Journal: Molecular Therapy. Methods & Clinical Development

Article Title: SERCA2a overexpression improves muscle function in a canine Duchenne muscular dystrophy model

doi: 10.1016/j.omtm.2024.101268

Figure Lengend Snippet: Evaluation of the T cell response in AAV.SERCA2a-injected and contralateral excipient-injected muscle of affected dogs Representative photomicrographs of H&E staining, CD8 (a marker for cytotoxic T cells), CD4 (a marker for helper T cells), and FoxP3 (a marker for regulatory T cells) immunohistochemistry staining and FLAG tag immunofluorescence staining. Right-column photomicrographs are high-magnification images of the boxed areas in the corresponding middle-column photomicrographs. Filled arrowhead, CD8+ T cells; open arrowhead, CD4+ T cells; arrow, FoxP3+ nuclei.

Article Snippet: Immunohistochemistry staining was used to detect CD4+ T cells, CD8+ T cells, and FoxP3+ nuclei, according to our established protocols., Specifically, CD4+ T cells were detected using a rat monoclonal IgG2a antibody against canine CD4 (1:1000; Bio-Rad, cat. no. MCA1038GA, clone YKIX 302.9).

Techniques: Injection, Staining, Marker, Immunohistochemistry, FLAG-tag, Immunofluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Optimization of cell culture conditions for generating canine CAR T cells . The impact of cell culture conditions, including T cell activation and T cell expansion, were evaluated to identify the optimal conditions. A Diagram of human B7-H3 CAR construct used to generate canine B7-H3 CAR T cells. In B , flow cytometry was used to determine expression of canine CD5 + T cell CD4 and CD8 subsets by day 10 CAR T cell cultures. In C the relative CAR T cell transduction efficiency was determined by immunostaining with recombinant L-protein. Canine T cells were activated with PHA, CD3/CD28 beads, or aAPCs, and expanded in rhIL-2 (top row) or rcIL-2 (bottom row), and the percentage of L-protein positive cells determined by flow cytometry. In D , CAR T cells were generated from n = 6 dogs (3 healthy, 3 osteosarcoma), and the mean ± SEM cell numbers and percentages of CAR T cells (red) and non-transduced T cells (blue) determined for each of the culture activation and expansion conditions (x-axis) determined, illustrating greatest CAR T generation when rhIL-2 was used for expansion. E Representative CAR T cell cytotoxicity study, using the canine OS cell line Abrams as the target, as described in Methods. Target killing over time determined for B7-H3 CAR T cells cultured in IL-2 + IL-21 (red) or IL-2 + IL-21 + IL-7 and IL15 (green), demonstrating superior killing activity of CAR T cells expanded in the 4-cytokine cocktail. Assays were run in triplicate, and statistical significance was determined using Dunn’s multiple means comparison test; *P < 0.05, *****, P < 0.0001, ns not significant

Article Snippet: The CAR T cells were further characterized using anti-canine CD5-FITC (cloneYKIX322.3, Bio-Rad), anti-canine CD8-alexa Fluor 647 (clone YCATE55.9, Bio-Rad), anti-canine CD4-Pacific Blue (clone YKIX302.9, Bio-Rad), anti-CD62L (clone FMC46, Bio-Rad), anti-CD45RA (clone CA21,4B3, Leukocyte Antigen Biology Laboratory).

Techniques: Cell Culture, Activation Assay, Construct, Flow Cytometry, Expressing, Transduction, Immunostaining, Recombinant, Generated, Activity Assay, Comparison

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

doi: 10.1007/s00262-024-03642-4

Figure Lengend Snippet: Functional in vitro activity of dual B7-H3-CXCR2 CAR T cells compared to single CAR B7-H3 CAR T cells . The ability of a chemokine receptor (CXCR2) to improve the activity of canine B7-H3 CAR T cells when the two molecules were co-expressed by transduced T cells was examined using in vitro studies. In A , the diagram of the dual B7-H3-CXCR2 construct is depicted. In B , the transduction efficiency of canine T cells with the dual CAR construct was evaluated by measuring L-protein binding (scFv detection) and with anti-huCXCR2 immunostaining, demonstrating expression of both molecules, along with the distribution of CD4 and CD8 CAR T cells. Flow cytometry dot plots showing percentage positive for each population. In C , to assess tumor target expression of a key chemokine (CXCL8) ligand for CXCR2, secretion of CXCL8 by 7 different canine OS tumor cell lines was measured, demonstrating that 6 of the 7 lines secreted large amounts of CXCL8 spontaneously. The ability of the huCXCR2 molecule to bind and become activated by canine CXCL8 was confirmed in D , using a transwell migration assay as described in Methods, with sustained migration of B7-H3-CXCR2 CAR T cells to a canine CXCL8 gradient. Serum containing medium was used as a positive control. In E , cytokine secretion following target Abrams cell engagement for 24 h was assessed, and revealed significantly greater production of both IFN-g and IL-2 with B7-H3-CXCR2 CAR T cells compared to B7-H3 CAR T cells, at equivalent E:T ratios (20:1)

Article Snippet: The CAR T cells were further characterized using anti-canine CD5-FITC (cloneYKIX322.3, Bio-Rad), anti-canine CD8-alexa Fluor 647 (clone YCATE55.9, Bio-Rad), anti-canine CD4-Pacific Blue (clone YKIX302.9, Bio-Rad), anti-CD62L (clone FMC46, Bio-Rad), anti-CD45RA (clone CA21,4B3, Leukocyte Antigen Biology Laboratory).

Techniques: Functional Assay, In Vitro, Activity Assay, Construct, Transduction, Protein Binding, Immunostaining, Expressing, Flow Cytometry, Transwell Migration Assay, Migration, Positive Control