Comparison of stable human Treg and Th clones by transcriptional profiling

Abstract

From cancerous and non-cancerous patients, we derived stable clones of CD4 1 Treg, defined as clones that expressed high CD25 at rest, were anergic in vitro, and suppressed the proliferation of co-cultured CD4 1 cells. A conserved region of FOXP3 intron 1 was demethylated in all Treg clones, whereas it was methylated in non-regulatory Th and CTL clones. In our panel of human clones, this stable epigenetic mark correlated better with suppressive activity than did FOXP3 mRNA or protein expression. We used expression microarrays to compare Treg and Th clones after activation, which is required for suppressive function. The transcriptional profile that is specific of activated Treg clones includes a TGF-b signature. Both activated Treg and Th clones produced the latent form of TGF-b. However, SMAD2 phosphorylation was observed after activation in the Treg but not in the Th clones, indicating that only activated Treg clones produced the bioactive form of TGF-b. A TGF-b signature was also displayed by a Th clone ''suppressed'' by a Treg clone. In conclusion, the hallmark of our panel of activated human Treg clones is to produce bioactive TGF-b which has autocrine actions on Tregs and can have paracrine actions on other T cells.

Effects of soluble extracts from Leishmania infantum promastigotes, Toxoplasma gondii tachyzoites on TGF-β mediated pathways in activated CD4+ T lymphocytes

“…4 shows that stimulation of activated CD25 þ cells with L. infantum or P. falciparum extracts, in the absence of any support of external TGF-b, induces phosphorylation of SMAD 2/3 proteins. In contrast, the same parasitic extracts do not activate SMAD 2/3 phosphorylation in activated CD25 À cells which express TGF-b receptor but do not express membranebound latent TGF-b [29,30].…”

“…TGF-b plays a key role in maintaining the balance between immunoprotection mediated by T cells and immunopathology either in experimental models of malaria and toxoplasma infection and in human diseases [5,29,30]. Macrophages and dendritic cells mostly account for TGF-b production following parasite uptake; nevertheless, the production of these cytokines by activated CD4 þ T cells following a direct stimulation with parasites or parasite products has also been reported [10,11,15].…”

Human Tumor Antigen-Specific Helper and Regulatory T Cells Share Common Epitope Specificity but Exhibit Distinct T Cell Repertoire

“…The converted DNA was then analyzed by real-time PCR for the methylated and the unmethylated forms of Foxp3 intron 1 as described previously (22). The results are expressed as percentages of unmethylated Foxp3 sequences, calculated as follows: % of demethylation = 2 ΔCt /(1 + 2 ΔCt ), wherein ΔCt = (Ct with methylated primers) 2 (Ct with unmethylated primers).…”

Targeting immunosuppression by Tregs with monoclonal antibodies against GARP

“…Analyzing their transcriptional profiles, we demonstrated that activated human Tregs, but not other T cells, produced active TGF-ß1. 2 A possible contribution of soluble TGF-ß1 to immunosuppression by Tregs was in line with the fatal autoimmune phenotype of Tgfb1 -/-mice, but not with the contact dependency of immunosuppression by Tregs. But then we observed that TGF-ß1-signaling in T cells co-cultured with Tregs was also contactdependent, suggesting that active TGF-ß1 was produced close to the Treg surface.…”

“…But then we observed that TGF-ß1-signaling in T cells co-cultured with Tregs was also contactdependent, suggesting that active TGF-ß1 was produced close to the Treg surface. 2 This prompted us to study the mechanisms of TGF-ß1 activation by Tregs. Indeed, most cells produce inactive forms of TGF-ß1 but very few activate the cytokine, via tightly regulated mechanisms that are cell-type specific.…”