Journal: bioRxiv
Article Title: Advancing CAR T-Cell Therapy: Simultaneously Attack Tumor and Immunosuppressive Cells in the Tumor Microenvironment
doi: 10.1101/2024.03.25.586653
Figure Lengend Snippet: (A) The CD163 + CD209 + immunophenotype of MDM-M2 macrophages was characterized and compared to that of their monocyte precursors (scatter plot). The PD-L1 expression on MDM-M2 macrophages was confirmed by immunostaining and analyzed using flow cytometry, with CD14 + monocytes serving as a negative control (histogram plots). (B) MC9999 CAR T-cells exhibited cytotoxicity against MDM-M2 macrophages but not CD14 + monocytes, as determined via a CD107a degranulation assay. (C) The release of granzyme B was significantly increased when MC9999 CAR T-cells were cultured with MDM-M2 macrophages. The data, plotted as mean ± SEM of triplicate sampling, are representative of three independent experiments and analyzed using the multiple T test (***, p < 0.001; ns, no significance). (D) The impedance-based killing assay demonstrated the direct killing of MDM-M2 macrophages by MC9999 CAR T-cells, as evidenced by a significant decrease in the cell index upon the addition of the CAR T-cells to cultured MDM-M2 macrophages. The data are representative of three independent experiments. (E) TAMs isolated from a surgically resected GBM tumor displayed the expected CD163 + CD209 + immunophenotype upon immunostaining. (F) The CD163 + CD209 + gated TAMs were highly positive for PD-L1 at the cell surface. CD14 + monocytes served as the negative control for both immunophenotypic characterization and PD-L1 staining. (G) Evaluation via the CD107a degranulation assay revealed the MC9999 CAR T-cells, derived from healthy donor T cells, elicited cytotoxicity against the TAMs extracted from GBM tumor.
Article Snippet: The analysis included staining for BUV395-CD14 (clone M5E2, BD Horizon, USA), APC-CD209 (clone DCN46, BD Pharmingen, USA), AF488-CD163 (clone MAC2-158, BD Pharmingen, USA), and BV650-PDL-1 (clone29E.2A3, Biolegend, USA).
Techniques: Expressing, Immunostaining, Flow Cytometry, Negative Control, Degranulation Assay, Cell Culture, Sampling, Isolation, Staining, Derivative Assay
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