Characterization of proteogenomic signatures of differentiation of CD4+ T cell subsets

Abstract

Functionally distinct CD4+ helper T (Th) cell subsets, including Th1, Th2, Th17, and regulatory T cells (Treg), play a pivotal role in the regulation of acquired immunity. Although the key proteins involved in the regulation of Th cell differentiation have already been identified how the proteogenomic landscape changes during the Th cell activation remains unclear. To address this issue, we characterized proteogenomic signatures of differentiation to each Th cell subsets by RNA sequencing and liquid chromatography-assisted mass spectrometry, which enabled us to simultaneously quantify more than 10,000 protein-coding transcripts and 8,000 proteins in a single-shot. The results indicated that T cell receptor activation affected almost half of the transcript and protein levels in a low correlative and gene-specific manner, and specific cytokine treatments modified the transcript and protein profiles in a manner specific to each Th cell subsets: Th17 and Tregs particularly exhibited unique proteogenomic signatures compared to other Th cell subsets. Interestingly, the in-depth proteome data revealed that mRNA profiles alone were not enough to delineate functional changes during Th cell activation, suggesting that the proteogenomic dataset obtained in this study serves as a unique and indispensable data resource for understanding the comprehensive molecular mechanisms underlying effector Th cell differentiation.

Keywords: CD4 T cell; RNA sequencing; immunology; liquid chromatography-assisted mass spectrometry; proteogenomics.

Figure 1.

A combination of global RNA-seq and proteome analyses revealed the effects of TCR stimulation on mRNA and protein expression profiles. (a) Overview of the experimental design. (b) List of cytokine conditions. (c and d) A scatter plot of gene or protein expression profiles by RNA-sequencing (c) or proteome analysis (d) compares in naive and Th0 cells. (e) Venn diagram showed overlaps and differences between 2.0-fold increased (Left panel) or decreased (Right panel) genes in naive and Th0 cells. (f) Bar plots showed fold changes of mRNA and protein expression immunity-related genes in Th0 cells relative to naive T cells.

Figure 2.

Protein expression analysis showed T cell activation status and Th cell subset signature. (a and b) A clustering heatmap depict the gene (a) or protein expression (b) in naive, Th0, Th1, Th2, Th17, and iTregs cells. (c) Pie chart showed the number of differentially expressed proteins in Th0, Th1, Th2, Th17, or iTregs cells relative to naive T cells (n = 4 per genotype). (d and e) Venn diagram showed overlaps and differences between 2.0-fold increased (d) or decreased (e) genes in Th0, Th1, Th2, Th17, or iTregs cells relative to naive T cells. (f) A scatter plot of protein expression profiles by proteome analysis compares in Th1, Th2, Th17, and iTregs cells relative to naive T cells.

Figure 3.

Cytokine responses caused the differences in RNA and protein expression in Th cell subsets compared to Th0 cells. (a and b) PCA plot of gene or protein expression profiles by RNA-sequencing (a) or proteome analysis (b), including Th0, Th1, Th2, Th17, and iTregs cells. (c and d) heat map depicts gene (c) or protein (d) expression of the T cell subset specific signature (n = 4 per genotype). (e and f) Venn diagram showed overlaps and differences between 2.0-fold increased (e) or decreased (f) proteins in Th1, Th2, Th17, or iTregs cells relative to Th0 cells. Genes or protein varied in each subset alone are listed at the bottom. (g–j) Venn diagram showed overlaps and differences between 2.0-fold increased genes in Th1 (g), Th2 (h), Th17 (i), and iTregs cells (j) as compared to Th0 cells.

Figure 4.

Cytokine stimulation caused uncorrelated expression of RNA and Protein in Th cell subsets. (a–d) A scatter plot of gene and protein expression profiles compares in Th1 (a), Th2 (b), Th17 (c), or iTregs cells (d) to Th0 cells. (e, g, i, and k) qRT-PCR analyses of the relative expression of Irf7 (e), Tnfsf8 (g), Pdcd1lg2 (i), or Ccr6 (k) in Th1, Th2, Th17, or iTregs cell compared to Th0 cells. Relative expression (normalized to Hprt) with s.d. is shown. (f, h, j, and l) Intracellular staining or surface staining of flow cytometry analysing of IRF7 (f), CD30L (h), PD-L2 (j), or CCR6 (l) in Th0, Th1, Th2, Th17, or iTregs cell. Mean fluorescence intensity (MFI) is shown. Summary data of three independent experiments of each protein expression are shown here. Data are means ± s.d. (n = 3 per each group biologically independent sample).