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. 2021 Oct 9;13(20):5039. doi: 10.3390/cancers13205039

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© 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

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Ide improves fitness of DNTs by inhibiting exhaustion and enhancing viability. (A) Expression of exhaustion markers, LAG3 and TIM3, on unstained (dotted line), ctrl-DNT (Red), and Ide-DNT (Black). Representative flow plot is shown (Left). The numbers represent the MFI value (Right). (B) The summary of results comparing the relative MFI of LAG3 and TIM3 on the ctrl-DNT and Ide-DNT from five donors. Horizontal bars represent the mean relative MFI and error bars show SEM. (C) Expression of the exhaustion-associated transcription factor gene, TOX, relative to the expression of the housekeeping gene, HPRT, determined by qPCR. Each paired symbol represents DNTs from one individual. (D) Production of TNFα and IFNγ by the ctrl-DNT and Ide-DNT with or without ex vivo stimulation. The ctrl-DNT and Ide-DNT were treated with a protein transport inhibitor, followed by stimulation using anti-CD3/CD28 activation beads for 4 h. Representative flow plot is shown (Left). The numbers represent the frequency in each gate. Bar graph shows the percentage of ctrl-DNT (black) and Ide-DNT (red) expressing TNFα, IFNγ, and TNFα and IFNγ (Right). The result shown is a summary of three experiments. (E) Frequency of viable ctrl-DNTs and Ide-DNTs on day 17 of expansion. The cell viability was determined with Annexin V and 7-AAD staining using flow cytometry. Flow plot shows a representative image of cell viability (Left). Dot plot shows summary of viability results using DNTs from five different donors (Right). Horizontal bars represent the mean, and the error bar shows SEM. (F) Viability of ctrl-DNTs and Ide-DNTs after co-culture with primary AML cells from seven different patients. Ctrl-DNTs and Ide-DNs were co-incubated with primary AML cells at 4:1 effector-to-target ratio for 2 h. Subsequently, the viability of DNTs (gated on CD45^high CD3⁺ CD33/CD34⁻) were determined using flow cytometry. (G) Expression of the anti-apoptotic gene, BCL2, on ctrl-DNT and Ide-DNTs relative to the expression of the housekeeping gene, HPRT, was determined by qPCR. Each paired symbol represents DNTs from one donor. * p < 0.05, ** p < 0.01, *** p < 0.001.

Ide improves fitness of DNTs by inhibiting exhaustion and enhancing viability. (A) Expression of exhaustion markers, LAG3 and TIM3, on unstained (dotted line), ctrl-DNT (Red), and Ide-DNT (Black). Representative flow plot is shown (Left). The numbers represent the MFI value (Right). (B) The summary of results comparing the relative MFI of LAG3 and TIM3 on the ctrl-DNT and Ide-DNT from five donors. Horizontal bars represent the mean relative MFI and error bars show SEM. (C) Expression of the exhaustion-associated transcription factor gene, TOX, relative to the expression of the housekeeping gene, HPRT, determined by qPCR. Each paired symbol represents DNTs from one individual. (D) Production of TNFα and IFNγ by the ctrl-DNT and Ide-DNT with or without ex vivo stimulation. The ctrl-DNT and Ide-DNT were treated with a protein transport inhibitor, followed by stimulation using anti-CD3/CD28 activation beads for 4 h. Representative flow plot is shown (Left). The numbers represent the frequency in each gate. Bar graph shows the percentage of ctrl-DNT (black) and Ide-DNT (red) expressing TNFα, IFNγ, and TNFα and IFNγ (Right). The result shown is a summary of three experiments. (E) Frequency of viable ctrl-DNTs and Ide-DNTs on day 17 of expansion. The cell viability was determined with Annexin V and 7-AAD staining using flow cytometry. Flow plot shows a representative image of cell viability (Left). Dot plot shows summary of viability results using DNTs from five different donors (Right). Horizontal bars represent the mean, and the error bar shows SEM. (F) Viability of ctrl-DNTs and Ide-DNTs after co-culture with primary AML cells from seven different patients. Ctrl-DNTs and Ide-DNs were co-incubated with primary AML cells at 4:1 effector-to-target ratio for 2 h. Subsequently, the viability of DNTs (gated on CD45^high CD3⁺ CD33/CD34⁻) were determined using flow cytometry. (G) Expression of the anti-apoptotic gene, BCL2, on ctrl-DNT and Ide-DNTs relative to the expression of the housekeeping gene, HPRT, was determined by qPCR. Each paired symbol represents DNTs from one donor. * p < 0.05, ** p < 0.01, *** p < 0.001.