Proteome analysis revealed the essential functions of protein phosphatase PP2A in the induction of Th9 cells

Abstract

Proteomic analysis identifies post-translational functions of proteins, which remains obscure in transcriptomics. Given the important functions of Th9 cells in anti-tumor immunity, we performed proteome analysis of Th9 cells to understand the involvement of proteins that might be crucial for the anti-tumor functions of Th9 cells. Here we performed a comprehensive proteomic analysis of murine Th0 and Th9 cells, and identified proteins that are enriched in Th9 cells. Pathway analysis identified an abundance of phosphoproteins in the proteome of Th9 cells as compared to Th0 cells. Among upregulated phosphoproteins, Ppp2ca (catalytic subunit of protein phosphatase, PP2A) was found to be highly enriched in Th9 cells. Although the role of PP2A has been shown to regulate the differentiation and functions of Th1, Th2, Th17 and Tregs, its role in the differentiation and functions of Th9 cells is not identified yet. Here we found that PP2A is required for the induction of Th9 cells, as PP2A inhibition leads to the suppression of IL-9 and expression of key transcription factors of Th9 cells. PP2A inhibition abrogates Th9 cell-mediated anti-tumor immune response in B16-OVA melanoma tumor model. Thus, we report that PP2A is essential for the differentiation and anti-tumor functions of Th9 cells. Cytokines such as TGF-β1 and IL-4 together skew naïve CD4 + T cells to IL-9-producing T helper (Th) cells, named as "Th9" cells 1,2. Th9 cells play a critical role in promoting anti-tumor immunity, allergic inflammation and autoimmune diseases 3,4. So far, different transcription factors have been reported to play a crucial role in the development of Th9 cells 5-11 , however, these factors have also been reported in other effector T cell subsets and are not lineage specific-transcription factors for Th9 cells. Understanding the generation of Th9 cells will allow modulating effector functions of these cells during the pathophysiology of asthma and anti-tumor immune responses. Thus, identification of biomarkers for Th9 cells is crucial for developing diagnostics and therapy for promoting the clinical outcomes for patients with Th9-mediated diseases. For deciphering the complete identity of a cell, quantitative proteomics is an important tool, as there is a discordance between transcriptome and proteome due to post-translational modifications and differential rates of protein synthesis. The mRNA level of a gene is poorly correlated with the protein levels in different conditions due to various above mentioned reasons 12. Therefore, despite of several transcriptome analysis, global proteome profiling has gained significant importance for mapping protein signatures at cellular level to unravel their precise functional characterization. There have been significant advancements in the development of proteome for different immune cells. Although the mass spectrometry studies on Th cells are rare, still few studies have reported the proteome analysis of different effector T cell subsets 13-17. Rautajoki et al. has demonstrated the differential...

Protein Phosphatase 2A: Role in T Cells and Diseases

“…Consistently, the siRNA-mediated knockdown of PP2A significantly inhibited IL-9 induction which functionally validated the positive regulation of Th9 cell differentiation by PP2A [ 73 ]. Further, the inhibition of PP2A significantly abrogated the antitumor immune response of Th9 cells by decreasing the frequency of IFN- γ -producing cytotoxic CD8 + T cells in the spleen, tumor-draining lymph nodes and tumor-infiltrating lymphocytes in B16-OVA melanoma model [ 73 ]. Thus, PP2A plays a critical role in the differentiation and antitumor functions of Th9 cells.…”

“…Transcriptomics studies have revealed an important role of several transcription factors in the development of Th9 cells and IL-9 induction [ 66 , 67 , 71 , 72 ]; however, the role of post-translational modifications in the expansion of Th9 cells remains unknown. Recently, it was shown that there was an abundance of phosphoproteins in the proteome of Th9 cells, among which PP2A was highly upregulated [ 73 ]. PP2A inhibition led to impaired differentiation of Th9 cells by downregulating the key transcription factors of Th9 cells such as Spi1 , Irf4 , Gata3 , Batf , Irf1 , Hif1α , and Foxo1 [ 73 ].…”

“…Recently, it was shown that there was an abundance of phosphoproteins in the proteome of Th9 cells, among which PP2A was highly upregulated [ 73 ]. PP2A inhibition led to impaired differentiation of Th9 cells by downregulating the key transcription factors of Th9 cells such as Spi1 , Irf4 , Gata3 , Batf , Irf1 , Hif1α , and Foxo1 [ 73 ]. Consistently, the siRNA-mediated knockdown of PP2A significantly inhibited IL-9 induction which functionally validated the positive regulation of Th9 cell differentiation by PP2A [ 73 ].…”

“…PP2A inhibition led to impaired differentiation of Th9 cells by downregulating the key transcription factors of Th9 cells such as Spi1 , Irf4 , Gata3 , Batf , Irf1 , Hif1α , and Foxo1 [ 73 ]. Consistently, the siRNA-mediated knockdown of PP2A significantly inhibited IL-9 induction which functionally validated the positive regulation of Th9 cell differentiation by PP2A [ 73 ]. Further, the inhibition of PP2A significantly abrogated the antitumor immune response of Th9 cells by decreasing the frequency of IFN- γ -producing cytotoxic CD8 + T cells in the spleen, tumor-draining lymph nodes and tumor-infiltrating lymphocytes in B16-OVA melanoma model [ 73 ].…”

PP2A and Its Inhibitors in Helper T-Cell Differentiation and Autoimmunity

“…Effect of PP2A on Th2 differentiation has been less studied, however the treatment with LB100 led to reduction in IL-4 expression and increase in IFNγ by Th2 cells ( 66 ). PP2A inhibition by okadaic acid or LB100 led to reduction in IL-9 production by Th9 cells ( 67 ).…”