Metabolic reprogramming augments potency of human pSTAT3–inhibited iTregs to suppress alloreactivity
Kelly Walton, Mario R. Fernandez, Elizabeth M. Sagatys, Jordan Reff, Jongphil Kim, Marie Catherine Lee, John V. Kiluk, Jane Yuet Ching Hui, David McKenna Jr., Meghan Hupp, Colleen Forster, Michael A. Linden, Nicholas J. Lawrence, Harshani R. Lawrence, Joseph Pidala, Steven Z. Pavletic, Bruce R. Blazar, Said M. Sebti, John L. Cleveland, Claudio Anasetti, Brian C. Betts
Kelly Walton, Mario R. Fernandez, Elizabeth M. Sagatys, Jordan Reff, Jongphil Kim, Marie Catherine Lee, John V. Kiluk, Jane Yuet Ching Hui, David McKenna Jr., Meghan Hupp, Colleen Forster, Michael A. Linden, Nicholas J. Lawrence, Harshani R. Lawrence, Joseph Pidala, Steven Z. Pavletic, Bruce R. Blazar, Said M. Sebti, John L. Cleveland, Claudio Anasetti, Brian C. Betts
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Research Article Immunology Transplantation

Metabolic reprogramming augments potency of human pSTAT3–inhibited iTregs to suppress alloreactivity

Abstract

Immunosuppressive donor Tregs can prevent graft-versus-host disease (GVHD) or solid-organ allograft rejection. We previously demonstrated that inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report that human pSTAT3–inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor antileukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing cutaneous lymphocyte-associated antigen, immunosuppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduced alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection and impaired infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provoked a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, cotreatment with coenzyme Q10 restored OxPhos in pSTAT3-inhibited iTregs and augmented their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3–inhibited iTregs to control alloreactive T cells.

Authors

Kelly Walton, Mario R. Fernandez, Elizabeth M. Sagatys, Jordan Reff, Jongphil Kim, Marie Catherine Lee, John V. Kiluk, Jane Yuet Ching Hui, David McKenna Jr., Meghan Hupp, Colleen Forster, Michael A. Linden, Nicholas J. Lawrence, Harshani R. Lawrence, Joseph Pidala, Steven Z. Pavletic, Bruce R. Blazar, Said M. Sebti, John L. Cleveland, Claudio Anasetti, Brian C. Betts

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Figure 1

Human pSTAT3–inhibited iTregs demonstrate superior suppressive potency.

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Human pSTAT3–inhibited iTregs demonstrate superior suppressive potency. ...
(A) Contour plots show the purity of the generated CD4+ iTregs after CD25 selection using magnetic bead isolation. The suppressive potency of purified iTregs generated over 5 days with (B) allogeneic DC stimulators or (C) CD3/CD28 beads while exposed to S3i-201 (50 μM) or DMSO was tested at different ratios of iTreg to T cell responders in alloMLRs. No S3i-201 or DMSO was added to the suppression assay. Data are shown as mean ± SEM. n = 4 independent experiments. (D) The suppressive potency of iTregs generated with STAT3 or nontargeted siRNA (mean ± SEM) is shown. Histograms shows STAT3 expression in the nontargeted siRNA–treated iTregs (orange, gMFI 2870) and STAT3 siRNA–treated iTregs (blue, gMFI 1705). One of 2 independent experiments is shown. Contour plots and box-and-whisker plots (max, min, median) show the frequency of (E and F) GARP+, (G and H) PD-1+, and (I–K) CD39+, LAG3+, or CTLA4+ iTregs (CD4+, CD127–, CD25+, Foxp3+) after expansion with S3I-201 or DMSO from up to 5 independent experiments. (L) Graph shows the suppressive potency (mean ± SEM) of pSTAT3-inhibited iTregs treated with anti–human PD-1, LAP/TGF-β mAb, CD39 inhibitor ARL67156, or control (PBS plus isotype) from 1 of 2 independent experiments. ANOVA (A, C, D, and L) or paired t test (F and H–K). *P < 0.05, **P = 0.001–0.01, ****P < 0.0001. iTregs, induced Tregs; pSTAT3, STAT3 phosphorylation.