Klf6 is required in thymic epithelial cells for normal thymus development [scATAC-seq]

Thymic epithelial cells (TEC) control T cell development and play essential roles in establishing self-tolerance. Transcription factors controlling TEC development are poorly characterized. We report that Klf6 plays a critical role in TEC development. Mice deficient for Klf6 in TEC had a hypoplastic thymus - evident from fetal stages into adulthood. Proliferation of TEC was not reduced, but a dramatic increase in the frequency of apoptotic TEC in fetal and adult thymus was observed. Among cortical TEC (cTEC), we found expansion of a previously unreported cTEC population expressing the transcription factor Sox10. Medullary TEC (mTEC) subsets were not equally impacted with Ccl21a+ mTEC I and Tuft-like mTEC IV being disproportionately affected. Consistent with these TEC defects, naïve conventional T cells and NKT cells were reduced in the spleen, and signs of autoimmunity were evident. Thus, Klf6 has a pro- survival role in TEC and is also required for differentiation of the mTEC I and mTEC IV populations of TEC in adult mice. In this work, we report that mice with Foxn1-Cre mediated ablation of Klf6 in TEC demonstrate thymic hypoplasia beginning from prenatal life and extending through adulthood. Guided by single-cell transcriptional profiling, we determined that loss of Klf6 increased programmed cell death of TEC in prenatal and adult mice. In adult mice, thymic Klf6 deficiency severely impacted the mTEC I and mTEC IV populations. In addition, Klf6 deficiency led to the expansion of a previously uncharacterized cTEC population expressing Sox10 that is present in wild-type mice at very low frequencies. We observed concordant reductions of the naïve aß T cell and iNKT pools in the periphery of young adult mice. Furthermore, we detected T cell infiltration in salivary and lacrimal glands, indicating defects in T cell tolerance. Overall design: scATAC-seq of Adult_KO_TEC and Adult_WT_TEC

胸腺上皮细胞（Thymic epithelial cells, TEC）调控T细胞发育，并在建立自身耐受过程中发挥关键作用。目前对调控TEC发育的转录因子的研究尚不充分。本研究证实Klf6在TEC发育中具有关键作用。TEC中Klf6缺陷的小鼠会出现胸腺发育不全，该表型从胎儿期持续至成年阶段。TEC的增殖并未受到抑制，但在胎儿及成年胸腺中，凋亡TEC的比例显著升高。在皮质胸腺上皮细胞（cortical TEC, cTEC）中，我们发现一个此前未被报道的、表达转录因子Sox10的cTEC亚群出现扩增。髓质胸腺上皮细胞（medullary TEC, mTEC）的各亚群受影响程度不均：Ccl21a+ mTEC I亚型与簇状样mTEC IV亚型受到的影响尤为显著。与上述TEC缺陷表型一致，初始常规T细胞与自然杀伤T细胞（NKT cells）在脾脏中的数量减少，且出现自身免疫相关症状。综上，Klf6在TEC中发挥促存活作用，同时也是成年小鼠体内mTEC I与mTEC IV亚群分化所必需的。

本研究中，我们报道了通过Foxn1-Cre介导TEC中Klf6基因敲除的小鼠，其胸腺发育不全从产前时期开始并持续至成年。借助单细胞转录组测序分析，我们证实Klf6缺失会增加产前及成年小鼠TEC的程序性细胞死亡。在成年小鼠中，胸腺Klf6缺陷会严重影响mTEC I与mTEC IV亚群。此外，Klf6缺陷会导致一个此前未被鉴定的、表达Sox10的cTEC亚群出现扩增，该亚群在野生型小鼠中仅以极低频率存在。我们观察到年轻成年小鼠外周中初始αβ T细胞与不变自然杀伤T细胞（iNKT）细胞池的一致性减少。进一步检测发现，唾液腺与泪腺中存在T细胞浸润，提示T细胞耐受存在缺陷。

实验整体设计：对成年Klf6敲除TEC（Adult_KO_TEC）与成年野生型TEC（Adult_WT_TEC）进行单细胞转座酶可及性测序（scATAC-seq）。