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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. 4 — Primary T cells were transduced (DN4.99 TCR-T cells) or not-transduced (T cells) with the lead DN4.99 TCR and assessed in vitro for the recognition and killing of CD1c-expressing acute leukemia or lymphoblastoid cell lines. a CD1c expression (black histograms) on 7 different acute leukemia (THP-1, MOLM-13, K562, NALM-6, CCRF-SB, MOLT-4) or lymphoblastoid (C1R) cell lines, as determined by flow cytometry measurement of labeling with anti-human CD1c monoclonal antibody (mAb). White histograms represent labeling with isotype-matched control mAb. Relative Fluorescence Intensity (RFI) was calculated as the ratio between intensity of labeling of the sample and control. b Recognition of CD1c-expressing acute leukemia or lymphoblastoid cell lines by DN4.99 TCR-transduced or non-transduced primary T cells following 48 h of co-culture at a 1:1 Effector:Target ratio with either 20 μg/ml of blocking anti-CD1c mAb (gray bars) or an equivalent concentration of mouse IgG1κ isotype control (black bars). IFN-γ secretion into supernatants was measured by ELISA. Data are represented as mean ± SD. Results are representative of 2 (for CCRF-SB, MOLT-4 and C1R-CD1c) and 4 (for THP-1-CD1c, MOLM-13-CD1c, K562-CD1c and NALM-6-CD1c) independent experiments with n = 3 replicates per experiments. ***P = 0.0001; **** P < 0.0001 determined by Ordinary one-way ANOVA followed by Tukey’s multiple comparison test. c, d Representative flow cytometry analysis to assess killing of THP-1-CD1c (c) and CCRF-SB (d) cells by DN4.99 TCR-T cells after 72 h of the same co-culture conditions of (b). e Killing of each target cell line in the same co-cultures determined as shown in (c, d), expressed as an Elimination Index as described in Methods. Gray bars represent co-cultures with anti-CD1c mAb, and black bars with the isotype control. Data are represented as mean ± SD. Results are representative of 2 (for K562-CD1c, CCRF-SB, MOLT-4 and C1R-CD1c) and 4 (for THP-1-CD1c, MOLM-13-CD1c and NALM-6-CD1c) independent experiments with n = 3 replicates per experiments. **P = 0.0071; ****P < 0.0001 determined by unpaired two-tailed Welch’s t-test.