Gene expression profiling of murine innate lymphoid cells

Innate lymphoid cells (ILCs) are a recently recognized heterogenous group of immune cells that are critical in orchestrating immunity and inflammation in the intestine, but whether ILCs influence immune responses or tissue homeostasis at other mucosal sites remains poorly characterized. Here we identify a population of lung-resident ILCs in mice and humans that expressed the alloantigen Thy-1 (CD90), interleukin 2 (IL-2) receptor a-chain (CD25), IL-7 receptor a-chain (CD127) and the IL-33 receptor subunit T1-ST2. Notably, mouse ILCs accumulated in the lung after infection with influenza virus, and depletion of ILCs resulted in loss of airway epithelial integrity, diminished lung function and impaired airway remodeling. These defects were restored by administration of the lung ILC product amphiregulin. Collectively, our results demonstrate a critical role for lung ILCs in restoring airway epithelial integrity and tissue homeostasis after infection with influenza virus. As part of this study, we performed gene expression profiling to examine how the transcriptional signatures compared between murine naïve group 2 lung ILC and group 3 splenic LTi cell populations. Group 2 ILCs from the lung (Lin- CD90+ CD25+ T1/ST2+) and Group 3 LTi cells from the spleen (Lin- CD90+ CD25+ CD4+) were sort-purified from naive wildtype C57BL/6 mice using BD FACS Aria. Four biological replicates were collected, each replicate containing 1.5 x 104 to 2 x104 cells sorted to a purity >97% from six pooled lungs (ILCs) or 10 pooled spleens (LTi cells). Cells were sorted directly into TRIzol LS (Invitrogen), then mRNA was isolated, amplified, labeled, and hybridized to Affymetrix GeneChip (Mouse Gene 1.0 ST).

固有淋巴细胞（innate lymphoid cells, ILCs）是近年来被发现的一类异质性免疫细胞，在调控肠道免疫应答与炎症反应中发挥关键作用，但目前对于固有淋巴细胞在其他黏膜部位是否影响免疫应答或组织稳态，相关研究仍较为匮乏。
本研究在小鼠与人类体内鉴定出一群肺驻留型固有淋巴细胞，该群细胞表达同种异体抗原Thy-1（CD90）、白细胞介素2（IL-2）受体α链（CD25）、IL-7受体α链（CD127）以及IL-33受体亚基T1-ST2。
值得注意的是，小鼠感染流感病毒后，肺部固有淋巴细胞会发生募集；而固有淋巴细胞的清除会导致气道上皮完整性丧失、肺功能下降以及气道重塑受损。通过给予肺驻留固有淋巴细胞的分泌产物双调蛋白（amphiregulin）可修复上述缺陷。
综上，本研究结果证实，肺驻留固有淋巴细胞在流感病毒感染后对气道上皮完整性的修复与组织稳态维持发挥关键作用。
作为本研究的一部分，我们开展了基因表达谱分析，以比较小鼠初始Ⅱ型肺固有淋巴细胞与脾脏Ⅲ型淋巴组织诱导（LTi）细胞的转录特征差异。
本研究通过BD FACSAria流式细胞分选仪，从未接触过抗原的野生型C57BL/6小鼠中分别分选纯化得到肺组织Ⅱ型固有淋巴细胞（Lin⁻ CD90⁺ CD25⁺ T1/ST2⁺）与脾脏Ⅲ型LTi细胞（Lin⁻ CD90⁺ CD25⁺ CD4⁺）。
共设置4个生物学重复，每个重复分别从6只小鼠的混合肺组织（用于分选固有淋巴细胞）或10只小鼠的混合脾脏（用于分选LTi细胞）中获取1.5×10⁴至2×10⁴个分选纯度＞97%的目标细胞。
分选得到的细胞被直接收集至TRIzol LS试剂（Invitrogen）中，随后进行mRNA提取、扩增、标记，并与Affymetrix基因芯片（Mouse Gene 1.0 ST）进行杂交。