A novel method for characterizing cell-cell interactions at single-cell resolution reveals unique immune signatures in blood T cell-monocyte complexes during infection (ATB scTCR-Seq)

We designed a novel method to study the transcriptome of single cells forming complexes in a high-throughput fashion, without the need for bioinformatic deconvolution. We applied this method to the study of T cells and monocytes forming complexes in blood isolated from individuals with active tuberculosis, with blood collected at diagnosis and after treatment. We found that the transcriptomic signature of T cells and monocytes forming complexes was associated with TCR/MHC-II immune synaptic features, enrichment for cytotoxic clonally expanded T cells and intermediate monocytes, and - especially at diagnosis - transcriptomic signatures indicating increased immune and metabolic activity. We also found a striking enrichment of “dual-expressing” cells (i.e., co-expressing T cells and monocyte marker genes) in complexes, suggesting RNA exchange occur during T cell-monocyte interactions. Thus, using our novel method, we identified that T cells and monocyte forming complexes in blood hold unique immune signatures distinct from singlets, and that they may be a previously overlooked valuable biomarker to monitor immune synaptic interactions during infection. PBMC were isolated, cryopreserved and thawed on the day of the experiment. T cell-monocyte complexes were identified by flow cytometry as live CD3+CD14+CD19- events and sorted in bulk. Cell sorting effectively disrupts the physical connection between cells forming complexes, with the vast majority of resulting cells being singlet CD3+ or singlet CD14+ cells remaining in suspension. The single-cell suspension was then used for droplet single-cell sequencing using the 10X genomics platform. ----------------------------------- Authors state the following concerning the raw data "privacy concerns".

本研究开发了一种全新方法，可在无需生物信息学反卷积（bioinformatic deconvolution）的前提下，以高通量方式对形成细胞复合物的单个细胞的转录组开展研究。我们将该方法应用于活动性结核病患者血液中形成复合物的T细胞与单核细胞的相关研究，血液样本分别采集于患者确诊时与治疗后。研究发现，形成复合物的T细胞与单核细胞的转录组特征与T细胞受体（TCR）/主要组织相容性复合体II类（MHC-II）免疫突触特征、细胞毒性克隆扩增T细胞及中间型单核细胞的富集显著相关；尤其在确诊阶段，还呈现出提示免疫与代谢活动增强的转录组特征。此外，我们还在复合物中观察到“双表达”细胞（即同时表达T细胞与单核细胞标志物基因的细胞）的显著富集，提示T细胞与单核细胞的相互作用过程中存在RNA交换现象。综上，借助本研究开发的全新方法，我们发现血液中形成复合物的T细胞与单核细胞拥有区别于单个细胞的独特免疫特征，且这类复合物或可作为此前被忽视的重要生物标志物，用于监测感染过程中的免疫突触相互作用。本研究在实验当日对外周血单个核细胞（PBMC）进行分离、冻存与复苏。通过流式细胞术将活细胞中CD3+CD14+CD19-的事件定义为T细胞-单核细胞复合物，并进行批量分选。细胞分选可有效破坏形成复合物的细胞间的物理连接，分选后绝大多数悬浮细胞为单个的CD3+ T细胞或CD14+单核细胞。随后将该单细胞悬浮液用于基于10X Genomics平台的液滴法单细胞测序。----------------------------------- 关于原始数据，作者就“隐私顾虑”作出如下说明：