RNA-sequencing of cTECs, mTECs, and thymocyte maturation subsets SM, M1 and M2 from the CD4 and CD8 lineages from WT and Psmb11-deficient mice [DSP405]

Purpose: Cortical thymic epithelial cells (cTECs) contain a unique type of proteasomes, thymoproteasomes. Indirect evidence suggests that the key role of PSMB11, a catalytic subunit of thymoproteasomes specific to cTECs, is to generate a unique repertoire of MHC I peptides. Notably, PSMB11-deficient mice display defective development of CD8 thymocytes. The objective of this study was to characterize the impact of PSMB11 on cTECs and thymocyte development. Since different types of proteasomes have non-redundant effects on gene expression, we hypothesized that thymoproteasomes should have a distinct impact on the transcriptome and thereby the function of cTECs. Results: We report that PSMB11 in cortical thymic epithelial cells has dramatic effects on cTECs on both CD4 and CD8 thymocyte populations. PSMB11 modulates the expression of 850 genes in cTECs, 582 in CD4 thymocytes and 284 in CD8 thymocytes. PSMB11-regulated cTEC genes are involved mainly in cell-cell adhesion, extracellular matric organization and thymocyte chemotaxis. PSMB11-deficient cTECs acquire features of mTECs and perturb thymocyte development. Deletion of PSMB11 causes a major cell stress in both CD4 and CD8 thymocyte populations. Of note, PSMB11-deficiency had no impact on medullary thymic epithelial cells (mTECs), which originate from progenitors that express PSMB11 early in ontogeny. Conclusion: We conclude that PSMB11 has pervasive effects on both CD4 and CD8 thymocytes via regulation of gene expression in cTECs. We performed RNA-sequencing in triplicate on cTECs, mTECs, and SM, M1 and M2 thymocytes from the CD4 and CD8 lineages, in order to identify differential gene expression between WT and Psmb11-deficient mice.研究目的：皮质胸腺上皮细胞（cortical thymic epithelial cells, cTECs）含有一类独特的蛋白酶体，即胸腺蛋白酶体（thymoproteasomes）。间接证据表明，仅在cTECs中特异性表达的胸腺蛋白酶体催化亚基PSMB11，其核心功能是生成独特的主要组织相容性复合体I（Major Histocompatibility Complex I, MHC I）肽段库。值得注意的是，PSMB11缺陷小鼠会出现CD8胸腺细胞发育缺陷。本研究旨在阐明PSMB11对cTECs以及胸腺细胞发育的影响。鉴于不同类型的蛋白酶体对基因表达具有非冗余调控作用，我们提出假说：胸腺蛋白酶体应对cTECs的转录组及其功能产生特异性影响。研究结果：本研究显示，皮质胸腺上皮细胞中的PSMB11对CD4和CD8两类胸腺细胞群体以及cTECs本身均具有显著调控作用。PSMB11可调控cTECs中850个基因、CD4胸腺细胞中582个基因以及CD8胸腺细胞中284个基因的表达。受PSMB11调控的cTECs基因主要参与细胞-细胞黏附、细胞外基质（extracellular matrix, ECM）组织以及胸腺细胞趋化过程。PSMB11缺陷的cTECs会获得髓质胸腺上皮细胞（medullary thymic epithelial cells, mTECs）的特征，并扰乱胸腺细胞发育。PSMB11的缺失会在CD4和CD8胸腺细胞群体中引发显著的细胞应激。值得关注的是，PSMB11缺陷对髓质胸腺上皮细胞（mTECs）并无影响，而mTECs源自胚胎发育早期即表达PSMB11的祖细胞。研究结论：综上，PSMB11可通过调控cTECs的基因表达，对CD4和CD8胸腺细胞产生广泛影响。为鉴定野生型（Wild Type, WT）与Psmb11缺陷小鼠之间的差异基因表达，我们对cTECs、mTECs以及CD4和CD8谱系的SM、M1、M2胸腺细胞进行了三次重复的RNA测序（RNA-sequencing）。