This renewal continues our optimization of regulatory T cell (Treg) infusion for creating transplant tolerance to prevent graft-vs-host disease (GVHD) in the clinic. Our first-in-human phase I Treg trials showed reduction but not elimination of GVHD. High Treg numbers were needed and suppression was variable. We propose to tackle both limitations by infusing high potency Tregs. Whereas protein kinase C theta (PKC-?) localization to the proximal immunological synapse (IS) is required for optimal Teffector function it is excluded from the IS in Tregs where it is located in a distal pole complex (DPC). Mouse or human Tregs treated with a clinically tested PKC-? inhibitor increased suppression by 300%. Phospho-proteomics show down-regulation of vimentin, a cytoskeletal filament preferentially expressed in Tregs, as well as targets involved in actin cytoskeletal dynamics and cell motility. Vimentin physically sequesters PKC-? in the DPC, believed to have negative regulatory function, and tethers mitochondria. PKC-? inhibitors and vimentin siRNA each caused DPC disruption and increased suppression and, upon adoptive transfer, had heightened potency in GVHD prevention and reduced host dendritic cell priming of alloantigen-specific Teffectors. These data point to the surprising conclusion that DPC disruption per se and not inhibitor targeting of PKC-? kinase activity was responsible for Treg super-suppressor function. Proliferating Tregs increase mitochondrial fatty acid ?- oxidation (FAO) to meet energy needs. Super-suppressor Tregs up-regulated fatty acid (FA) uptake and increased oxidative phosphorylation and FAO without altering glycolysis. Increased suppression was associated with down-regulation of the mTOR component, mTORC2, that affects cytoskeletal dynamics and mitochondrial anchoring, with no mTORC1 effects. Intracellular metabolomics of DPC disrupted Tregs indicate significant effects on FA metabolism and de novo FA biosynthesis, leading to the central hypothesis that the cytoskeleton structure restrains Treg function by regulating mitochondrial function and FAO. Thus the DPC disrupted Tregs have a super-suppressive phenotype with coordinately regulated attributes of signaling, metabolism and function.
In aim 1 A, we hypothesize mitochondrial fusion is induced by Treg DPC disruption and gain-or-loss-of function fusion will alter super-suppression.
In aim 1 B, we hypothesize that DPC disruption increases mitochondrial motility to the IS to modulate calcium signaling by altering mitochondria-cytoskeletal tethering.
In aim 2 A, we will determine whether Treg loss-of-function for FA uptake, de novo synthesis or mitochondrial metabolism precludes super-suppressor function.
In aim 2 B, we hypothesize that key aim 2A metabolic proteins sequestered by vimentin or actin fibers must be dispersed to drive Treg super-suppression.
In aim 3, we hypothesize super-suppressor mechanisms identified above will translate into in vivo suppressor capacity in localized colitis and systemic multi-organ system inflammatory GVHD models. These data will provide important biological insights for translating Treg PKC-? inhibitor and related treatments into the clinic.

Public Health Relevance

Our team of experts will develop novel approaches and gain biological insights into immune system control by Tregs. Our findings will have broad implications for the use of Tregs in controlling adverse immune responses leading to translational applications to harness the full power of Tregs for hematopoietic stem cell and solid transplantation as well as autoimmunity settings.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Transplantation, Tolerance, and Tumor Immunology Study Section (TTT)
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El Kassar, Nahed
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University of Minnesota Twin Cities
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Sarhan, Dhifaf; Hippen, Keli L; Lemire, Amanda et al. (2018) Adaptive NK Cells Resist Regulatory T-cell Suppression Driven by IL37. Cancer Immunol Res 6:766-775
Owen, David L; Mahmud, Shawn A; Vang, Kieng B et al. (2018) Identification of Cellular Sources of IL-2 Needed for Regulatory T Cell Development and Homeostasis. J Immunol 200:3926-3933
Zitzer, Nina C; Snyder, Katiri; Meng, Xiamoei et al. (2018) MicroRNA-155 Modulates Acute Graft-versus-Host Disease by Impacting T Cell Expansion, Migration, and Effector Function. J Immunol 200:4170-4179
Habtetsion, Tsadik; Ding, Zhi-Chun; Pi, Wenhu et al. (2018) Alteration of Tumor Metabolism by CD4+ T Cells Leads to TNF-?-Dependent Intensification of Oxidative Stress and Tumor Cell Death. Cell Metab 28:228-242.e6
Hippen, K L; O'Connor, R S; Lemire, A M et al. (2017) In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines. Am J Transplant 17:3098-3113
Purroy, Carolina; Fairchild, Robert L; Tanaka, Toshiaki et al. (2017) Erythropoietin Receptor-Mediated Molecular Crosstalk Promotes T Cell Immunoregulation and Transplant Survival. J Am Soc Nephrol 28:2377-2392
McKenna Jr, David H; Sumstad, Darin; Kadidlo, Diane M et al. (2017) Optimization of cGMP purification and expansion of umbilical cord blood-derived T-regulatory cells in support of first-in-human clinical trials. Cytotherapy 19:250-262
Fuchs, Anke; Gliwi?ski, Mateusz; Grageda, Nathali et al. (2017) Minimum Information about T Regulatory Cells: A Step toward Reproducibility and Standardization. Front Immunol 8:1844
Kean, Leslie S; Turka, Laurence A; Blazar, Bruce R (2017) Advances in targeting co-inhibitory and co-stimulatory pathways in transplantation settings: the Yin to the Yang of cancer immunotherapy. Immunol Rev 276:192-212
Zanin-Zhorov, Alexandra; Kumari, Sudha; Hippen, Keli L et al. (2017) Human in vitro-induced regulatory T cells display Dlgh1dependent and PKC-? restrained suppressive activity. Sci Rep 7:4258

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