Swan Gregory A, Fujii Chika, Guzynski Mia E, Page Sheridan M, Meyers Isabelle V, Penev Yordan P, Littleton Sejiro, Azzahra Adinda, Richardson Christine, Sarafova Sophia D
Integrative Immunobiology Department, Duke University, Durham, NC, United States.
Biology Department, Davidson College, Davidson, NC, United States.
Front Immunol. 2025 Jan 15;15:1469402. doi: 10.3389/fimmu.2024.1469402. eCollection 2024.
The regulation of expression during T-cell development and immune responses is essential for proper lineage commitment and function in the periphery. However, the mechanisms of genetic and epigenetic regulation are complex, and their interplay not entirely understood. Previously, we demonstrated the need for CD4 upregulation during positive selection to ensure faithful commitment of MHC-II-restricted T cells to the CD4 lineage. In this study, we investigate whether a conserved region, here called NCE, that is proximal to the Cd4 silencer and contains E4m has the required developmental-stage-specific canonical enhancer function and TCR responsiveness to mediate the CD4 upregulation required to prevent lineage errors.
To investigate the role of NCE, transient transfection of reporter plasmids was performed in thymoma cell lines arrested at the double-positive (DP, CD4CD8) and intermediate (INT, CD4CD8) stages of development. CRISPR/Cas9-mediated deletion of the coreNCE/E4m region was carried out in these cell lines to assess its impact on CD4 surface expression, re-expression rates, and TCR signaling responsiveness. To avoid developmental alterations from direct manipulation of the endogenous locus , BAC-transgenic reporter mice were generated with the locus modified to express EGFP in the presence or absence of NCE. EGFP mRNA levels were measured via RT-qPCR, and EGFP fluorescence was analyzed in post-selection thymocytes.
Our experiments demonstrate that NCE by itself can function as an enhancer at the INT, but not the DP stage of development. Furthermore, CRISPR/Cas9-mediated deletion of coreNCE/E4m resulted in reduced CD4 surface levels, slower re-expression rates, and reduced TCR signaling responsiveness in INT cells, but not in DP cells. , NCE-sufficient transgenic mice exhibited upregulation of Cd4 reporter EGFP mRNA levels at the INT stage and a corresponding upregulation of EGFP fluorescence, whereas NCE-deficient mice showed a significant loss of reporter EGFP mRNA and no detectable EGFP production in any post-selection thymocytes.
This study demonstrates that the canonical enhancer function of coreNCE/E4m is essential for CD4 upregulation following positive selection. The NCE region, with its developmental-stage-specific activity and its known epigenetic regulatory capabilities, ensures faithful lineage commitment to the CD4 lineage.
T细胞发育和免疫反应过程中的表达调控对于外周正确的谱系定向和功能至关重要。然而，遗传和表观遗传调控机制复杂，它们之间的相互作用尚未完全了解。此前，我们证明了在阳性选择过程中上调CD4对于确保MHC-II限制性T细胞忠实地定向到CD4谱系是必要的。在本研究中，我们调查了一个保守区域（此处称为NCE），它靠近Cd4沉默子且包含E4m，是否具有所需的发育阶段特异性的典型增强子功能和TCR反应性，以介导防止谱系错误所需的CD4上调。
为了研究NCE的作用，在停滞于双阳性（DP，CD4CD8）和中间（INT，CD4CD8）发育阶段的胸腺瘤细胞系中进行了报告质粒的瞬时转染。在这些细胞系中进行CRISPR/Cas9介导的coreNCE/E4m区域缺失，以评估其对CD4表面表达、重新表达率和TCR信号反应性的影响。为了避免因直接操纵内源性基因座而导致的发育改变，构建了BAC转基因报告小鼠，其基因座经修饰后在有或无NCE的情况下表达EGFP。通过RT-qPCR测量EGFP mRNA水平，并在选择后的胸腺细胞中分析EGFP荧光。
我们的实验表明，NCE自身在发育的INT阶段而非DP阶段可作为增强子发挥作用。此外，CRISPR/Cas9介导的coreNCE/E4m缺失导致INT细胞中CD4表面水平降低、重新表达率减慢以及TCR信号反应性降低，但在DP细胞中未出现这种情况。此外，NCE充足的转基因小鼠在INT阶段Cd4报告基因EGFP mRNA水平上调，EGFP荧光相应上调，而NCE缺陷小鼠在任何选择后的胸腺细胞中报告基因EGFP mRNA显著缺失且未检测到EGFP产生。
本研究表明coreNCE/E4m的典型增强子功能对于阳性选择后CD4上调至关重要。NCE区域具有发育阶段特异性活性及其已知的表观遗传调控能力，可确保忠实地定向到CD4谱系。
