The development and maintenance of immunosuppressive CD4 + regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis 1 , type I diabetes 2 , rheumatoid arthritis and graft versus host disease, 4 several fundamental differences in human versus mouse Treg biology 5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion 6 .Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4 + T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans 7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into naïve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and naïve T cells have different reported polarization requirements and plasticities 8 , pre-sorting of the initial T cell population into CD45RA + and CD45RO + subsets can be used to examine these discrepancies. Consistent with others, our CD25 Hi CD45RA -iTregs express high levels of FoxP3 9 , GITR and CTLA-4 11 and low levels of CD127 12 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells. Video LinkThe video component of this article can be found at http://www.jove.com/video/3738/ Protocol Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) from Buffy CoatThis procedure can be scaled down for smaller volumes of blood. Dilution of whole blood samples is 1:1 in PBS.

INTRODUCTION A dysregulation of regulatory T cells (Tregs) could play a major role in the pathogenesis of bronchial asthma. Sex-dependent differences as well as the impact of hormonal changes in the incidence and severity of asthma are widely recognized. Emerging evidence suggests that asthma symptoms are alleviated in female patients taking hormone oral contraceptives (OCs). The impact of OCs on the generation of induced Tregs (iTregs) was assessed in a cohort of female patients with asthma. METHODS Thirteen patients were included in this pilot study. During three distinct phases of their menstrual cycles we measured exhaled nitric oxide (eNO) levels, forced expiratory volume at 1s (FEV1s), asthma control test (ACT) score, sex steroid hormone levels in serum, natural Tregs in peripheral blood and the ability of CD4+ T cells to generate iTregs ex vivo. RESULTS The luteal serum levels of estradiol and progesterone negatively correlated with the proportion of iTregs generated ex vivo in patients not taking OCs. In addition, physiological doses of estradiol and progesterone prevented the acquisition of a suppressor T cell phenotype in vitro. Interestingly, patients taking OCs had reduced serum sex hormone levels associated with higher iTreg induction, a better ACT score and a tendency toward lower eNO levels. CONCLUSIONS Our results identify an impact of sex hormones in the capacity of T cells to polarize towards a regulatory phenotype and suggest the regulation of peripheral T cell lineage plasticity as a potential mechanism underlying the beneficial effects of OCs in women with asthma.

Peripherally induced Treg (iTregs) are being recognized as a functional and physiologically relevant T cell compartment. Understanding the molecular basis of their development is a necessary step before the therapeutic potential of iTreg manipulation can be exploited. In this study, we report that the differentiation of primary human T cells to suppressor iTregs involves the relocation of key proximal TCR signaling elements to the highly active IL-2-Receptor (IL2-R) pathway. In addition to the recruitment of Lymphocyte-specific protein tyrosine kinase (Lck) to the IL2-R complex, we identified the dissociation of the voltage gated K+ channel Kv1.3 from the TCR pathway and its functional coupling to the IL2-R. The regulatory switch of Kv1.3 activity in iTregs may constitute an important contributing factor in the signaling rewiring associated with the development of peripheral human iTregs and sheds new light upon the reciprocal cross-talk between the TCR and IL2-R pathways.

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