Quantitative Proteomics Analysis of the Nuclear Fraction of Human CD4+ Cells in the Early Phases of IL-4-induced Th2 Differentiation

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.

A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation

“…Most of the already existing studies regarding T-cell biology are often conducted in Jurkat T-cell lines instead of primary T cells, focusing on proteomic events during activation close to the TCR, located in lipid rafts (12)(13)(14). Other studies focused on T-cell subproteomes within the early stages of T-cell differentiation and investigated proteomic changes in the nucleus of activated human cord blood CD4 ϩ T cells after interleukin-4 stimulation (15) or focused on changes of the global phosphoproteome of human primary T cells in response to 5 min of TCR activation with ␣CD3 (16). In vitro manipulated T cells were previously analyzed such as 7-day cultures of in vitro differentiated T helper 1 and T helper 2 cells (17), however, the surface proteome of human naive CD4 ϩ T cells and how these proteins change during the early time window of ␣CD3/␣CD28 activation has not been investigated so far.…”

Serum Proteomes Distinguish Children Developing Type 1 Diabetes in a Cohort With HLA-Conferred Susceptibility

“…For iTRAQ labeling, the samples were processed in accordance with the manufacturer's protocol for 8plex reagents (AB Sciex, Framingham, MA) and then fractionated using strong cation exchange chromatography as previously described (20). Samples from 26 children were compared using the iTRAQ method, applying 27 paired/ cross-referenced 8plex iTRAQ labeling schemes of the samples.…”

Quantitative Phosphoproteomic Analysis Reveals a Role for Serine and Threonine Kinases in the Cytoskeletal Reorganization in Early T Cell Receptor Activation in Human Primary T Cells

“…Because of the usage of primary cells, isobaric mass tags (iTRAQ) (16,17) were employed to measure differences in the phosphorylation profile after TCR stimulation.…”