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: These aAPCs were conjugated with anti-canine CD3 (clone CA17.2A12, Bio-Rad) and anti-canine CD28 (clone 5B8, Invitrogen) at a 1:1 ratio as previously described [ ].
Techniques: Cell Culture, Activation Assay, Construct, Flow Cytometry, Expressing, Transduction, Immunostaining, Recombinant, Generated, Activity Assay, Comparison
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