Circulating NK cells establish tissue-residency upon acute infection of skin and mediate accelerated effector responses to secondary infection. Circulating NK cells establish tissue-residency upon acute infection of skin and mediate accelerated effector responses to secondary infection

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues; these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigated whether circulating conventional NK (cNK) cells can develop into long-lived, tissue-resident cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi tissue-resident (trNK) cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK arose from IFN-g-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissueresidency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny Overall design: scRNA-seq, cell hashing and CITE-seq. ILC1s and NK cells were sorted as individual, hash-tagged populations before pooling into one reaction. Before sorting, cells were labeled with oligonucleotide-tagged antibodies for cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq). List of Hashtags and CITE seq antibody used: Ms.Hashtag_1 ACCCACCAGTAAGAC, Ms.Hashtag_2 GGTCGAGAGCATTCA, Ms.Hashtag_3 CTTGCCGCATGTCAT, Ms.Hashtag_4 AAAGCATTCTTCACG, Ms.Hashtag_5 CTTTGTCTTTGTGAG. HuMs.CD11b TGAAGGCTCATTTGT, Ms.CD45 TGGCTATGGAGCAGA, Ms.CD45.2 CACCGTCATTCAACC, HuMsRt.CD27 CAAGGTATGTCACTG, Ms.CD69 TTGTATTCCGCCATT, Ms.CD127 GTGTGAGGCACTCTT, Ms.CD103 TTCATTAGCCCGCTG, HuMs.KLRG1 GTAGTAGGCTAGACC, Ms.CD49b CGCGTTAGTAGAGTC, Ms.Ly49H CCAGTAGGCTTATTA. We multiplexed Skin d8 with hashtag_1, Skin d25 with hashtag_2, Salivary Gland d25 with hashtag_3, Spleen d8 with hashtag_4, and Spleen d25 with hashtag_5.

自然杀伤（Natural killer, NK）细胞既存在于血液循环系统中，也可定居于各类组织内；这两类细胞群体展现出截然不同的发育需求，且被认为在个体发生层面存在差异。本研究旨在探讨循环型常规NK（circulating conventional NK, cNK）细胞在急性感染后是否可分化为长寿的组织驻留NK细胞。研究发现，皮肤的病毒与细菌感染可招募cNK细胞，并使其分化为Tcf1hiCD69hi组织驻留NK（tissue-resident NK, trNK）细胞，这类细胞与人体组织中的CD56brightTCF1hi NK细胞具有高度相似的转录谱特征。皮肤trNK细胞源自分泌干扰素-γ的效应细胞，且需要转录调控因子Blimp1的限制性表达，以优化依赖Tcf1的trNK细胞形成过程。当发生二次感染时，trNK细胞可快速获得效应功能，介导加速的NK细胞应答。综上，cNK细胞可通过一种促进组织驻留的机制重新分布并永久定位于既往感染部位，该机制与个体发生阶段NK细胞及固有淋巴细胞1型（ILC1）定植组织所依赖的Hobit调控通路截然不同。

整体实验设计：采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）、细胞条形码（cell hashing）及转录组与表位联合测序（cellular indexing of transcriptomes and epitopes by sequencing, CITE-seq）技术。实验前，将ILC1与NK细胞按单个细胞群体分选并标记哈希条形码，随后将标记后的细胞混合至同一反应体系中。分选前，细胞已用寡核苷酸标记的抗体进行染色，以用于CITE-seq的转录组与表位联合索引测序。所用哈希条形码及CITE-seq抗体列表如下：
Ms.Hashtag_1 ACCCACCAGTAAGAC，Ms.Hashtag_2 GGTCGAGAGCATTCA，Ms.Hashtag_3 CTTGCCGCATGTCAT，Ms.Hashtag_4 AAAGCATTCTTCACG，Ms.Hashtag_5 CTTTGTCTTTGTGAG。
HuMs.CD11b TGAAGGCTCATTTGT，Ms.CD45 TGGCTATGGAGCAGA，Ms.CD45.2 CACCGTCATTCAACC，HuMsRt.CD27 CAAGGTATGTCACTG，Ms.CD69 TTGTATTCCGCCATT，Ms.CD127 GTGTGAGGCACTCTT，Ms.CD103 TTCATTAGCCCGCTG，HuMs.KLRG1 GTAGTAGGCTAGACC，Ms.CD49b CGCGTTAGTAGAGTC，Ms.Ly49H CCAGTAGGCTTATTA。
本研究将感染后第8天的皮肤样本与哈希条形码1复合标记，感染后第25天的皮肤样本与哈希条形码2复合，感染后第25天的唾液腺样本与哈希条形码3复合，感染后第8天的脾脏样本与哈希条形码4复合，感染后第25天的脾脏样本与哈希条形码5复合。