The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus.

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4(+)CD25(+) naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.

Relevance of nuclear receptor expression in a Tchreg cell line, HOZOT: RXRα and PPARγ negatively regulate IFN-γ production.

Nuclear receptors (NRs) have recently received much attention for their newly discovered roles in T cell development, as exemplified by RARα (Treg cells) and RORγt (Th17 cells). In previous studies, we characterized a new type of T cell subset, designated as Tchreg (cytotoxic, helper, and regulatory T) cells, in terms of its cytokine signature. In this study, we investigated the expression and functional relevance of NRs in Tchreg cells by performing mRNA profiling of HOZOT, a cord blood-derived Tchreg cell line. We identified eleven inducible and eight constitutively expressed NRs in HOZOT. Among these NRs, RXRα and PPARγ showed features of signature NRs of Tchreg cells because they were selectively expressed in HOZOT compared with other T cell subsets. These NRs exhibited contrasting expression patterns, as RXRα was independent of anti-CD3/28 antibody stimulation while PPARγ was stimulated-dependent. Upon agonist treatment, both proteins translocated to the nucleus and inhibited IFN-γ production through binding to the promoter region of the IFN-γ gene. These results provide new insight into the roles of RXRα and PPARγ in T cell biology, especially in their biological relevance in Tchreg cells.

Marked induction of CD4+CD8+ T cells with multifunctional properties by coculturing CD2+ cells with xenogeneic stromal cells.

A number of T cell subsets have been identified, and the in vitro characterization of these subsets largely depends on an appropriate induction system for each one. In previous studies, we characterized a unique T cell line, HOZOT, which possessed a CD4+CD8+ double positive (DP) phenotype and multifunctional properties including cytotoxic, helper, and regulatory functions. Therefore, this T cell subset has been termed Tchreg cells. In this study, we established a new method of Tchreg cell induction, which consists of three steps and provides more efficient and reproducible results. By using a purified T cell population, the efficiency of Tchreg generation was profoundly increased, and the use of purified CD2+ cells rather than CD3+ cells was shown to be superior. One surprising finding was that at the initial step, stromal cell stimulation induced DP T cells more efficiently than dendritic cells or anti-T cell receptor stimulation, indicating a distinct antigen presenting cell (APC) ability of stromal cells as distinguished from conventional APCs. Even in subsequent steps, the presence of stromal cells was required to maintain the DP phenotype. In the second step, addition of stromal cell-conditioned (but not unconditioned) autologous CD14+ cells instead of interleukin-2 was beneficial for subsequent expansion of Tchreg cells. The improved three-step culture method described here represents a step forward in efforts to achieve clinical application and a good tool for elucidating how Tchreg cells, especially CD4+CD8+ T cells, are generated.

Interleukin-8 and RANTES are signature cytokines made by HOZOT, a new type of regulatory T cells.

Distinct cytokine production profiles define the effector functions of both helper and regulatory T cells. Recently, we established novel cytotoxic regulatory T (Treg) cell lines, HOZOT, which have been characterized as IL-10-producing T cells. In this study, we further characterized HOZOT by performing comprehensive analyses of cytokines produced by HOZOTs in order to identify a signature cytokine profile. Using DNA microarrays, we compared the gene expression profiles of HOZOT-4, a representative HOZOT cell line, under three different conditions. Seven genes, including IL-8, IL-10, IL-13, MIP-1alpha, and MIP-1beta, were identified as inducible cytokines when stimulated with stromal cells or anti-CD3/CD28 antibodies. Twelve genes, including IL-2, IL-3, IL-4, IL-22, CCL1, and lymphotactin, were categorized as antibody stimulation-responsive but stromal cell-non-responsive. Three genes, IL-32, RANTES, and CCL23, were constitutively expressed irrespective of stimulation condition. Among these cytokines, we focused on two chemokines, IL-8 and RANTES for further studies, and found that only HOZOT produced both of them at considerable levels whereas other T cell subsets, including Tregs and helper T cells, did not. Kinetic and inhibition experiments revealed contrasting properties for the two chemokines. IL-8 was induced only after stimulation, whereas RANTES mRNA and protein accumulated to high levels even before stimulation. Interestingly, IL-8 mRNA was induced by cycloheximide treatment and RANTES showed reduced mRNA but increased protein expression by antibody stimulation. As a whole, the unique cytokine signature profile consisting of Th1, Th2, and cytolytic T cell cytokines as well as Treg cytokines reflect the multifunctional nature of HOZOT. In particular, the dual production of IL-8 and RANTES by distinct mechanisms is a hallmark of HOZOT.

T cell hyporesponsiveness induced by oral administration of ovalbumin is associated with impaired NFAT nuclear translocation and p27kip1 degradation.

Oral tolerance is an important physiological component of the immune system whereby the organism avoids dangerous reactions such as hypersensitivity to ingested food proteins and other luminal Ags which may cause tissue damage and inflammation. In addition, it has been shown in animal models and in humans that oral tolerance can be applied to controlling undesired immune responses, including autoimmune diseases, allergies, and organ transplant rejections. However, the molecular mechanisms of oral tolerance have been poorly defined. In this study, we investigated the molecular basis underlying the hyporesponsiveness of orally tolerant CD4 T cells using a TCR transgenic mouse system in which oral tolerance was induced by long-term feeding with high dose Ag. We demonstrate that the hyporesponsive state of the CD4 T cells was maintained by a selective impairment in the TCR-induced calcium/NFAT signaling pathway and in the IL-2R-induced degradation of p27(kip1) and cell cycle progression. Thus, physiological mucosal tolerance is revealed to be associated with a unique type of T cell hyporesponsiveness which differs from previously described anergic T cells.