Organism-level analysis of vaccination reveals networks of protection across tissues

To study the response of single lung and liver memory CD8+ T cells to reactivation, mice vaccinated subcutaneously with MVA (10^7 PFUs/mouse) were subsequently challenged intranasally with: (1) PBS; (2) WR (10^7 PFUs/mouse); or (3) Vaccinia virus-derived B8R20-27 peptide (20 µg/mouse). Sixteen hours after challenge, single-cell suspensions were generated from lung or liver and lymphocytes were enriched using Percoll density centrifugation prior to immunostaining and cell sorting. Single memory CD8+ T cells from lung and liver were profiled using plate-based or droplet-based single-cell RNA-seq. Overall design: Three datasets were generated from single memory CD8+ T cells isolated from lung or liver using plate-based (2,423 cells) or droplet-based (7,292) single-cell RNA-seq methods, which included: (i) 947 cells from lung of MVA-vaccinated mice challenged with PBS, WR or B8R20-27 peptide (plate-based – experiment 1); (ii) 1,476 cells from lung or liver of MVA-vaccinated mice challenged with PBS or WR (plate-based – experiment 2); and (iii) 7,292 cells from lung of MVA-vaccinated mice challenged with PBS or WR (droplet-based – experiment 3). Libraries from each experiment were sequenced as a single pool on an Illumina NextSeq500 sequencer.

为探究单一肺脏与肝脏来源的记忆性CD8+ T细胞（memory CD8+ T cells）的再激活应答，研究人员对小鼠经皮下接种改良痘苗病毒安卡拉株（MVA，10^7 PFU/只），随后经鼻腔实施攻毒处理，攻毒分组如下：(1) 磷酸盐缓冲液（PBS）；(2) 痘苗病毒WR株（WR，10^7 PFUs/只）；或(3) 痘苗病毒来源的B8R20-27肽（20 µg/只）。
攻毒后16小时，研究人员从肺脏或肝脏制备单细胞悬液，并在免疫染色与细胞分选前，采用Percoll密度梯度离心法富集淋巴细胞。随后，采用基于平板（plate-based）或基于液滴（droplet-based）的单细胞RNA测序（single-cell RNA-seq）技术，对肺脏与肝脏来源的单一记忆性CD8+ T细胞进行测序分析。
整体实验设计：本数据集共包含3组来自肺脏或肝脏的记忆性CD8+ T细胞测序数据，分别通过基于平板（2423个细胞）或基于液滴（7292个细胞）的单细胞RNA测序方法获得，具体如下：(i) 947个细胞来自经PBS、WR或B8R20-27肽攻毒的MVA免疫小鼠肺脏（基于平板测序——实验1）；(ii) 1476个细胞来自经PBS或WR攻毒的MVA免疫小鼠的肺脏与肝脏（基于平板测序——实验2）；(iii) 7292个细胞来自经PBS或WR攻毒的MVA免疫小鼠肺脏（基于液滴测序——实验3）。各组实验构建的测序文库均作为单个混合池，在Illumina NextSeq500测序仪上完成测序。