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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL118979-07
Application #
9905424
Study Section
Transplantation, Tolerance, and Tumor Immunology Study Section (TTT)
Program Officer
El Kassar, Nahed
Project Start
2014-04-15
Project End
2022-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
7
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Pediatrics
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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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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