Single-cell RNA sequencing of IkbkbV203I Tregs from spleen and bone marrow

Foxp3+ regulatory T cells (Tregs) typically suppress immune responses to prevent autoimmunity and aberrant inflammatory responses. In addition, tissue resident Tregs contribute to homeostasis and repair of non-lymphoid parenchyma. We generated a mouse model of a human inborn error of immunity arising from a gain-of-function missense mutation inIkbkband observed cell-intrinsic expansion of Tregs that maintained expression of Foxp3 and CD25. Remarkably, a subset of Tregs in this model expressed IL-17. We show by scRNA seq that NF-kB regulates specifically the expansion of a subset tissue Tregs, previously names skin NLT. Mice homozygous for theIkbkbmutation developed psoriasis, while homozygous mice also developed features of psoriatic arthritis. Skin NLTs were enriched at sites of pathology, and disease was dependent on the presence of pathological Tregs. Thus, we identify canonical NF-kB as a regulator tissue Treg differentiation and that this pathway can explain aberrant tissue healing responses that underpin psoriasis and psoriatic arthritis. FACS-sorted FOXP3-GFP+ Tregs from bone marrow and spleen of wild type and IkbkbV203I homozygous mice. Single gene expression libraries were preprared by the Biomolecular Resource Facility at JCSMR, ANU using Chromium Next GEM Single cell 5' kit v2 Dual Index (10X Genomics) according to manufacturer's instructions. The library was sequenced in S1 Flowcell with 2x50bp paired-end reads by NovaSeq 6000 sequencer (Illumina). The 10X Cell ranger package (10X Genomics, v6.0.1) was used to process data and align to mouse genome (mm10).Foxp3+调节性T细胞（Foxp3+ regulatory T cells，Tregs）通常通过抑制免疫应答，防止自身免疫与异常炎症反应的发生。此外，组织驻留Tregs还可参与非淋巴组织实质的稳态维持与损伤修复。 我们构建了一种由Ikbkb基因功能获得性错义突变引发的人类原发性免疫缺陷病小鼠模型，观察到Tregs发生细胞自主性扩增，且持续表达Foxp3与CD25。值得注意的是，该模型中存在一类表达IL-17的Tregs亚群。我们通过单细胞RNA测序（scRNA seq）证实，核因子κB（NF-κB）可特异性调控此前被命名为皮肤非淋巴组织（skin non-lymphoid tissue，NLT）的组织驻留Tregs亚群的扩增。 携带Ikbkb突变的纯合小鼠会出现银屑病表型，同时还会表现出银屑病关节炎的相关特征。皮肤NLTs在病理部位富集，且疾病进程依赖于致病性Tregs的存在。综上，我们证实经典NF-κB通路是组织Treg分化的调控因子，且该通路可解释支撑银屑病与银屑病关节炎发病的异常组织愈合应答。 我们从野生型与IkbkbV203I纯合小鼠的骨髓及脾脏中分选得到FOXP3-GFP+ Tregs。澳大利亚国立大学约翰·柯廷医学研究学院（JCSMR）生物分子资源设施中心采用Chromium Next GEM单细胞5'双索引试剂盒v2（10X Genomics），按照制造商提供的操作说明制备了单细胞基因表达文库。该文库使用NovaSeq 6000测序仪（Illumina）在S1流动槽中以2×50bp双端读长模式进行测序。我们使用10X Cell ranger软件包（10X Genomics，v6.0.1）对测序数据进行处理，并将序列比对至小鼠基因组（mm10）。