Rhesus Macaque Natural CD4 Regulatory T Cells Exhibit Decreased Proliferation But Enhanced Suppression After Pulsing with Sirolimus

The Emory Transplant Center, Department of Surgery, Emory University School of Medicine, Atlanta, GA, USA.
American Journal of Transplantation (Impact Factor: 5.68). 02/2012; 12(6):1441-57. DOI: 10.1111/j.1600-6143.2011.03963.x
Source: PubMed


Although regulatory T cells (Tregs) suppress allo-immunity, difficulties in their large-scale production and in maintaining their suppressive function after expansion have thus far limited their clinical applicability. Here we have used our nonhuman primate model to demonstrate that significant ex vivo Treg expansion with potent suppressive capacity can be achieved and that Treg suppressive capacity can be further enhanced by their exposure to a short pulse of sirolimus. Both unpulsed and sirolimus-pulsed Tregs (SPTs) are capable of inhibiting proliferation of multiple T cell subpopulations, including CD4(+) and CD8(+) T cells, as well as antigen-experienced CD28(+) CD95(+) memory and CD28(-) CD95(+) effector subpopulations. We further show that Tregs can be combined in vitro with CTLA4-Ig (belatacept) to lead to enhanced inhibition of allo-proliferation. SPTs undergo less proliferation in a mixed lymphocyte reaction (MLR) when compared with unpulsed Tregs, suggesting that Treg-mediated suppression may be inversely related to their proliferative capacity. SPTs also display increased expression of CD25 and CTLA4, implicating signaling through these molecules in their enhanced function. Our results suggest that the creation of SPTs may provide a novel avenue to enhance Treg-based suppression of allo-immunity, in a manner amenable to large-scale ex vivo expansion and combinatorial therapy with novel, costimulation blockade-based immunosuppression strategies.

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    • "There are precedents for the loading of cells with hydrophobic drugs, with Pessina et al. [35] reporting that sufficient amounts of the anti-cancer drug paclitaxel were adsorbed by MSCs in a 24-h incubation to reduce tumor growth in vivo and Orange et al. [28] finding that FK-506eloaded dendritic cells could inhibit autoimmune arthritis. A rapid partition of ISDs into cells would appear to explain our observations of increased suppression by brief pretreatments of MSCs, fibroblasts, APCs and HUVECs and may explain at least part of the increased suppression reported for rapamycintreated endothelia and Tregs [30] [36]. It is well documented that rapamycin partitions into blood cells [25], and measurement of rapamycin in our treated MSCs showed sufficient levels of drug even after extensive washing to inhibit T-cell proliferation in co-culture. "
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    ABSTRACT: Multipotent mesenchymal stromal cells (MSCs) are distinguished by their ability to differentiate into a number of stromal derivatives of interest for regenerative medicine, but they also have immunoregulatory properties that are being tested in a number of clinical settings. We show that brief incubations with rapamycin, everolimus, FK506 or cyclosporine A increase the immunosuppressive potency of MSCs and other cell types. The treated MSCs are up to 5-fold more potent at inhibiting the induced proliferation of T lymphocytes in vitro. We show that this effect probably is due to adsorption of the drug by the MSCs during pre-treatment, with subsequent diffusion into co-cultures at concentrations sufficient to inhibit T-cell proliferation. MSCs contain measurable amounts of rapamycin after a 15-min exposure, and the potentiating effect is blocked by a neutralizing antibody to the drug. With the use of a pre-clinical model of acute graft-versus-host disease, we demonstrate that a low dose of rapamycin-treated but not untreated umbilical cord-derived MSCs significantly inhibit the onset of disease. The use of treated MSCs may achieve clinical end points not reached with untreated MSCs and allow for infusion of fewer cells to reduce costs and minimize potential side effects. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Sep 2015 · Cytotherapy
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    • "It has been reported that combination of high dose IL-2, rapamycin, anti-CD3 and (or) anti-CD28 beads or monoclonal antibodies and artificial APCs (aAPC) can expand Treg massively in mice and humans [10] [11] [12]. In contrast to these studies in mice and humans, few reports have addressed expansion of Treg from non-human primates (NHP), -important pre-clinical models in organ transplantation [13] [14] [15] [16] [17] [18]. Given the challenge of manufacturing Treg on a large scale and the potential advantage of banking these cells, cryopreservation may facilitate their clinical application [19]. "
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    ABSTRACT: We expanded flow-sorted Foxp3(+) cynomolgus monkey regulatory T cells (Treg) >1000-fold after three rounds of stimulation with anti-CD3 mAb-loaded artificial antigen-presenting cells, rapamycin (first round only) and IL-2. The expanded Treg maintained their expression of Treg signature markers, CD25, CD27, CD39, Foxp3, Helios, and CTLA-4, as well as CXCR3, which plays an important role in T cell migration to sites of inflammation. In contrast to expanded effector T cells (Teff), expanded Treg produced minimal IFN-γ and IL-17 and no IL-2 and potently suppressed Teff proliferation. Following cryopreservation, thawed Treg were less viable than their freshly-expanded counterparts, although no significant changes in phenotype or suppressive ability were observed. Additional rounds of stimulation/expansion restored maximal viability. Furthermore, adoptively-transferred autologous Treg expanded from cryopreserved second round stocks and labeled with CFSE or VPD450 were detected in blood and secondary lymphoid tissues of normal or immunosuppressed recipients at least two months after their systemic infusion. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Feb 2015 · Cellular Immunology
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    • "Many of the studies investigating the role of mTOR in T regs have relied on the use of rapamycin (also known as sirolimus), which selectively inhibits mTORC1 at low doses but can also inhibit mTORC2 at higher doses (Delgoffe et al., 2011). Unlike conventional T cells, T regs are resistant to rapamycin-induced apoptosis (Strauss et al., 2009) and hence this drug can selectively block pro-inflammatory T cells while preserving T regs (Battaglia et al., 2006; Qu et al., 2007; Lu et al., 2010; Zuber et al., 2011) and their suppressive function (Singh et al., 2012). These data support the conclusion that activation of T regs does not require strong activity of the PI3K pathway. "
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    ABSTRACT: The relative activity of regulatory versus conventional CD4(+) T cells ultimately maintains the delicate balance between immune tolerance and inflammation. At the molecular level, the activity of phosphatidylinositol 3-kinase (PI3K) and its downstream positive and negative regulators has a major role in controlling the balance between immune regulation and activation of different subsets of effector CD4(+) T cells. In contrast to effector T cells which require activation of the PI3K to differentiate and mediate their effector function, regulatory T cells rely on minimal activation of this pathway to develop and maintain their characteristic phenotype, function, and metabolic state. In this review, we discuss the role of the PI3K signaling pathway in CD4(+) T cell differentiation and function, and focus on how modulation of this pathway in T cells can alter the outcome of an immune response, ultimately tipping the balance between tolerance and inflammation.
    Full-text · Article · Aug 2012 · Frontiers in Immunology
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