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. 2020 May 8;11:2283. doi: 10.1038/s41467-020-16160-5

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Cell-lysis activity of single-input and bispecific CD8⁺ CAR-T cells against K562 targets engineered to express either BCMA or CS1. Cells were seeded at an effector-to-target (E:T) ratio of 2:1, where effector-cell seeding was based on CAR⁺ T-cell count. The fraction of viable K562 cells left after a 20-h coincubation was quantified by fluorescence imaging of target cells using IncuCyte. All bispecific CARs in this panel contained a short extracellular spacer. b Proliferation of single-input and bispecific BCMA/CS1 CD8 + CAR-T cells upon antigen stimulation. CAR-T cells were stained with CellTrace Violet (CTV) dye. CTV median fluorescence intensity (MFI) was quantified by flow cytometry after a 5-day coincubation with parental (BCMA⁻/CS1⁻), BCMA⁺, or CS1⁺ K562 target cells at a 2:1 E:T ratio. CARs containing huLuc63 paired with dAPRIL, J22.9-xi, and huJ22.9-xi were subsequently eliminated from the panel based on poor cytotoxicity and/or T-cell proliferation. c Cytotoxicity of reduced bispecific CAR-T-cell panel upon repeated antigen challenge. CD8⁺ CAR-T cells were coincubated with K562 target cells at a 1:1 E:T ratio and rechallenged every 2 days with fresh target cells. Viable target-cell count was quantified by flow cytometry 2 days after each target-cell addition. ‘C#’ denotes the challenge number and ‘D#’ denotes the number of days post challenge. d Characterization of the remaining K562 target-cell populations after four challenges from c reveals differing antigen preference among the panel of bispecific CARs. Values shown are the means of technical triplicate samples, with error bars indicating +1 standard deviation (SD). P-values were calculated by unpaired two-tailed Student’s t-test; n.s. not statistically significant (p > 0.05); *p < 0.05; **p < 0.01, with Bonferroni correction for multiple comparisons applied. P-values in c were calculated for the final time point for each construct, relative to the top-performing CAR, c11D5.3-Luc90. Source data are provided as a Source Data File. P-values in a and b can be referenced in the Source Data File.