American Society of Hematology

Figure 2.

$Loss of Tyr260 results in resistance to CAR T cells by preventing surface expression of CD19. (A) Percentage of Jurkat cells engineered to express either WT or CD19ΔY260 that were killed by CAR T cells. Cocultures were established at several effector-to-target ratios, and measurements were taken after 48 hours. (B) Jurkat cells engineered to express either WT or ΔY260 with a GFP marker were combined with nonengineered CD19− GFP− Jurkats at a ratio of 1:1, and these mixed cell pools were combined with CD19 CAR T cells. Data represent composition of cocultures after 48 hours. (C-D) Jurkat cells were engineered to express either WT or ΔY260 CD19 with a GFP marker, and binding of anti-CD19 antibodies SJ25C1 (C) and HIB19 (D) was evaluated by flow cytometry. (E-F) Jurkat cells were engineered to express either WT or ΔY260 CD19 that was linked to an N-terminal FLAG tag, and expression of CD19 and FLAG was determined by extracellular staining (E) or intracellular staining (F) and analysis by flow cytometry. (G) Jurkat cells engineered with nothing (CD19−), WT, or ΔY260 CD19 underwent fractionated cellular lysis. Lysates from the whole cell (WCL) or cytoplasmic or membrane compartments were separated by electrophoresis, and membranes were stained for CD19 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). (H) Peptide:N-glycosidase (PNGase) F–treated whole-cell lysates from Jurkats expressing either WT or ΔY260 CD19 were probed for CD19 or GAPDH (loading control).$

Loss of Tyr260 results in resistance to CAR T cells by preventing surface expression of CD19. (A) Percentage of Jurkat cells engineered to express either WT or CD19ΔY260 that were killed by CAR T cells. Cocultures were established at several effector-to-target ratios, and measurements were taken after 48 hours. (B) Jurkat cells engineered to express either WT or ΔY260 with a GFP marker were combined with nonengineered CD19⁻ GFP⁻ Jurkats at a ratio of 1:1, and these mixed cell pools were combined with CD19 CAR T cells. Data represent composition of cocultures after 48 hours. (C-D) Jurkat cells were engineered to express either WT or ΔY260 CD19 with a GFP marker, and binding of anti-CD19 antibodies SJ25C1 (C) and HIB19 (D) was evaluated by flow cytometry. (E-F) Jurkat cells were engineered to express either WT or ΔY260 CD19 that was linked to an N-terminal FLAG tag, and expression of CD19 and FLAG was determined by extracellular staining (E) or intracellular staining (F) and analysis by flow cytometry. (G) Jurkat cells engineered with nothing (CD19⁻), WT, or ΔY260 CD19 underwent fractionated cellular lysis. Lysates from the whole cell (WCL) or cytoplasmic or membrane compartments were separated by electrophoresis, and membranes were stained for CD19 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control). (H) Peptide:N-glycosidase (PNGase) F–treated whole-cell lysates from Jurkats expressing either WT or ΔY260 CD19 were probed for CD19 or GAPDH (loading control).

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