CD4⁺CD25⁺FOXP3⁺ T regulatory cells (Tregs) are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. Rapamycin has been shown to promote tolerance in experimental models and to favor CD4⁺CD25⁺ Treg-dependent suppression. We recently reported that rapamycin allows in vitro expansion of murine CD4⁺CD25⁺FoxP3⁺ Tregs, which preserve their suppressive function. In the current study, we show that activation of human CD4⁺ T cells from healthy subjects in the presence of rapamycin leads to growth of CD4⁺CD25⁺FOXP3⁺ Tregs and to selective depletion of CD4⁺CD25⁻ T effector cells, which are highly sensitive to the antiproliferative effect of the compound. The rapamycin-expanded Tregs suppress proliferation of both syngeneic and allogeneic CD4⁺ and CD8⁺ T cells. Interestingly, rapamycin promotes expansion of functional CD4⁺CD25⁺FOXP3⁺ Tregs also in type 1 diabetic patients, in whom a defect in freshly isolated CD4⁺CD25⁺ Tregs has been reported. The capacity of rapamycin to allow growth of functional CD4⁺CD25⁺FOXP3⁺ Tregs, but also to deplete T effector cells, can be exploited for the design of novel and safe in vitro protocols for cellular immunotherapy in T cell-mediated diseases.

Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c⁺ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c⁺ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c⁺ DCs was closely correlated with high CD14 expression levels observed in CD1c⁺ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

Th2 cells play a key role in directing immune responses against helminths. Additionally, Th2 cells are crucial for many types of allergic reactions. Whereas the molecular mechanisms underlying the differentiation of other types of Th cells are well understood, Th2 differentiation is still a controversial topic. IL‐4 and its downstream transcription factor signal transducer and activator of transcription (STAT)6 are well‐known key mediators in Th2 differentiation. The fact that Th2 cells themselves are the most potent source of IL‐4 suggests that additional mechanisms promoting the initiation of Th2 differentiation exist. This article gives an overview on STAT6‐dependent and ‐independent mechanisms involved in the process of Th2 polarization, including Notch, mTORC2, IL‐2/STAT5, and Wnt. Furthermore, we emphasize the role of STAT6 not only as a transcriptional activator promoting Th2 development, but also in fine‐tuning alternative signaling pathways which are involved in the initiation of Th2 polarization.

Eosinophils are attracted to sites of allergic inflammation by a number of chemoattractants including eotaxin-1. This chemokine can be secreted from epithelial cells and fibroblasts after IL-4 and TNF-α stimulation in a synergistic fashion. TNF-α activated gene expression at the transcriptional level in a STAT6-dependent manner, because: 1) eotaxin-1 promoter luciferase constructs were TNF-α inducible in STAT6-defective HEK293 cells only on cotransfection of STAT6 expression vector, an effect that was partially mediated by activation-induced binding of NF-κB proteins to a composite STAT6/NF-κB element; 2) reporter constructs defective in STAT6 DNA binding did not respond to TNF-α stimulation; 3) eotaxin-1 protein secretion was detected only in STAT6-transfected HEK293 cell supernatants on TNF-α treatment; and 4) a trans-dominant negative STAT6 protein inhibited TNF-α-induced eotaxin-1 secretion in primary fibroblasts. TNF-α inducibility of the IL-8 and monocyte chemoattractant protein-1 genes was not dependent on STAT6 expression in the same experimental systems. The inducing effect of IL-4 and IL-13 was also mediated by STAT6. The synergistic effect of IL-4 and TNF-α observed at the RNA and the protein level was not seen at the promoter level. The data demonstrate that both IL-4 and TNF-α induce eotaxin-1 expression at the level of transcription via a STAT6-mediated pathway.