Transcriptome characterization at the single cell level of viral specific CD8+ T cells

<p>Transcriptome-level characterization of the immune response during viral infection can reveal key mechanisms that underpin the activation and status of cytotoxic CD8+ T Cells (CTL). Once stimulated with antigen CTLs proliferate and differentiate generating a heterogeneous progeny. The advent of single cell analyses, has enabled tracking of the evolving CTLs in human samples during viral infections. We quantified the single cell transcriptome of 81 CTLs identified from a subject with chronic HCV infection. All cells are specific for HLA-I A0201 restricted epitope CINGVCWTV. Single CTLs were obtained from PBMC and from cell line derived from the same patient (unstimulated and following antigen re-stimulation). We analysed the difference between the three groups of single cells using a list of genes previously associated with CTL functions. Genes associated with cytotoxic response (IFN-g, perforin, granzyme B) were highly expressed in cell lines, but not in PBMC derived CTLs. Using unsupervised clustering analysis we identified co-expression clusters within each group. By filtering out the cell-cycle related genes (a major co-founder), we revealed that cells from the restimulated cell line showed a significantly higher level of heterogeneity (p-value <0.0001). The chemokine cell receptors (CCL4, CXCR4) were among the most variable genes.</p

Average values of key parameters for each eye: AU-ROC = area under the receiver operator characteristic curve, Ideal Thresh = ideal threshold, Illum sd = image illumination variation and Tot Movt = maximum frame movement during video of three cardiac cycles.

<p>Average values of key parameters for each eye: AU-ROC = area under the receiver operator characteristic curve, Ideal Thresh = ideal threshold, Illum sd = image illumination variation and Tot Movt = maximum frame movement during video of three cardiac cycles.</p

Schematic diagram of the technical sequence for objectively analysing the video frames, calculating pulsation amplitudes and generating heat maps of amplitude.

<p>Schematic diagram of the technical sequence for objectively analysing the video frames, calculating pulsation amplitudes and generating heat maps of amplitude.</p

Receiver operator curve for Observer 2 with all data from all eyes analysed together.

<p>Receiver operator curve for Observer 2 with all data from all eyes analysed together.</p

A diagram showing 2 cluster waveforms from different optic disk regions over three cardiac cycles (A) with video frames taken during diastole (B) and systole (C).

<p>A heat map with colour scale (D) is shown with observer 1 manual outline of pulsating region overlaid upon the heat map (E). Objective detection (F) with threshold amplitude set at 5 units is shown in yellow where this was in agreement with observer, in green without agreement from observer and in red where the observer noted pulsation but amplitude values were less than 5 units.</p

Additional file 1: Figure S1. of A method for near full-length amplification and sequencing for six hepatitis C virus genotypes

Successful amplification of near full-length HCV genomes from multiple genotypes. Amplicons (~9.2 kb) were run on an agarose gel (0.8 %) and visualized on a Gel Doc molecular imager (Bio-Rad); M represents the DNA marker HyperLadder 1 (Bioline). Lanes 1 to 8 represent GT1a amplicons. Lanes 9 to 11 represent GT1b amplicons. Lanes 12 to 16 represent GT3a amplicons. All amplicons were purified and successfully sequenced on the Illumina platform, including the faintly visible band in lane 7. (PDF 2448 kb