Table 4_Direct and indirect RANK and CD40 signaling regulate the maintenance of thymic epithelial cell frequency and properties in the adult thymus.xlsx

Medullary thymic epithelial cells (mTECs) play a crucial role in suppressing the onset of autoimmunity by eliminating autoreactive T cells and promoting the development of regulatory T cells in the thymus. Although mTECs undergo turnover in adults, the molecular mechanisms behind this process remain unclear. This study describes the direct and indirect roles of receptor activator of NF-κB (RANK) and CD40 signaling in TECs in the adult thymus. Flow cytometric and single-cell RNA-seq (scRNA-seq) analyses suggest that the depletion of both RANK and CD40 signaling inhibits mTEC differentiation from CCL21+ mTEC progenitors to transit-amplifying TECs in the adult thymus. Unexpectedly, this depletion also exerts indirect effects on the gene expression of TEC progenitors and cortical TECs. Additionally, the expression levels of AP-1 genes, which enable the further subdivision of TEC progenitors, are up-regulated following the depletion of RANK and CD40 signaling. Overall, our data propose that RANK and CD40 signaling cooperatively maintain mature mTEC frequency in the adult thymus and sustain the characteristics of TEC progenitors through an indirect mechanism.

胸腺髓质上皮细胞（Medullary thymic epithelial cells, mTECs）在胸腺内发挥关键功能：通过清除自身反应性T细胞、促进调节性T细胞发育，抑制自身免疫疾病的发生。尽管成人胸腺中的mTECs存在细胞更新现象，但该过程背后的分子机制目前仍不明确。本研究解析了NF-κB受体激活剂（receptor activator of NF-κB, RANK）与CD40信号通路在成人胸腺上皮细胞中的直接与间接调控作用。流式细胞术与单细胞RNA测序（single-cell RNA-seq, scRNA-seq）分析结果显示，同时阻断RANK与CD40信号通路，会抑制成人胸腺内CCL21阳性mTEC祖细胞向过渡扩增型胸腺上皮细胞的分化进程。出乎意料的是，该信号通路联合缺失还会对胸腺上皮细胞祖细胞与皮质胸腺上皮细胞（cortical TECs）的基因表达产生间接影响。此外，介导胸腺上皮细胞祖细胞进一步分型的AP-1家族基因的表达水平，在RANK与CD40信号通路联合缺失后出现上调。综上，本研究数据表明，RANK与CD40信号通路可协同维持成人胸腺内成熟mTEC的细胞丰度，并通过间接机制维持胸腺上皮细胞祖细胞的固有特性。