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Abstract
T-helper cells are differentiated from CD4+ T cells and are traditionally characterized by inflammatory or immunosuppressive responses in contrast to cytotoxic CD8+ T cells. Mass-spectrometry studies on T-helper cells are rare. In this study, we aimed to identify the proteomes of human Th1 and Th1/Th17 clones derived from intestinal biopsies of Crohn's disease patients and to identify differentially expressed proteins between the two phenotypes. Crohn's disease is an inflammatory bowel disease, with predominantly Th1- and Th17-mediated response where cells of the "mixed" phenotype Th1/Th17 have also been commonly found. High-resolution mass spectrometry was used for protein identification and quantitation. In total, we identified 7401 proteins from Th1 and Th1/Th17 clones, where 334 proteins were differentially expressed. Major differences were observed in cytotoxic proteins that were overrepresented in the Th1 clones. The findings were validated by flow cytometry analyses using staining with anti-granzyme B and anti-perforin and by a degranulation assay, confirming higher cytotoxic features of Th1 compared with Th1/Th17 clones. By testing a larger panel of T-helper cell clones from seven different Crohn's disease patients, we concluded that only a subgroup of the Th1 cell clones had cytotoxic features, and these expressed the surface markers T-cell-specific surface glycoprotein CD28 and were negative for expression of natural killer group 2 member D.
Highlights
The CD4ϩ T cells called T-helper (Th)1 cells are important constituents of the immune system
The two Th1 clones analyzed by mass spectrometry had a much higher expression of cytotoxic proteins such as GZMs and PRF compared with the Th1/Th17 clones (Table I)
These findings altogether imply identification of CD28ϩ NKG2D- Th1 phenotype as a cytotoxic CD4ϩ T cell, a phenotype that is currently not described in literature as cytotoxic
Summary
The CD4ϩ T cells called T-helper (Th) cells are important constituents of the immune system. Quantitative Proteomics Reveal Cytotoxic CD4؉ T Cells response to IL-12 and IL-4 signaling has been studied [16, 17]. These studies all used in vitro differentiation of naive CD4ϩ T cells from human blood by cytokine supplementation of the growth medium (16 –20), while we here investigate in vivo differentiated Th cell clones isolated from gut-derived biopsies. Crohn’s disease is an intestinal disease where exaggerated Th1 and Th17 responses appear to be important [23], and the purpose of this study was to gain increased understanding of the complexity of these CD4ϩ T cell subsets. In addition to quantifying protein differences between Th1 and Th1/Th17, we wanted to examine the reproducibility of Th cell clones by common variations in cell culture expansion
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