Abstract
We used stable isotope labeling with 4-plex iTRAQ (isobaric tags for relative and absolute quantification) reagents and LC-MS/MS to investigate proteomic changes in the nucleus of activated human CD4+ cells during the early stages of Th2 cell differentiation. The effects of IL-4 stimulation upon activated naïve CD4+ cells were measured in the nuclear fractions from 6 and 24 h in three biological replicates, each using pooled cord blood samples derived from seven or more individuals. In these analyses, in the order of 800 proteins were detected with two or more peptides and quantified in three biological replicates. In addition to consistent differences observed with the nuclear localization/expression of established human Th2 and Th1 markers, there were changes that suggested the involvement of several proteins either only recently reported or otherwise not known in this context. These included SATB1 and among the novel changes detected and validated an IL-4-induced increase in the level of YB1. This unique data set from human cord blood CD4+ T cells details an extensive list of protein determinations that compares with and complements previous data determined from the Jurkat cell nucleus.
Highlights
As a response to antigen encounter and their cytokine environment, naïve CD4ϩ cells differentiate into functionally distinct T helper (Th)1 cell subsets, the best characterized of
The importance of IL-4 and signal transducer and activator of transcription 6 (STAT6) for the induction of the Th2 response is well documented, and the transcription factor GATA3 plays an important role in several stages of Th2 cell development where it is required for the regulation of several Th2specific cytokines [25,26,27]
Protein Identification and Relative Quantification—In this study, we investigated changes in the nuclear proteome of human naïve T helper cells in the early phases of Th2 cell differentiation using iTRAQ technology
Summary
As a response to antigen encounter and their cytokine environment, naïve CD4ϩ cells differentiate into functionally distinct T helper (Th) cell subsets, the best characterized of. These interleukin; iTRAQ, isobaric tags for relative and absolute quantification; MMTS, methyl methanethiosulfonate; SATB1, special AT-rich sequence-binding protein 1; SCX, strong cation exchange; STAT, signal transducer and activator of transcription; TCF7, transcription factor 7; YB1, nuclease-sensitive element-binding protein 1 (Y boxbinding protein 1); GO, gene ontology; WB, Western blotting; Erk, extracellular signal-regulated kinase; Ct, threshold cycle; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3diol; MAPK, mitogen-activated protein; siRNA, short interfering RNA; c-MAF, Transcription factor Maf; GATA3, trans-acting T-cell-specific transcription factor GATA-3; TBX21, T-box transcription factor TBX21; ThP, T helper cell precursor. We used 4-plex iTRAQ reagents [33] to compare the abundances of proteins in the nuclear fractions of activated CD4ϩ cells and those activated and IL-4-stimulated (at 6 and 24 h) With these measurements, we aimed to identify protein abundance changes associated with Th2 cell differentiation with potential mechanistic relevance to the early phases of this process. Further evaluations were made in terms of known protein interactions and functions and GO annotation in general
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