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. 2021 Aug 11;12:4844. doi: 10.1038/s41467-021-25223-0

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© The Author(s) 2021

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. 8 — DN4.99 TCR-transduced T (DN4.99 TCR-T) cells restrict NALM-6-CD1c leukemia progression in vivo. a NOD.Cg-Prkdcscid IL- 2rgtm1Wjl/SzJ (NSG) mice (n = 30) received intravenous (i.v.) injection of 5 × 10⁵ NALM-6 CD1c co-expressing secreted LUCIA luciferase. Two days later, the mice were sub-lethally irradiated, followed 24 h later by the transfer of 10⁷ primary 70% CD8⁺/30% CD4⁺ DN4.99 TCR-T cells (red lines/dots), control T cells (black line/squares) transduced with the CD1c-restricted, mycobacterial lipid-specific DL15A31 TCR (DL153A1 TCR-T cells; Supplementary Fig. 4 and ref. ¹⁰) or vehicle (gray lines/triangles). n = 10 mice/group. After 7 days, DN4.99 TCR-T cells were transferred a second time, without prior irradiation, into the same mice. b Tumor progression was monitored in peripheral blood by a bioluminescence assay. RLU, relative light unit. Data are represented as mean ± SEM. ****P < 0.0001 determined on AUC by Ordinary one-way ANOVA followed by Tukey’s multiple comparison test. c DN4.99 TCR-T cells reduce the number of circulating NALM-6-CD1c leukemia cells. The frequency of human CD19⁺ tumor cells was determined by flow cytometry in the blood of mice at day+17 from tumor injection. The percentage of the tumor cells was normalized based on the percentage of mouse CD45^- cells. *P = 0.0101; ***P = 0.0003; ****P < 0.0001. d Presence of DN4.99 TCR-T cells in the bone marrow of NALM-6 CD1c bearing mice, determined by flow cytometry 4 days after their adoptive transfer. DN4.99 TCR-T cells were identified as mouse (m)TCRb⁺ cells in the human (h)CD45⁺ mouse (m)CD45^- cells^. e Kaplan-Meier survival curves show a significant increase in the survival of mice receiving DN4.99 TCR-T cells compared to control groups. **P = 0.0014 determined by log-rank (Mantel–Cox) test. f Reduced CD1c expression on NALM-6-CD1c cells that survive DN4.99 TCR-T cell, as determined by flow cytometry labeling (MFI, Mean Fluorescence Intensity) of human CD19⁺ NALM-6-CD1c cells in the circulation at day+17 from tumor injection. **P = 0.0015; ****P < 0.0001. Data in (c) and (f) are represented as mean ± SD and P-values were determined by unpaired two-tailed t test followed by Mann–Whitney test. Results are representative of 3 independent experiments with independent T cell lines giving comparable results.