Regulatory T-cells (Treg) play a critical role in preventing autoimmune and alloimmune reactions. Clinical trials demonstrated that Treg adoptive transfer significantly reduced but did not uniformly eliminate grades II-IV graft-vs-host disease (GVHD).
Treg more dominantly rely on fatty acid oxidation (FAO) as a fuel source, in contrast to glycolytic conventional T-cells. We hypothesized that increasing FAO would augment Treg efficacy by increasing potency and persistence, thereby ultimately decreasing the Treg cell number required to enhance Treg-mediated GVHD reduction. The FAO and fatty acid synthesis (FAS) pathways compete for acetyl-CoA use. Acetyl-CoA carboxylase-1 (ACC1) skews acetyl-CoA toward FAS and results in downstream inhibition of CPT1a, the rate-limiting enzyme in FAO. ACC1 deletion or inhibition skews naive CD4 T-cell toward FAO, favoring Treg over Th17 differentiation.
To precisely determine the role of ACC1 on in vivo Treg-mediated suppression of GVHD, we tested the effects of a 2-hour treatment of murineTreg with ND630, a pharmacological inhibitor of ACC1 currently in clinical trials for hepatic steatosis, or Treg-specific ACC1 knockout (KO). ACC1 inhibition or KO increased in vitro Treg suppressive function, and when infused into mice with established bronchiolitis obliterans (BOS) and chronic GVHD (cGVHD), ACC1KO Treg were more potent than wild-type Treg in reversing BOS pulmonary resistance (FigA), a finding consistent in each of 3 replicate transplants.
Mechanistically, both ND630 treated Treg and ACC1KO Treg demonstrated an increased oxygen consumption rate (OCR), a surrogate for mitochondrial oxidative phosphorylation (OXPHOS), a critical source of ATP for Treg. This was associated with an increase in uptake of BoDipyC16, a synthetic fatty acid, suggesting exogenous fatty acids may drive the increase in OXPHOS through FAO. IACS, an OXPHOS inhibitor, abrogated OCR augmentation and precluded increased suppressive function after ND630 treatment, indicating a critical role for OXPHOS in driving Treg suppressor function. To directly evaluate effects on Treg mitochondria, where OXPHOS occurs, Treg were stained with MitoTracker dyes that measure mitochondrial mass and membrane potential, two key surrogate measurements for mitochondrial function. Compared to controls, ND630 treated and ACC1KO Treg each showed increased mitochondrial mass and membrane potential, as well as enhanced mitochondrial polarization (membrane potential to mass ratio), which is required for full Treg functionality. Expansion and scanning electron microscopic analysis of Treg mitochondria revealed enhanced mitochondrial fusion and elongation after ND630 treatment ( FigB). This mitochondrial structural change boosts OXPHOS, consistent with a direct effect of ACC1 inhibition/KO on mitochondrial fitness.
In light of these mitochondrial morphology changes, we hypothesized that directly modifying the mitochondria itself, without inhibiting ACC1, might result in augmented Treg function. Consistent with this hypothesis, a 2-hour treatment with the combination of M1 (mitochondrial fusion promoter) and mDIVI1 (mitochondrial fission inhibitor), significantly enhanced Treg function in vitro, and enhanced Treg OCR to a similar degree as ND630. Electron microscopy confirmed that M1/mDIVI1 treatment resulted in a similar degree of mitochondrial fusion as ND630 treatment. Similar to ACC1KO Treg, M1/mDIVI1 treated Treg were more potent than control Treg in reversing established cGVHD. To further explore mitochondrial fusion effects on Treg function, we used siRNA to knockdown the critical mitochondrial fusion protein mitofusin 1 (MFN1). A 50% decrease in MFN1 protein significantly decreased Treg in vitro functionality and OCR, and treatment of MFN1 knockdown Treg with ND630 had no functional or metabolic effect (FigC), pointing to mitochondrial fusion as a critical component of the mechanism for ACC1 inhibition/KO.
In summary, ACC1 inhibition or deletion amplifies murine Treg potency resulting in superior metabolic fitness, and greater efficacy in treating cGVHD mice with established BOS, in part through enhancing mitochondrial fusion. Since ND630 is already under investigation in patients, ACC1 inhibition with ND630 could be readily translatable for clinical trials with a goal of utilizing Tregs with inhibited or deleted ACC1 to improve GVHD therapy.
Disclosures
Blazar:BlueRock Therapeutics: Current Employment, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Current Employment, Research Funding.
