Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells

Abstract

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.

Evidence of the immunomodulatory role of dual PI3K/mTOR inhibitors in transplantation: an experimental study in mice

“…Early work from our group characterized the transition into functional iTregs as a tightly regulated and sequential process. This occurs in response to weak TCR stimulation in the presence of IL‐2 and TGF‐β only after the cell undergoes several rounds of cell division, and it entails a fine‐tuned reconfiguration of the PI3K/AKT/mTOR pathway . This is consistent with the “two‐step differentiation model” proposed by Guo et al .…”

“…We demonstrated in early studies that primary human naïve CD4 + T‐cells differentiate into suppressor Tregs in the presence of TGF‐ß and IL‐2 only after undergoing multiple rounds of cell division . In light of our results, we questioned whether the antiproliferative action of rapamycin and dual inhibitors was compatible with the IL‐2‐dependent differentiation of naïve T‐cells into iTregs.…”

Mitochondrial and cytosolic roles of PINK1 shape induced regulatory T‐cell development and function

“…3A). In association with these results, the PINK1 −/− T cells proliferated less, which is of particular interest as the acquisition of suppressor ability is associated with multiple rounds of proliferation [20,21] (Fig. 3B).…”

Glycosylation‐dependent interaction between CD69 and S100A8/S100A9 complex is required for regulatory T‐cell differentiation

“…Both hPBMCs and CD4 + naive T cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen/Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco/Life Technologies), 2 mM L‐glutamine, 125 U/ml penicillin, and 125 μg/ml streptomycin in a humidified atmosphere at 37°C with 5% CO 2 for 24 h before experimental treatment and analysis. For stimulation of immune response, freshly isolated hPBMCs were treated with phorbol‐12‐myristate‐13‐acetate (PMA) at 1 ng/ml or LPS at 1 μg/ml (both from Sigma‐Aldrich, St. Louis, MO, USA) for 24 h. T reg lineage was generated according to previously established protocols with minor modifications (25, 26). In brief, freshly enriched CD4 + naive T cells were cultured for 5 d in RPMI 1640 medium as above mentioned except in the presence of 10 μg/ml anti‐CD3 (eBioscience), 2 μg/ml anti‐CD28 (eBioscience), 100 U/ml IL‐2 (PeproTech, Rocky Hill, NJ, USA), and 1 ng/ml TGF‐β (PeproTech).…”