American Society of Hematology

Figure 1

$Figure 1. An anti-CD19 CAR gives T cells the ability to specifically recognize CD19. (A) The CAR incorporated an anti-CD19 single chain Fv (scFv), part of the CD28 costimulatory molecule, and the cytoplasmic part of the CD3ζ molecule. (B) PBMCs from healthy allogeneic donors were activated with an anti-CD3 monoclonal antibody and transduced with gammaretroviruses encoding an anti-CD19 CAR. The cells were infused after 8 days of culture. (C) Expression of the CAR was detected on the surface of the infusion cells of patient 1 on day 7 of culture by staining with anti–antigen-binding fragment (Fab) antibodies. Staining with isotype-matched control antibodies is also shown. The plots are gated on CD3+ lymphocytes. (D) The infusion cells of patient 1 were stained for CD45RA and CC chemokine receptor 7 (CCR7). Staining with isotype-matched control antibodies is also shown. The staining was performed on day 7 of culture, and the plots are gated on CAR-expressing CD3+ lymphocytes. (E) On day 8 of culture, infusion cells of patient 1 were cultured for 4 hours with either the CD19+ cell line CD19-K562 or the negative-control cell line NGFR-K562 that does not express CD19. CD19-specific upregulation of CD107a occurred. Plots are gated on CD3+ lymphocytes. Similar results were obtained with the infusion cells of all patients treated. (F-H) A representative example of CD19-specific cytokine production by anti-CD19-CAR T cells is shown. Infusion cells of patient 4 were stimulated with either CD19-K562 cells or NGFR-K562 cells for 6 hours on day 8 of culture. Intracellular cytokine staining followed by flow cytometry revealed that large fractions of the T cells produced interferon γ (IFN-γ), tumor necrosis factor (TNF), and interleukin (IL) 2 in a CD19-specific manner. Plots are gated on CD3+ lymphocytes.$

An anti-CD19 CAR gives T cells the ability to specifically recognize CD19. (A) The CAR incorporated an anti-CD19 single chain Fv (scFv), part of the CD28 costimulatory molecule, and the cytoplasmic part of the CD3ζ molecule. (B) PBMCs from healthy allogeneic donors were activated with an anti-CD3 monoclonal antibody and transduced with gammaretroviruses encoding an anti-CD19 CAR. The cells were infused after 8 days of culture. (C) Expression of the CAR was detected on the surface of the infusion cells of patient 1 on day 7 of culture by staining with anti–antigen-binding fragment (Fab) antibodies. Staining with isotype-matched control antibodies is also shown. The plots are gated on CD3⁺ lymphocytes. (D) The infusion cells of patient 1 were stained for CD45RA and CC chemokine receptor 7 (CCR7). Staining with isotype-matched control antibodies is also shown. The staining was performed on day 7 of culture, and the plots are gated on CAR-expressing CD3⁺ lymphocytes. (E) On day 8 of culture, infusion cells of patient 1 were cultured for 4 hours with either the CD19⁺ cell line CD19-K562 or the negative-control cell line NGFR-K562 that does not express CD19. CD19-specific upregulation of CD107a occurred. Plots are gated on CD3⁺ lymphocytes. Similar results were obtained with the infusion cells of all patients treated. (F-H) A representative example of CD19-specific cytokine production by anti-CD19-CAR T cells is shown. Infusion cells of patient 4 were stimulated with either CD19-K562 cells or NGFR-K562 cells for 6 hours on day 8 of culture. Intracellular cytokine staining followed by flow cytometry revealed that large fractions of the T cells produced interferon γ (IFN-γ), tumor necrosis factor (TNF), and interleukin (IL) 2 in a CD19-specific manner. Plots are gated on CD3⁺ lymphocytes.

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