American Society of Hematology

Figure 4.

$GPR56 is dynamically upregulated by CAR-T cells upon target recognition. (A) Schematic visualizing the experimental setup: Peripheral blood mononucleated cells (PBMCs) from 4 healthy donors were first activated, activated T cells (ATCs) were then transduced with a retroviral vector comprising a CD33.CAR construct. On day 15 of production, CAR-T cells were coincubated with the AML cell line HL60, expressing CD33 on the surface, or with HL60 cells, in which CD33 was knocked out using CRISPR/Cas9 (HL60 CD33 KO). (B) Representative FACS plots showing CD27 and GPR56 expression on CAR-T cells after activation and transduction, but without contact to leukemia cells (upper left), after 5-day coculture with CD33+ HL60 (upper right), after coculture with HL60 CD33 KO cells (lower left), and on nontransduced cells after coculture with HL60 CD33+ cells (lower right). Note that GPR56 upregulation occurs exclusively when CAR-T cells carrying the CD33.CAR were incubated with HL60 CD33+ cells. This suggests that GPR56 upregulation occurs only upon antigen recognition by the T cell receptor. (C) Statistical analysis of the experiment shown in 4B. ∗∗∗ P < .0005. (D) Percentage of GPR56+CD27+ fractions (blue) and CD15+ HL60 cells (orange) in the 4 individual donors during the 5 serial 5-day challenges. (E) (Top) Experimental setup of sorting experiment; CAR-T cells were challenged once with HL60 for 5 days. Then, cultures were sorted for CD8+ and the following 4 quadrants: GPR56+CD27–, GPR56+CD27+, GPR56–CD27+, and GPR56–CD27–. Subsequently, equal numbers of sorted cells were re-exposed to HL60 cells for 5 days and subsequently analyzed for surface marker expression. All fractions were capable of eliminating HL60 WT cells except for 1 culture with double-negative cells (data not shown), confirming that all fractions contained the CAR construct. (Lower left) Representative FACS plots of the sorted CAR-T cells after re-exposure to target cells. The label above the plots indicates the originally sorted phenotype. (Lower right) Stacked bar graph showing the distribution of the 4 quadrants in the 4 different conditions. Mean and standard deviation of the 4 donors are shown. The x-axis labels indicate the originally sorted fraction. Colors of bars indicate the output phenotype according to the legend. nt, nontransduced; wt, wild type.$

GPR56 is dynamically upregulated by CAR-T cells upon target recognition. (A) Schematic visualizing the experimental setup: Peripheral blood mononucleated cells (PBMCs) from 4 healthy donors were first activated, activated T cells (ATCs) were then transduced with a retroviral vector comprising a CD33.CAR construct. On day 15 of production, CAR-T cells were coincubated with the AML cell line HL60, expressing CD33 on the surface, or with HL60 cells, in which CD33 was knocked out using CRISPR/Cas9 (HL60 CD33 KO). (B) Representative FACS plots showing CD27 and GPR56 expression on CAR-T cells after activation and transduction, but without contact to leukemia cells (upper left), after 5-day coculture with CD33⁺ HL60 (upper right), after coculture with HL60 CD33 KO cells (lower left), and on nontransduced cells after coculture with HL60 CD33⁺ cells (lower right). Note that GPR56 upregulation occurs exclusively when CAR-T cells carrying the CD33.CAR were incubated with HL60 CD33⁺ cells. This suggests that GPR56 upregulation occurs only upon antigen recognition by the T cell receptor. (C) Statistical analysis of the experiment shown in 4B. ∗∗∗ P < .0005. (D) Percentage of GPR56⁺CD27⁺ fractions (blue) and CD15⁺ HL60 cells (orange) in the 4 individual donors during the 5 serial 5-day challenges. (E) (Top) Experimental setup of sorting experiment; CAR-T cells were challenged once with HL60 for 5 days. Then, cultures were sorted for CD8⁺ and the following 4 quadrants: GPR56⁺CD27^–, GPR56⁺CD27⁺, GPR56^–CD27⁺, and GPR56^–CD27^–. Subsequently, equal numbers of sorted cells were re-exposed to HL60 cells for 5 days and subsequently analyzed for surface marker expression. All fractions were capable of eliminating HL60 WT cells except for 1 culture with double-negative cells (data not shown), confirming that all fractions contained the CAR construct. (Lower left) Representative FACS plots of the sorted CAR-T cells after re-exposure to target cells. The label above the plots indicates the originally sorted phenotype. (Lower right) Stacked bar graph showing the distribution of the 4 quadrants in the 4 different conditions. Mean and standard deviation of the 4 donors are shown. The x-axis labels indicate the originally sorted fraction. Colors of bars indicate the output phenotype according to the legend. nt, nontransduced; wt, wild type.

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