Engagement of ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells. Engagement of ICOS in tissues promotes establishment of CD8+ tissue-resident memory T cells

Recirculating and tissue-resident memory CD8+ T cells provide distinct modes of immune protection, yet the signals that dictate differentiation of these populations are ill-defined. In particular, the interactions within tissues that promote generation of resident memory T cells (TRM) are unclear. Here, we show that the inducible costimulatory molecule ICOS, well known to regulate differentiation of CD4+ T cell populations, is required for CD8+ TRM but not recirculating memory subsets. Furthermore, ICOS engagement during CD8+ T cell recruitment to non-lymphoid tissues is critical for efficient TRM establishment: ICOS/ICOS-L interactions are dispensable throughout CD8+ T cell priming and for TRM maintenance, while ICOS-L expression by radioresistant cells is key for optimal TRM generation. This role for ICOS depends on its ability to signal through PI3K. Together, our data illustrate that specific local costimulatory cues promote production of tissue-resident populations, with potential implication for therapeutic manipulation. Overall design: CD8+ P14 T cells from WT or Icos knockout (KO) mice were mixed, co-adoptively transferred into recipient mice, and the mice were infected with LCMV the following day. CD8+ T cells were isolated from recipient mouse spleen, small intestine intraepithelial lymphocytes (SI-IEL), and kidney in two different cohorts (i.e. day 5 or day 32 post infection). Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) single cell RNA sequencing (scRNA-seq) analysis was completed. Cells were stained with antibody-oligo derived tags (ADTs) against CD45.2 and ICOS to identify cells derived from WT and Icos KO mice, and three hashtag oligos (HTOs) to identify cells derived from different tissues. For each timepoint, cells from different tissues were mixed at equal proportion and captured in the same library using the 10x Genomics Chromium Single Cell 3' (ver. 3.1) reagent kits, generating three independent gene expression (GEX) and feature barcode (ADT and HTO) sequencing libraries. The metadata.txt (representing 14 samples) contains each sample information and additional details.

循环性与组织驻留记忆CD8+ T细胞（CD8+ T cells）可介导不同模式的免疫保护，但调控这两类细胞亚群分化的信号机制仍未明确。其中，促进组织驻留记忆T细胞（tissue-resident memory T cells，TRM）生成的组织内相互作用尚不清晰。本研究证实，诱导性共刺激分子ICOS（inducible costimulatory molecule ICOS）——其调控CD4+ T细胞亚群分化的功能已得到充分阐明——是CD8+ TRM生成所必需的，但对循环性记忆亚群并非必需。进一步研究发现，在CD8+ T细胞募集至非淋巴组织的过程中，ICOS的共刺激信号对高效建立TRM至关重要：ICOS/ICOS-L的相互作用在CD8+ T细胞致敏阶段以及TRM维持过程中均非必需，而辐射抗性细胞表达的ICOS-L对优化TRM生成具有关键作用。ICOS的这一功能依赖于其通过PI3K传导信号的能力。综上，本研究数据表明，特定的局部共刺激信号可促进组织驻留细胞亚群的生成，这一发现为相关治疗干预提供了潜在思路。
实验整体设计：将野生型（WT）或Icos基因敲除（KO）小鼠来源的CD8+ P14 T细胞混合后，通过共同过继转移至受体小鼠体内，并于次日用淋巴细胞脉络丛脑膜炎病毒（Lymphocytic choriomeningitis virus, LCMV）感染受体小鼠。在两个不同的实验队列（即感染后第5天和第32天）中，分别从受体小鼠的脾脏、小肠上皮内淋巴细胞（SI-IEL）以及肾脏中分离CD8+ T细胞。本研究完成了转录组与表位测序联用（cellular indexing of transcriptomes and epitopes by sequencing, CITE-seq）的单细胞RNA测序（single cell RNA sequencing, scRNA-seq）分析：使用针对CD45.2和ICOS的抗体寡核苷酸标签（antibody-oligo derived tags, ADTs）染色细胞，以区分野生型与Icos敲除小鼠来源的细胞；同时使用3组hashtag寡核苷酸（hashtag oligos, HTOs）标记不同组织来源的细胞。对于每个时间点，将不同组织来源的细胞按等比例混合，使用10x Genomics Chromium单细胞3'端（版本3.1）试剂试剂盒在同一文库中完成捕获，最终构建3组独立的基因表达（gene expression, GEX）与特征条形码（feature barcode，包含ADT与HTO）测序文库。metadata.txt文件（包含14个样本）记录了所有样本的相关信息与补充细节。