Transcriptome profiling of CTLs regulated by rapamycin using RNA-Seq

Abstract

Memory programming of cytotoxic T cells (CTLs) by inflammatory cytokines can be regulated by mammalian target of rapamycin (mTOR). We have shown that inhibition of mTOR during CTL activation leads to the enhancement of memory, but the molecular mechanisms remain largely unknown. Using high-throughput RNA-Seq, we identified genes and functions in mouse CTLs affected by mTOR inhibition through rapamycin. Of the 43,221 identified transcripts, 184 transcripts were differentially expressed after rapamycin treatment, corresponding to 128 annotated genes. Of these genes, 114 were downregulated and only 14 were upregulated. Most importantly, 50 of them are directly related to cell death and survival. In addition, several genes such as CD62L are related to migration. Furthermore, we predicted downregulation of transcriptional regulators based on the total differentially expressed genes, as well as the subset of apoptosis-related genes. Quantitative PCR confirmed the differential expressions detected in RNA-Seq. We conclude that the regulatory function of rapamycin may work through inhibition of multiple genes related to apoptosis and migration, which enhance CTL survival into memory.

Figure 1

Enhancement of CTL memory by rapamycin. Purified OT1 cells were stimulated with antigen+B7+IL-12 in the presence of rapamycin at 250 ng/ml as (Li et al. 2011). Programmed CTLs were harvested at 72 hrs, and transferred into naïve B6 mice at 10⁶/mouse through tail injection (Li et al. 2011). (A) Memory OT1 cells in spleen at day 40 after transfer. (B) Representative staining of spleen samples from (A). ***, P<0.001.

Figure 2

Volcano plot of differentially expressed transcripts by rapamycin. The 183 significantly differentially expressed protein-coding transcripts (represented in red), as determined by Cuffdiff default settings with normalization. X-axis values are log2 (fold change) and y-axis values are the −log10 of q-value. q-values indicate FDR-adjusted p-value for multiple-testing.

Figure 3

Significantly differentially expressed genes identified by Cuffdiff with differential fold change.

Figure 4

Network analysis of apoptosis related genes regulated by rapamycin. Network was composed through IPA. Green: significantly downregulated. Red: significantly upregulated.

Figure 5

Validation of RNA-Seq data using quantitative RT-PCR. (A) qRT-PCR analysis of 10 selected downregulated genes and 5 upregulated genes. Fold change (y-axis) were generated by comparing rapamycin treated sample to control. The data represent mean of three individual samples plus SEM. The experiment was repeated twice with similar results. (B) The Log2 Fold of Average qRT-PCR (x-axis) were plotted against Log2 Fold Change values of RNA sequencing(y-axis), (Pearson correlation values >0.92)