Transcriptomic analysis of liver CD8+ T cells

Sorted single CD8+T cells expressing CD14 from human liver for SMARTSeq2.

Livers processed: Kucykowicz et al STAR Prot 2022:pubmed.ncbi.nlm.nih.gov/35516846/

Published: Pallett et al Nature 2022
Tissue CD14+CD8+T-cells are reprogrammed by myeloid cells and modulated by LPS

The liver is bathed in bacterial products including LPS transported from the intestinal portal vasculature, but maintains a state of tolerance exploited by persistent pathogens and tumours1,2,3,4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+T-cells compartmentalised in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins; CD14+CD8+T-cells preferentially accumulate within the donor pool in liver allografts, amongst hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+T-cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10/IL-2 production directly ex vivo, and an enhanced capacity to rapidly produce antiviral/anti-tumour cytokines upon TCR engagement. This CD14+CD8+T-cell profile can be recapitulated by acquisition of membrane proteins, including the LPS receptor complex, from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T-cells in vitro. CD14+CD8+T-cells express integrins and chemokine receptors favouring interactions with the local stroma, which can promote their induction through CXCL12. LPS can also increase the frequency of CD14+CD8+T-cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+T-cells with immunomodulatory capacity.

For the analysis of the single cell transcriptome two intrahepatic perfusate samples were isolated and frozen at -80 'C for storage prior to transportation to Newcastle, UK. Once thawed in pre-warmed HBSS containing 0.001% DNaseI, intrahepatic leukocytes were sorted by FACS to index classical mononuclear phagocytes (MNP) defined as Singlets, CD3-CD56-CD19-CD14hiHLA-DR+ or CD14± CD8+T-cells as follows: Singlets, CD3+CD56-CD19-CD4-CD8+Vï¡7.2- and split in to CD14± based on an isotype control. Single cells were sorted into 96-well skirted LoBind plates (Eppendorf) containing 5 µL lysis buffer (TCL buffer [Qiagen], supplemented with 1% b-mercaptoethanol [Sigma Aldrich; Merck]) on a BD Bioscience FACSFusion. The sorted plates were subsequently immediately sealed, centrifuged, and placed on dry ice to flash-freeze the cell lysate and stored at -80 'C.

A modified SMART-seq2 protocol57 was performed on the single flow cytometry sorted-cells as previously described58. After cDNA generation, libraries were prepared (384 cells per library) using the Illumina Nextera XT kit (Illumina). Each library was sequenced to achieve a minimum depth of 1-2 million raw reads per cell using an Illumina HiSeq 4000 using v. 4 SBS chemistry to generate 75-bp paired-end reads.EGA study EGAS00001006885

用于SMARTSeq2测序的、从人肝脏中分选得到的表达CD14的单个CD8+T细胞。

样本肝脏处理方法参考Kucykowicz等人2022年发表于《STAR Protocols》的研究：pubmed.ncbi.nlm.nih.gov/35516846/

本数据集已发表于Pallett等人2022年发表于《Nature》的研究：《组织中CD14+CD8+T细胞可被髓系细胞重编程并受脂多糖（LPS）调控》

肝脏沐浴于源自肠道门静脉血管的细菌产物（包括LPS）之中，却能维持免疫耐受状态，这一状态可被持续性病原体与肿瘤所利用1,2,3,4。介导这种免疫耐受、同时又允许向免疫激活或免疫病理状态转换的细胞基础，仍有待进一步阐明，以期为肝脏疾病的免疫治疗提供新的思路。本研究发现，人肝脏中存在一定比例的CD8+T细胞可同时表达CD14与其他典型髓系膜蛋白；CD14+CD8+T细胞优先富集于肝移植供体群体、肝脏病毒特异性及肿瘤浸润性免疫应答中，以及肝硬化腹水中。CD14+CD8+T细胞表现出更高的周转速率、激活状态与组成性免疫调节特征，在离体状态下即可高水平产生稳态IL-10与IL-2，且在T细胞受体（TCR）结合后可快速产生抗病毒/抗肿瘤细胞因子的能力显著增强。该CD14+CD8+T细胞表型可通过从单核吞噬细胞获取包括LPS受体复合物在内的膜蛋白得以重现，体外实验证实这一过程可增强TCR重定向T细胞的肿瘤杀伤能力。CD14+CD8+T细胞表达整合素与趋化因子受体，利于与局部基质发生相互作用，而CXCL12可通过该途径促进其诱导生成。LPS还可在体内外增加CD14+CD8+T细胞的频率，并使其功能偏向于产生趋化性与再生性细胞因子。综上，肠-肝轴中的细菌产物与组织基质因子可通过驱动具有免疫调节能力的CD8+T细胞获得髓系特性，从而调控肝脏免疫。

针对该单细胞转录组的分析，我们获取了两份肝内灌流液样本，于-80℃冷冻保存，随后转运至英国纽卡斯尔。将样本置于含0.001% DNaseI的预热HBSS中解冻后，通过荧光激活细胞分选（FACS）对肝内白细胞进行分选：经典单核吞噬细胞（MNP）的门控策略为：活细胞单态、CD3-CD56-CD19-CD14hiHLA-DR+；CD14± CD8+T细胞的门控策略为：活细胞单态、CD3+CD56-CD19-CD4-CD8+Vγ9Vδ2-，并基于同型对照区分CD14±亚群。将单个细胞分选至96孔裙边低吸附板（Eppendorf）中，每孔预先加入5 μL裂解缓冲液（含1% β-巯基乙醇的Qiagen TCL缓冲液，Sigma Aldrich; Merck供货），分选操作在BD Bioscience FACSFusion流式细胞仪上完成。分选完成后立即密封板盖、离心，并置于干冰中快速冷冻细胞裂解物，随后保存于-80℃。

我们采用经改良的SMART-seq2方案57，对经流式分选的单个细胞进行测序，具体步骤如先前文献58所述。完成cDNA合成后，使用Illumina Nextera XT试剂盒（Illumina）构建文库（每个文库包含384个细胞）。采用Illumina HiSeq 4000平台，以v.4 SBS化学试剂进行测序，每个细胞的原始测序深度不低于100-200万条reads，生成75 bp双端测序读段。本研究的欧洲基因组学档案（EGA）编号为EGAS00001006885。