GP96 is a GARP chaperone and controls regulatory T cell functions
Yongliang Zhang, … , Bei Liu, Zihai Li
Yongliang Zhang, … , Bei Liu, Zihai Li
Published January 20, 2015
Citation Information: J Clin Invest. 2015;125(2):859-869. https://doi.org/10.1172/JCI79014.
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Research Article Immunology

GP96 is a GARP chaperone and controls regulatory T cell functions

Abstract

Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ–producing and IL-17–producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane–associated TGF-β (mLTGF-β). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-β and resulted in inefficient production of active TGF-β. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.

Authors

Yongliang Zhang, Bill X. Wu, Alessandra Metelli, Jessica E. Thaxton, Feng Hong, Saleh Rachidi, Ephraim Ansa-Addo, Shaoli Sun, Chenthamarakshan Vasu, Yi Yang, Bei Liu, Zihai Li

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Figure 6

GP96-null Tregs lose FOXP3 expression and convert to IFN-γ–producing ex-FOXP3 T cells.

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GP96-null Tregs lose FOXP3 expression and convert to IFN-γ–producing ex-...
(A) FOXP3 and GP96 expression in Tregs from WT and KO mice was determined by Western blot. Data represent 2 independent experiments. (B) Mean fluorescence intensity (MFI) of FOXP3 stain of Tregs from Het and KO mice. Data are represented as mean ± SEM. (C) Surface expression of β2 integrin (CD18) on Tregs and Teff cells from WT and KO mice was determined by flow cytometry. Numbers represent percentages of CD18– cells in the gated populations. (D) CD4+CD25+ Tregs from WT and KO mice were stimulated with plate-bound antibodies against CD3 and CD28 plus IL-12 for 3 days, followed by IC staining for FOXP3 and IFN-γ. Numbers indicate percentages of indicated cells in the entire population. Two independent experiments were performed with similar findings. (E and F) FACS-sorted CD4+FOXP3GFP+ Tregs (2 × 105) from 3-week-old WT or KO mice were transferred to NOD Rag–/– mice. IC FOXP3 levels in CD4 T cells from the peripheral blood were then analyzed 4 and 6 weeks after the adoptive transfer. The percentage of FOXP3– cells within CD4+ cells (mean ± SEM) was plotted in F. (G and H) IFN-γ levels (percentages) in CD4 T cells from splenocytes of the mice in E and F above were analyzed. Data in C–F represent 4 independent experiments. Two-tailed Student’s t test was used for statistical analysis between groups.