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
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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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Available from: Karnail Singh, Jun 13, 2014

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

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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.
    Cellular Immunology 02/2015; 295(1). DOI:10.1016/j.cellimm.2015.02.006 · 1.92 Impact Factor
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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.
    Frontiers in Immunology 08/2012; 3:245. DOI:10.3389/fimmu.2012.00245
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    • "Animal studies have shown that treatment with IL-2 increases regulatory T cell proliferation and survival (Rabinovitch et al., 2002; Tang et al., 2008). Combination with rapamycin, which stabilizes the expression of Forkhead box P3 (FoxP3) and enhances suppression (Battaglia et al., 2006; Singh et al., 2012), may promote tolerance in these autoimmune and potentially alloimmune settings. "
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    Frontiers in Immunology 07/2012; 3:198. DOI:10.3389/fimmu.2012.00198
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