post-transduction. The percentage of GFP + cells in the cultures without MnTBAP (None, control) is set at 100% (n = 4 independent experiments). Statistical analysis in (D) was performed using paired t test. *p < 0.05; ***p < 0.0001.

Article Snippet: After washing with PBS, sections were blocked with PBS with 10% goat serum for 30 min and then incubated with 1/40 dilution of a rat monoclonal primary antibody directed against CD8a (Thermo Fisher Scientific) for 1–2 h at RT.

Techniques: Transduction, Flow Cytometry, Expressing, Fluorescence, Control, MANN-WHITNEY

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer

doi: 10.1016/j.omtm.2019.06.011

Figure Lengend Snippet: Temporary In Vivo Inhibition of the Immune System by MnTBAP (A) Schema of OVA peptide immunization within the MnTBAP treatment window. C57BL/6 mice received i.p. multi-injections of MnTBAP or an equivalent volume of PBS (80 mg/kg daily during 5 days, day 0 to day 4). Two hours after the first i.p. injection of MnTBAP or PBS, mice received s.c. immunizations of 100 μg each of class I and class II OVA peptides or PBS emulsified with IFA. Splenic cells were harvested 8 days after immunization (day 8) to measure CD4 + and CD8 + T cell responses by IFN-γ ELISPOT, following in vitro stimulation with OVA 323–339 and OVA 257–264 peptides, respectively. (B) OVA-specific CD4 + (left panel) and CD8 + (right panel) T cell responses. MnTBAP (open symbols) or PBS (closed symbols) i.p. injections and OVA peptide (circles) or PBS (triangles) s.c. immunizations. Each symbol represents IFN-γ spot-forming units (duplicate measures) in each mouse. Horizontal bars indicate the average values. (C) Schema of OVA peptide immunization outside the MnTBAP treatment window. Immunizations were as described earlier, with one difference: that mice received s.c. injections of OVA peptides or PBS, emulsified with IFA, 21 days after the first i.p. injection of MnTBAP or PBS. Splenic cells were harvested 8 days after immunization (day 29) to measure CD4 + and CD8 + T cell responses by IFN-γ ELISPOT, following in vitro stimulation with OVA 323–339 and OVA 257–264 peptides, respectively. (D) OVA-specific CD4 + (left panel) and CD8 + (right panel) T cell responses. MnTBAP (open symbols) or PBS (closed symbols) i.p. injections and OVA peptide (circles) or PBS (triangles) s.c. immunizations. Each symbol represents IFN-γ spot-forming units (duplicate measures) of each mouse. Horizontal bars indicate the average values. Data represent one experiment with five mice per group. (E–G) MnTBAP effect on cell-surface levels of CD3 (E), CD4 (F), and CD8 (G) cell populations in the spleen. C57BL/6 mice received i.p. multi-injections with MnTBAP or with an equivalent volume of PBS (day 0 to day 4). At days 4, 8, and 16, spleens were harvested and analyzed for cell-surface CD3, CD4, and CD8 expression by flow cytometry. Geometric means of fluorescence for each receptor are expressed relative to the cells from the control PBS-treated mice (PBS, arbitrarily set at 100%). Results are from 2 independent experiments. All statistical analyses were performed using the Mann-Whitney test. **p < 0.005.

Article Snippet: After washing with PBS, sections were blocked with PBS with 10% goat serum for 30 min and then incubated with 1/40 dilution of a rat monoclonal primary antibody directed against CD8a (Thermo Fisher Scientific) for 1–2 h at RT.

Techniques: In Vivo, Inhibition, Injection, Enzyme-linked Immunospot, In Vitro, Expressing, Flow Cytometry, Fluorescence, Control, MANN-WHITNEY

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Temporary Reduction of Membrane CD4 with the Antioxidant MnTBAP Is Sufficient to Prevent Immune Responses Induced by Gene Transfer

doi: 10.1016/j.omtm.2019.06.011

Figure Lengend Snippet: MnTBAP Counteracts the Adverse Effects of rAAV-Mediated i.m. Gene Delivery (A) Protocol outline. C57BL/6 mice received i.p. multi-injections of MnTBAP or an equivalent volume of PBS (80 mg/kg daily during 5 days). Two hours after the first injection of MnTBAP, mice were administered PBS or rAAV1_CMV_SGCA_HY vector (2.5 × 10 9 vg per mouse) in the left TA. Four days post-vector injection, mice were intravenously injected with 1.5 mg L-012, a chemical agent that reacts with ROS to produce light. Fifteen days post-rAAV-vector injection, muscle integrity, transgene and CD8a mRNA expression, and transgene-specific T cell responses were analyzed. (B) Histological analysis of injected TA muscle from PBS- or MnTBAP-treated mice, 15 days following injection of rAAV vector or control PBS. Microscopy analysis of cryosections of rAAV-injected muscles after H&E staining (left panel) or anti-CD8 staining followed by enzymatic detection (right panel) was performed. Images are representative of one experiment out of 2, with 3–4 mice per group. Scale bars, 500 μm. (C) High magnifications of selected areas shown in (D). Scale bars, 150 μm. (D and E) Analysis of CD8a and SGCA transgene expression by RT-PCR in injected TA muscle 15 days following injection of rAAV vector or control PBS. Levels of CD8a (D) and SGCA (E) mRNA are expressed in arbitrary units (AU) relative to the endogenous murine acidic ribosomal phosphoprotein (PO) mRNA (n = 2; 3–4 mice per group). (F and G) Analysis of transgene-specific T cell responses: 8 days (upper panels) and 15 days (lower panels) post-injection of vector, splenic cells were harvested to measure the frequency of transgene specific CD4 + (F) and CD8 + (G) T cells by IFN-γ ELISPOT. Dots represent individual mice. Horizontal bars indicate the average values. All statistical analyses were performed using the Mann-Whitney test. *p < 0.05; **p < 0.005.

Article Snippet: After washing with PBS, sections were blocked with PBS with 10% goat serum for 30 min and then incubated with 1/40 dilution of a rat monoclonal primary antibody directed against CD8a (Thermo Fisher Scientific) for 1–2 h at RT.

Techniques: Injection, Plasmid Preparation, Expressing, Control, Microscopy, Muscles, Staining, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: The Canadian Journal of Cardiology

Article Title: Effect of interleukin-15 on the course of myocarditis in Coxsackievirus B3-infected BALB/c mice

doi:

Figure Lengend Snippet: Immunohistochemical evaluation

Article Snippet: According to the staining method described by Kühl et al ( 20 ), specific monoclonal rat antimouse primary antibodies directed against the leukocyte surface antigens CD4, CD8a, CD11a and CD11b (Becton Dickinson PharMingen, USA), and CD3 and CD54/ICAM-1 (Dianova, Germany) were embedded for 60 min. Unbound antibodies were eluted twice with phosphate-buffered saline (PBS) for 5 min each.

Techniques: Immunohistochemical staining, Control

Journal: The Canadian Journal of Cardiology

Article Title: Effect of interleukin-15 on the course of myocarditis in Coxsackievirus B3-infected BALB/c mice

doi:

Figure Lengend Snippet: CD11b staining reflecting infiltration of macrophages in the myocardium. A In the myocardium of a normal, sham-infected mouse, no significant infiltrating cells are found. B Infiltrating CD11b-positive cells (arrows) in the heart of a Coxsackivirus B3 (CVB3)-infected mouse. C After treatment with interleukin (IL)-15, cellular infiltrates are significantly reduced. The white arrow marks a single CD11b-positive cell. D Treatment with IL-15 fusion protein did not decrease the number of cellular infiltrates compared with CVB3-infected animals. The arrows indicate a cluster of CD11b cellular infiltrates

Article Snippet: According to the staining method described by Kühl et al ( 20 ), specific monoclonal rat antimouse primary antibodies directed against the leukocyte surface antigens CD4, CD8a, CD11a and CD11b (Becton Dickinson PharMingen, USA), and CD3 and CD54/ICAM-1 (Dianova, Germany) were embedded for 60 min. Unbound antibodies were eluted twice with phosphate-buffered saline (PBS) for 5 min each.

Techniques: Staining, Infection