概要: During host-pathogen encounters, the complex interactions between different immune cell-types can determine the outcome of infection. Advances in single cell RNA-seq (scRNA-seq) allow to probe this complexity of immunity, and afforded the basis for deconvolution algorithms that infer cell-type compositions from bulk RNA-seq measurements. However, immune activation, an important aspect of immune surveillance, is not represented in current algorithms. Here, using scRNA-seq of human peripheral blood cells infected with Salmonella, we developed a novel deconvolution algorithm to infer dynamic immune states from bulk measurements. We applied our dynamic deconvolution algorithm both to cohorts of healthy individuals challenged ex vivo with Salmonella and to cohorts of tuberculosis patients during different stages of disease. We revealed cell-type specific immune responses associated not only with ex vivo infection phenotype but also with clinical disease stage. We propose that our approach provides a predictive power to identify risk for disease, and can be applied to comprehensively study human infection outcome.
项目整体设计: Frozen PBMCs from healthy individual were defrosted and infectd ex vivo with Salmonella enterica serovar Typhimurium.
Venous blood was drawn from the cubital vein of volunteers and PBMCS were isolated as described in Li et al. Cell 2016. The cells were counted and frozen until used. A day before experiment, the cells where defrosted, suspended in medium (RPMI 1640 with L- Glutamine supplemented with 10% heat inactivated fetal bovine serum and 1mM sodium pyruvate) and plated on untreated plates. A day after, the cells were collected from the dish. To avoid cell lost, the dish was washed with medium and the remaining cells were added to the collected cells. The cells were then manually counted with trypan blue.
处理方案:
Salmonella strain used in this study was derived from the wild-type strain SL1344 containing GFP (pFPV25.1; Addgene). Cultures of Salmonella were grown in Luria-Bertani (LB) medium at 37℃ for 16 hours and used for PBMCs infection at MOI 5 for the exposed cells. After 30 min of internalization, the cells were washed and suspended with media containing 50 ug/ml gentamicin to eliminate Salmonella that were not internalized. The cells were incubated for 4 hours at 37℃ in 5% CO2 in non-treated cell culture plates.
提取方案:
4 hours after infection, the cells were washed with PBS, resuspended in 120ul of 5mM EDTA and incubated 5min in RT to detach cells that might have attached the dish. The EDTA was then washed and the cells were counted with trypan blue, suspended with 0.04% BSA in PBS and directly used for single-cell sequencing by the Chromium Single Cell 3'Reagent version 2 kit and Chromium Controller (10X Genomics, CA, USA) as previously described at Zheng, G. X. Y. et al Nature Communications 2017.
建库方案:
Libraries were prepared by the Chromium Single Cell 3' Reagent version 2 kit and Chromium Controller (10X Genomics, CA, USA) as previously described at Zheng, G. X. Y. et al Nature communications 2017.
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
Forward
测序平台:
ILLUMINA
测序仪型号:
Illumina NextSeq 500
链特异性:
Specific
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文章
Predicting bacterial infection outcomes using single cell RNA-sequencing analysis of human immune cells.
