Tregs restrain dendritic cell autophagy to ameliorate autoimmunity
Themis Alissafi, … , Ken Cadwell, Panayotis Verginis
Themis Alissafi, … , Ken Cadwell, Panayotis Verginis
Published June 5, 2017
Citation Information: J Clin Invest. 2017;127(7):2789-2804. https://doi.org/10.1172/JCI92079.
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Research Article Autoimmunity Immunology

Tregs restrain dendritic cell autophagy to ameliorate autoimmunity

Abstract

Design of efficacious Treg-based therapies and establishment of clinical tolerance in autoimmune diseases have proven to be challenging. The clinical implementation of Treg immunotherapy has been hampered by various impediments related to the stability and isolation procedures of Tregs as well as the specific in vivo targets of Treg modalities. Herein, we have demonstrated that Foxp3+ Tregs potently suppress autoimmune responses in vivo through inhibition of the autophagic machinery in DCs in a cytotoxic T-lymphocyte–associated protein 4–dependent (CTLA4-dependent) manner. Autophagy-deficient DCs exhibited reduced immunogenic potential and failed to prime autoantigen-specific CD4+ T cells to mediate autoimmunity. Mechanistically, CTLA4 binding promoted activation of the PI3K/Akt/mTOR axis and FoxO1 nuclear exclusion in DCs, leading to decreased transcription of the autophagy component microtubule-associated protein 1 light chain 3β (Lc3b). Human DCs treated with CTLA4-Ig, a fusion protein composed of the Fc region of IgG1 and the extracellular domain of CTLA4 (also known as abatacept, marketed as Orencia), demonstrated reduced levels of autophagosome formation, while DCs from CTLA4-Ig–treated rheumatoid arthritis patients displayed diminished LC3B transcripts. Collectively, our data identify the canonical autophagy pathway in DCs as a molecular target of Foxp3+ Treg–mediated suppression that leads to amelioration of autoimmune responses. These findings may pave the way for the development of therapeutic protocols that exploit Tregs for the treatment of autoimmunity as well as diseases in which disturbed tolerance is a common denominator.

Authors

Themis Alissafi, Aggelos Banos, Louis Boon, Tim Sparwasser, Alessandra Ghigo, Kajsa Wing, Dimitrios Vassilopoulos, Dimitrios Boumpas, Triantafyllos Chavakis, Ken Cadwell, Panayotis Verginis

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Figure 6

CTLA4-mediated nuclear exclusion of FoxO1 in DCs.

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CTLA4-mediated nuclear exclusion of FoxO1 in DCs. (A) Western blot for C...
(A) Western blot for CD86 following immunoprecipitation of p85-containing complexes in DCs from Foxp3+ Treg–transferred MOG35–55–immunized Rag1–/– mice. (B and D) WT or Akt1–/– BMDCs were treated with LPS and CTLA4-Ig or IgG for 12 or 20 hours (B and E–J). (B) mRNA expression of Foxo1 and Lc3b. *P = 0.0214; **P = 0.0214; †P = 0.0023. (C) Schematic representation of Lc3b proximal promoter with FoxO1-binding sites (A1 and A2). (D) ChIP analysis of the promoter of Lc3b gene for FoxO1-binding sites. *P = 0.0359; †P = 0.0489. Statistical significance using paired t test. (E) Western blot for FoxO1, tubulin, and TBP from cytoplasmic (CEs) and nuclear (NEs) extracts. Relative intensity of FoxO1. *P = 0.0405. (F) FoxO1 (red), and DAPI (blue). Scale bar: 10 μm. Relative intensity of nuclear FoxO1. **P < 0.0001. (G) Western blot for LC3 and actin. Relative intensity of LC3II/LC3I using 2-way ANOVA. *P = 0.05; †P = 0.0291; **P = 0.0069. (H) LC3 (red), LAMP-1 (green), p62 (silver white), and DAPI (blue). Scale bar: 10 μm. LC3 and p62 puncta/cell are depicted. ***P < 0.0001; *P = 0,0142; †P = 0.027. (I) FoxO1 (red) and Dapi (blue). Scale bar: 10 μm. Relative intensity of nuclear FoxO1. ***P < 0.0001. (J) BMDCs were transfected with FoxO1WT or FoxO1AAA. LC3 and p62 puncta/cell are depicted. Scale bar: 5 μm. ***P < 0.0001. Results are expressed as mean ± SEM. n = 4–5 mice. One representative experiment of 3 or 4 is shown. For B, E, F, and H–J, statistical significance was obtained by unpaired Student’s t test.