MEF2D sustains activation of effector Foxp3+ Tregs during transplant survival and anticancer immunity
Eros Di Giorgio, … , Ulf H. Beier, Wayne W. Hancock
Eros Di Giorgio, … , Ulf H. Beier, Wayne W. Hancock
Published August 13, 2020
Citation Information: J Clin Invest. 2020;130(12):6242-6260. https://doi.org/10.1172/JCI135486.
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Research Article Immunology Oncology

MEF2D sustains activation of effector Foxp3+ Tregs during transplant survival and anticancer immunity

Abstract

The transcription factor MEF2D is important in the regulation of differentiation and adaptive responses in many cell types. We found that among T cells, MEF2D gained new functions in Foxp3+ T regulatory (Treg) cells due to its interactions with the transcription factor Foxp3 and its release from canonical partners, like histone/protein deacetylases. Though not necessary for the generation and maintenance of Tregs, MEF2D was required for the expression of IL-10, CTLA4, and Icos, and for the acquisition of an effector Treg phenotype. At these loci, MEF2D acted both synergistically and additively to Foxp3, and downstream of Blimp1. Mice with the conditional deletion in Tregs of the gene encoding MEF2D were unable to maintain long-term allograft survival despite costimulation blockade, had enhanced antitumor immunity in syngeneic models, but displayed only minor evidence of autoimmunity when maintained under normal conditions. The role played by MEF2D in sustaining effector Foxp3+ Treg functions without abrogating their basal actions suggests its suitability for drug discovery efforts in cancer therapy.

Authors

Eros Di Giorgio, Liqing Wang, Yan Xiong, Tatiana Akimova, Lanette M. Christensen, Rongxiang Han, Arabinda Samanta, Matteo Trevisanut, Tricia R. Bhatti, Ulf H. Beier, Wayne W. Hancock

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Figure 1

Foxp3 controls Mef2d transcription and influences MEF2D protein-protein interactions.

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Foxp3 controls Mef2d transcription and influences MEF2D protein-protein ...
(A) qPCR results in Treg and Teff cells from WT mice (n = 4, t test). (B) Immunoblots of the indicated proteins in Treg and Teff (Te) cells from WT mice. (C) Diagram of phosphorylation sites retained or lost in MEF2Dα1 and -α2 isoforms. (D) qPCR in freshly isolated WT Teffs and Tregs or cells cultured under activating conditions for 24 hours (1:1 ratio anti-CD3/anti-CD28 mAb–coated beads); n = 6, Tukey’s multiple-comparison test. (E) Immunoblots of the indicated proteins in the same cells as described in D. (F) Densitometric analysis relative to E; n = 2, t test relative to Teffs. (G) qPCR results of Mef2d (pan, α1, α2) expression in freshly isolated WT Teffs and Tregs or cultured under activating conditions for 60 hours in the presence of 5 μM scrambled or Foxp3 antisense (as) oligonucleotide; n = 3, Tukey’s multiple-comparison test. (H) Immunoblots of the indicated proteins in the same cells as described in G. (I) Densitometric analysis relative to H. n = 2, t test relative to scrambled. (J) HEK293T cells were transfected with 1 μg each of tagged constructs encoding Foxp3, MEF2D, and p300; lysates were pulled down with anti-Myc or IgG Ab (1 μg). The membrane was probed with biotinylated anti-Foxp3 mAb, anti-HA for p300, and anti-Myc for MEF2D. (K) HEK293T cells were transfected with 1 μg each of FLAG-Foxp3 and the depicted deletion mutants of MEF2D-GFP. Lysates were pulled down with anti-GFP Ab (1 μg). (L) Lysates from freshly isolated or 24-hour anti-CD3/anti-CD28–stimulated Teffs and Tregs were pulled down with anti-MEF2D or IgG Ab (1 μg). (M) Lysates from freshly isolated Tregs were pulled down with anti-MEF2Dα1 or anti-MEF2Dα2 or IgG Ab (1 μg). (N) HEK293T cells were transfected with 1 μg each of tagged constructs encoding Foxp3, MEF2Dα1, MEF2Dα2, and Hdac9. Lysates were pulled down with anti-MEF2Dα1, -Mα2, or IgG Ab (1 μg). (O) Lysates from freshly isolated Tregs were pulled down with anti-Foxp3 or IgG Abs (1 μg); 1/50 input included in all the IPs. Asterisks in panels K, L, and O indicate heavy chains of IgG Abs used in pull-down experiments. *P < 0.05; **P < 0.01; ***P < 0.005.