Gene Expression
Nebulas
Gene Expression NebulasSummary: Human Natural Killer (NK) cells in peripheral blood perform many functions but classification of specific subsets has been a long-standing problem. Here, we report single-cell RNA sequencing of NK cells from healthy CMV-negative donors, comparing gene expression in unstimulated and IL-2 activated cells. Unsupervised clustering identified seven NK cell subsets. Three resembled well-described populations, CD56brightCD16-, CD56dimCD16+CD57- and CD56dimCD16+CD57+. CD56dimCD16+CD57- cells sub-divided to include a population with higher chemokine mRNA and increased frequency of KIR expression. Three novel human blood NK cell populations were identified as: a population of type I interferon responding NK cells which were CD56neg; a population exhibiting an in-vivo cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2, and finally, a small population, with low ribosomal expression, down-regulation of oxidative phosphorylation and high levels of immediate early response genes indicative of cellular activation. Human cytomegalovirus positive donors also included a higher frequency of adaptive NK cells. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analysing NK cell responses in health and disease.
Overall Design: scRNAseq of sorted NK cells from one healthy individual
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| Growth Protocol: | Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood collected into EDTA-coated tubes (fisher scientific) using density gradient centrifugation (Ficoll-Paque Plus; Amersham Pharmacia Biotech). Primary human NK cells were isolated using negative magnetic bead selection (Miltenyi Biotec or Stemcell Technologies). |
| Treatment Protocol: | - |
| Extract Protocol: | Individually-barcoded single cell RNA-seq libraries were prepared using the Chromium Single Cell Controller |
| Library Construction Protocol: | Chromium Single Cell 3'Library & Gel Bead Kit v2 kit (10x Genomics, Pleasanton, CA). All steps were carried out according to the manufacturers'instructions. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific |