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. 2023 Jul 27;14:4524. doi: 10.1038/s41467-023-40170-8

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a OT-I cells isolated from wild type OT-I;Ptpn2^fl/fl (n = 5–6) versus OT-I;Lck-Cre;Ptpn2^fl/fl (n = 4) mice were stimulated with 1 nM of the cognate ovalbumin (OVA) peptide antigen SIINFEKL (N4) in the presence of vehicle (1% v/v DMSO) or Compound 182 (CMP-182; 1 μM) for 48 h and CD25 and CD69 mean fluorescent intensities (MFIs) in CD8⁺ OT-I cells were determined by flow cytometry. b OT-I cells (n = 6) were activated with 1 nM N4 for 16 h and rested in media supplemented with IL-2 (5 ng/ml) and IL-7 (0.2 ng/ml) for 2 days. On day 3, T cells were pre-treated with vehicle or 1 μM CMP-182 overnight and stimulated with 1 nM N4 plus/minus 1 μM CMP-182 in the presence of GolgiPlug™/GolgiStop™. IFN-γ⁺, TNF⁺ or Granzyme B⁺ (GRZMB) CD8⁺ T cells were analyzed by flow cytometry. c OT-I T cells isolated from wild-type OT-I; Ptpn2^fl/fl (n = 4) versus OT-I;Lck-Cre; Ptpn2^fl/fl (n = 4) mice were stained with CTV and stimulated with 0.5 nM N4 plus/minus 1 μM CMP-182 for 3 days and CTV dilution monitored by flow cytometry. d Naive CD8⁺ T cells (C57BL6) (n = 3) were preincubated with 10 μM CMP-182 for 4 h, the TCR cross-linked with α-mouse CD3ε (5 μg/ml) and goat anti-hamster IgG (20 μg/ml) and Y418 phosphorylated and activated SFK (p-SFK) MFIs determined by flow cytometry. e OT-I cells (C57BL/6) (n = 4) were stimulated with 1 nM N4 for 16 h, incubated plus/minus of 1 μM CMP-182 for 1 h and Y694 phosphorylated STAT-5 (p-STAT-5) in CD8⁺ OT-I cells assessed by flow cytometry. f OT-I cells (C57BL/6) (n = 6) were stimulated with 1 nM N4 for 16 h, incubated plus/minus 1 μM CMP-182 for 1 h, and then stimulated with IL-2 (5 ng/ml) plus/minus 1 μM CMP-182 and p-STAT-5 MFIs in CD8⁺ OT-I cells determined by flow cytometry. In (a–f) representative results (means ± SEM) from at least two independent experiments are shown. Significances in (a, c) were determined using a 1-way ANOVA Test and in (b, e) using a 2-tailed Mann-Whitney U Test. In (d, f) significances were determined using a 2-way ANOVA Test.