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. 2024 Dec 30;14:31838. doi: 10.1038/s41598-024-82957-9

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. 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-nc-nd/4.0/.

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Fig. 1 — AMBRA1 expression is upregulated during CD8⁺ T cell differentiation to CTLs. (A) Workflow of in vitro CD8⁺ T cell differentiation starting from human CD8⁺ T cells purified from buffy coats of healthy donors. Freshly isolated CD8⁺ T cells (day 0) were activated with anti-CD3/CD28 coated beads in the presence of IL-2 and expanded for 5–7 days to generate CTLs. (B) RT-qPCR analysis of AMBRA1 mRNA and (C) immunoblot analysis of AMBRA1 protein levels in CD8⁺ T cells collected at days 0, 2, 5 and 7 after stimulation. 18S was used for normalization in RT-qPCR analysis. Actin was used as loading control. The migration of molecular mass markers is indicated. The histograms show the quantification of AMBRA1 expression during CTL differentiation (n_donor 3, one sample t test, day 0 value = 1). (D) RT-qPCR analysis of human TBET and RUNX3 mRNA in CD8⁺ T cells at days 0, 2, 5 and 7 after stimulation. 18S was used for normalization (n_donor = 3, one-way ANOVA test, day 0 value = 1). Data are shown as mean fold ± SD, day 0 value = 1. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.

Inline graphic — AMBRA1 expression is upregulated during CD8⁺ T cell differentiation to CTLs. (A) Workflow of in vitro CD8⁺ T cell differentiation starting from human CD8⁺ T cells purified from buffy coats of healthy donors. Freshly isolated CD8⁺ T cells (day 0) were activated with anti-CD3/CD28 coated beads in the presence of IL-2 and expanded for 5–7 days to generate CTLs. (B) RT-qPCR analysis of AMBRA1 mRNA and (C) immunoblot analysis of AMBRA1 protein levels in CD8⁺ T cells collected at days 0, 2, 5 and 7 after stimulation. 18S was used for normalization in RT-qPCR analysis. Actin was used as loading control. The migration of molecular mass markers is indicated. The histograms show the quantification of AMBRA1 expression during CTL differentiation (n_donor 3, one sample t test, day 0 value = 1). (D) RT-qPCR analysis of human TBET and RUNX3 mRNA in CD8⁺ T cells at days 0, 2, 5 and 7 after stimulation. 18S was used for normalization (n_donor = 3, one-way ANOVA test, day 0 value = 1). Data are shown as mean fold ± SD, day 0 value = 1. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001.