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. 2023 Jun 22;14:3728. doi: 10.1038/s41467-023-39295-7

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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 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 — a Representative gating strategy for one healthy donor (HD) and one NBEAL2-deficient patient on CD4 + T cells. Tregs were defined as CD4⁺CD25⁺CD127_low T cells. b Representative histograms of CTLA-4 and FOXP3 expression on CD25⁺CD127_low Treg for one healthy donor (HD) and one NBEAL2-deficient patient. c Normalized CTLA-4 mean of fluorescence intensity (MFI) in CD25⁺CD127_low Treg from controls (N = 6) and NBEAL2-deficient patients (N = 7). Mean ± SEM is represented. d Gating strategy to define CTLA-4 positive T cells after activation. The gating was tuned using a control isotype antibody. e Normalized CTLA-4 mean of fluorescence intensity (MFI) of activated T cells from controls (N = 4) and NBEAL2-deficient patients (N = 8). Mean ± SEM is represented. Two-tailed p-value is determined with a non-parametric Mann–Whitney test. f, g Diagram of the primary structure of NBEAL2 (f) and LRBA (g) proteins. Positions targeted by the CRISPR guide RNA used are indicated. h, i NBEAL2 (h) and LRBA (i) immunoblotting after CRISPR-Cas9 experiment on activated T cells. WT: refers to cells that underwent nucleofection without CRISPR/Cas9 in the solution. Ctrl: refers to cells nucleofected with a scramble guide and the Cas9. AAVS1: refers to cells transfected with a the Cas9 and a guide targeting an intronic position of the genome. Other cell lysates correspond to NBEAL2-CRISPR/Cas9 treatment (f) and LRBA-CRISPR/Cas9 treatment (g). Ku-70 is used as a loading control. j Normalized CTLA-4 MFI after CRISPR-Cas9 knockdown (KD) experiment for each mRNA guide (6 for NBEAL2; 5 for LRBA) used on three independent healthy donors activated T cells, over 2 independent experiments. Mean ± SEM is represented. Controls correspond to the three conditions Ctrl, WT, and AAVS1 described in h and i for each donor tested. Non-parametric two-sided Kruskal-Wallis test corrected with Dunn’s test for multiple comparisons was used to compare each group. k Immunoprecipitation (IP) of CTLA-4 in activated (+) or unstimulated (−) T cells from healthy donors. NBEAL2, LRBA, and CTLA-4 were immunoblotted on input fractions and on the IP fractions, as indicated (blots representative of N = 3 experiments). Source data are provided as a Source Data file.