The present study indicates that increased peripheral immune tolerance may be an independent risk factor for lung, colorectal, and ER-negative breast cancer, whereas its role on the development of prostate and ER-positive breast tumors remains uncertain.

BackgroundThe FOXP3 gene is the master regulator for T regulatory cells and is under tight DNA methylation control at the Treg specific demethylated region (TSDR) in its first intron. This said, methylation of its promoter region, the significance of which is unknown, has also been associated with various immune-related disease states such as asthma, food allergy, auto-immunity and cancer. Here, we used induced T regulatory cells (iTreg) as a target cell population to identify candidate hypomethylated CpG sites in the FOXP3 gene promoter to design a DNA methylation quantitative assay for this region.FindingsThree CpG sites at the promoter region showed clear demethylation pattern associated with high FOXP3 expression after activation in presence of TGFβ and were selected as primary targets to design methylation-dependent RT-PCR primers and probes. We then examined the methylation of this ‘inducible-promoter-demethylated-region’ (IPDR) in various FOXP3+ T cell subsets. Both naïve and memory thymic-derived Treg cells were found to be fully demethylated at both the IPDR and TSDR. Interestingly, in addition to iTregs, both CD25− and CD25lo conventional memory CD4+CD45RA− T cells displayed a high fraction of IPDR demethylated cells in absence of TSDR demethylation.ConclusionThis implies that the fraction of memory T cells should be taken in account when interpreting FOXP3 promoter methylation results from clinical studies. This approach, which is available for testing in clinical samples could have diagnostic and prognostic value in patients with immune or auto-inflammatory diseases.

Adoptive transfer of regulatory T cells (FOXP3 + Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after ex vivo expansion. Methods : We isolated bona fide Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR Vβ diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their in vitro and in vivo potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. Results : LAP + repurification results in >90% LAP + FOXP3 + Tregs, leaving behind FOXP3 - and FOXP3 + nonTregs within the LAP - population. After 4-week expansion, the LAP + Tregs were >1 billion cells, highly suppressive and anergic in vitro , >90% demethylated in the TSDR and able to maintain TCR Vβ diversity. In the xGVHD model, exogenous CD25 - PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP + Tregs increased median survival to 47 days, while the LAP parent (CD25 + ) and LAP - nonTregs had median survival of 39 and 31 days, respectively. Conclusions : These preclinical data together provide evidence that LAP + Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP + Tregs to prevent or treat GVHD and other autoimmune diseases.

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