Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity
Yan Xiong, … , Philip A. Cole, Wayne W. Hancock
Yan Xiong, … , Philip A. Cole, Wayne W. Hancock
Published January 9, 2020
Citation Information: J Clin Invest. 2020;130(4):1830-1842. https://doi.org/10.1172/JCI131375.
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Research Article Immunology Oncology

Inhibiting the coregulator CoREST impairs Foxp3+ Treg function and promotes antitumor immunity

Abstract

Foxp3+ Tregs are key to immune homeostasis, but the contributions of various large, multiprotein complexes that regulate gene expression remain unexplored. We analyzed the role in Tregs of the evolutionarily conserved CoREST complex, consisting of a scaffolding protein, Rcor1 or Rcor2, plus Hdac1 or Hdac2 and Lsd1 enzymes. Rcor1, Rcor2, and Lsd1 were physically associated with Foxp3, and mice with conditional deletion of Rcor1 in Foxp3+ Tregs had decreased proportions of Tregs in peripheral lymphoid tissues and increased Treg expression of IL-2 and IFN-γ compared with what was found in WT cells. Mice with conditional deletion of the gene encoding Rcor1 in their Tregs had reduced suppression of homeostatic proliferation, inability to maintain long-term allograft survival despite costimulation blockade, and enhanced antitumor immunity in syngeneic models. Comparable findings were seen in WT mice treated with CoREST complex bivalent inhibitors, which also altered the phenotype of human Tregs and impaired their suppressive function. Our data point to the potential for therapeutic modulation of Treg functions by pharmacologic targeting of enzymatic components of the CoREST complex and contribute to an understanding of the biochemical and molecular mechanisms by which Foxp3 represses large gene sets and maintains the unique properties of this key immune cell.

Authors

Yan Xiong, Liqing Wang, Eros Di Giorgio, Tatiana Akimova, Ulf H. Beier, Rongxiang Han, Matteo Trevisanut, Jay H. Kalin, Philip A. Cole, Wayne W. Hancock

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Figure 8

Effects of CoREST complex inhibitor on human Tregs.

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Effects of CoREST complex inhibitor on human Tregs. (A) Human healthy do...
(A) Human healthy donor Tregs were incubated with corin (1 μM) for 2.5 hours, washed twice, and incubated with CFSE-labeled, anti-CD3ε microbead-stimulated healthy donor PBMCs for 5–6 days. Representative data show impaired Treg suppressive function for CD4+ responder cells (CD8+ responder cells are shown in Supplemental Figure 8). (B) After suppression assays, cells were stained with fixable live/dead marker Zombie Yellow and Foxp3, cells were gated into CD4+CFSE–Zombie+ (= dead Treg), CD4+CFSE–Zombie–Foxp3– (= live exTreg), and CD4+CFSE–Zombie–Foxp3+ (= live Treg), to evaluate Treg stability (loss of Foxp3) and survival (% of Zombie– cells). (C) Statistical analysis of data shown in A; Tregs from 5 healthy donors and Tregs and responder PBMCs from 3 healthy donors were tested in 5 independent experiments (total of 12 suppression assays). Treg abilities to suppress divisions of CD4+ and CD8+ responders were analyzed separately; 1 sample t test with theoretical mean = 1. (D) Statistical analysis of data from B; data from 6 assays were pooled, Wilcoxon’s signed ranked test. (E) Cells were stained for Zombie Yellow, Foxp3, and CTLA-4. Viable CD4+CFSE–Zombie–Foxp3+ Tregs were gated and evaluated for CTLA-4 expression (MOF, percentage of positive cells) and Foxp3 MOF. Data were pooled from 6 assays. Wilcoxon’s signed ranked test. (F–J) Healthy donor PBMCs (from 5 different donors in 3 experiments) were incubated with corin (1 μM) and stimulated overnight with CD3ε/CD28 mAb-coated beads (1.3 beads/cell). (F) TIGIT and GITR expression tended to be decreased in viable CD4+Foxp3+ Tregs, but without significance, whereas CD127 expression significantly increased in the presence of corin. (G) Representative example of Foxp3 expression in PBMCs and (H) Foxp3 MOF in viable CD4+Foxp3+ Tregs. (I and J) Statistics showing (I) decreased Treg numbers and (J) decreased MOF of Foxp3 in human Tregs treated with corin. All tests in F through I are 1-sample t tests with Bonferroni’s correction for multiple comparisons, whereas data shown in J were evaluated by unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are shown as mean ± SD.