Plasma membrane proteomes of differentially matured dendritic cells identified by LC–MS/MS combined with iTRAQ labelling

Abstract

Dendritic cells (DCs) play a pivotal role in polarising Th lymphocyte subsets but it is unclear what molecular events occur when DCs generate Th2-type responses. Here, we analysed plasma membrane-enriched fractions from immature, pro-Th1 and pro-Th2 DCs and used a combination of iTRAQ labelling and LC–MS/MS to quantify changes in the proteomes. Analysis was performed on triplicate biological samples and changes verified by flow cytometry. MHC class II molecules and CD29 were up-regulated in pro-Th1 DCs whilst CD18 and CD44 were up-regulated in pro-Th2 DCs. One of the most down-regulated molecules in pro-Th1 DCs was YM-1 whilst the greatest decrease in pro-Th2 DCs was NAP-22. Other molecules up-regulated in pro-Th2 DC compared to pro-Th1 DCs included some potentially involved in protein folding during antigen processing (clathrin and Rab-7), whilst other non-membrane proteins such as enzymes/transporters related to cell metabolism (malate dehydrogenase, pyruvate kinase, and ATPase Na+/K+) were also recorded. This suggests that pro-Th2 DCs are more metabolically active while pro-Th1 DCs have a mature ‘end state’. Overall, although several molecules were preferentially expressed on pro-Th2 DCs, our proteomics data support the view of a ‘limited maturation’ of pro-Th2 DCs compared to pro-Th1 DCs.

Immunomodulatory Glycan Lacto-N-Fucopentaose III Requires Clathrin-Mediated Endocytosis To Induce Alternative Activation of Antigen-Presenting Cells

“…We found that the ability of LNFPIII-NGC to alternatively activate APCs that drive CD4 ϩ Th2 responses is dependent on APC endocytosis of LNFPIII-NGC, as APCs treated with dynasore failed to drive CD4 ϩ Th2 responses. These results are similar to an earlier study suggesting that DC2s generated by activation with SEA had a distinct phenotype that included upregulation of clathrin and the endosomal marker Rab5 (54). The study by Ferret-Bernard et al strengthens our observation that dynamin/clathrin-mediated endocytosis of LNFPIII-NGC induces Th2-type immune responses (54).…”

Transgelin-2 in immunity: Its implication in cell therapy

“…Although transgelin‐2 has been mainly studied within T cells, B cells, and macrophages, there have been other immune cell types that have been reported to express transgelin‐2, such as dendritic cells (DCs). A proteomic analysis of differentially matured DCs demonstrated that transgelin‐2 expression was highly upregulated in some DC subpopulations . Moreover, it may be also worth mentioning that the DC cytoskeleton promotes T‐cell adhesion and activation via ICAM‐1 avidity alteration .…”

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

“…The quantification is performed on MS/MS level by comparing the intensities of reporter ions (114-117 for 4-component kit and 113-119 and 121 for 8-component kit). iTRAQ technology coupled with MDLC-MS has been broadly utilized in quantitative proteomics as a means of screening the potential biomarkers [118][119][120][121][122][123][124]. However, the labeling processes were all performed offline.…”