Abstract
While genetic traits and epigenetic modifications mainly encode cell type-specific effector functions, the eventual outcome is also prone to modulation by post-transcriptional regulation mechanisms. T cells are a powerful model for the investigation of such modulatory effects, as common precursor cells may differentiate either to helper CD4+ T cells or cytotoxic CD8+ cells, which elicit distinct functionalities upon TCR-stimulation. Human primary CD4+ and CD8+ T cells were purified from three individual donors and activated with anti-CD3/CD28 antibodies. Associated proteome alterations were analyzed by high-resolution mass spectrometry using a label-free shotgun approach. Metabolic activation was indicated by upregulation of enzymes related to glycolysis, NADH production, fatty acid synthesis, and uptake as well as amino acid and iron uptake. Besides various inflammatory effector molecules, the mitochondrial proteins CLUH, TFAM, and TOMM34 were found specifically induced in CD4+ T cells. Investigation of overrepresented conserved transcription binding sites by the oPOSSUM software suggested interferon type I inducer IRF1 to cause many of the observed proteome alterations in CD4+ T cells. RT qPCR demonstrated the specific induction of IRF1 in CD4+ T cells only. While the interferon regulatory factor IRF4 was found induced in both T cell subtypes at protein and mRNA level, IRF9 and the type I interferon-induced proteins IFIT1, IFIT3, and MX1 were only found induced in CD4+ T cells. As oxidative stress enhances mitochondrial DNA-dependent type I interferon responses, the present data suggested that mitochondrial activities regulate those cell type-specific signaling pathways. Indeed, we detected mitochondrial superoxide formation predominantly in CD4+ T cells via FACS analysis with MitoSOX™ and confirmed this observation by live cell imaging with confocal microscopy. As interferon signaling regulates important features such as resistance regarding immune checkpoint blockade therapy, the present data may identify potential new targets for the efficient control of highly relevant immune cell properties.
Highlights
T cell receptor (TCR) stimulation has a profound effect on cells resulting in the induction of multiple defense mechanisms dependent on the cell type
Several mitochondrial proteins related to maintenance and import, i.e., clustered mitochondria protein homolog (CLUH), TFAM, and TOMM34 were found to be up-regulated in activated CD4+ T-cells, while this induction was nearly absent in CD8+ T cells
Parallel analyses of RNA levels of the respective three genes independently confirm the specificity of the proteome analysis results and suggest that expression of these factors is regulated at the transcriptional level following T cell activation
Summary
T cell receptor (TCR) stimulation has a profound effect on cells resulting in the induction of multiple defense mechanisms dependent on the cell type. Virtually all inflammatory responses somehow involve changes in protein expression, making proteome profiling a powerful tool to investigate inflammatory mechanisms as already established in our lab (Bileck et al, 2014; Slany et al, 2016; Bileck et al, 2017; Tahir et al, 2017). The immune system seems to be able to tune its metabolic needs and responses much more than previously anticipated (Buck et al, 2017). Optimal metabolic management is essential for immune cells in order to unfold their whole functionality on demand. Metabolic cues are involved in the shaping of the response, e.g. the polarization and function of different T-helper cell subsets (Almeida et al, 2016) and can be used to manipulate immune responses (Sen, 2000). Numerous studies indicate that mitochondrial adaptations to metabolic stress may affect or even cause tumorigenesis (Seyfried et al, 2014), as suggested by us in case of chronic lymphocytic leukemia (Mayer et al, 2018)
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