Background/Purpose: Mechanistic target of rapamycin (mTOR) activation has emerged as a prominent mediator of pro-inflammatory T-cell expansion and regulatory T (Treg) cell depletion in systemic lupus erythematosus (SLE). While Treg dysfunction has been documented in SLE, it is unknown i) whether the mTOR pathway is activated within Tregs, ii) what pro-inflammatory cues regulate mTOR in Tregs, and iii) how mTOR controls their function.

Methods: Naïve CD4⁺ T cells from matched SLE and healthy control (HC) subjects were cultured for 3 days in the presence of anti-CD3/CD28, TGF-β, and IL-2 with or without IL-6, IL-21, or rapamycin. Next, CD4⁺CD25⁺ Treg function was determined in the presence or absence of IL-21 or IL-2 by assessing the % suppression of proliferation of CFSE-stained autologous CD4⁺CD25^– responder T cells cultured for 5 days in the presence of anti-CD3 and irradiated PBMCs. In some experiments, Tregs were expanded in vitro for 4 weeks with or without rapamycin before coculture. Upon Treg isolation, -polarization, and -suppression assay, expression of FOXP3, GATA-3, and CTLA-4 in the Treg were determined by flow cytometry. Using lysates of naïve CD4⁺ T cells cultured under Treg-polarizing conditions with or without IL-21, or SLE Tregs expanded in the presence or absence of rapamycin, phosphorylation of STAT3 at tyrosine 705, Akt at Serine 473, S6K1 at Threonine 389, and 4E-BP1 at Threonine 37 and 46, FOXP3, and LC3 were detected by immunoblotting. Cytokine secretion by CD3⁺ T cells cultured in the presence or absence or rapamycin was determined by LUMINEX assay. Statistical analyses were done using Student’s t-test.

Results: SLE Treg exhibits increased mTOR complex 1 (mTORC1) and 2 (mTORC2) activities, but diminished autophagy, GATA-3 and CTLA-4 expression (% CTLA-4⁺ cells among CD4⁺CD25⁺FOXP3⁺ cells, HC: 6.96±0.79%, SLE: 5.11±0.75%; p=0.034), and function. IL-21, but not IL-6, activates mTORC1 and mTORC2, inhibits autophagy, and abrogates Treg differentiation and function (% suppression with or without IL-21, HC: 38.21±3.78%, 46.63±4.90%; p=0.013, SLE: 18.99±4.95%, 30.25±4.48%; p=0.042) although both of these cytokines phosphorylate STAT3 to comparable levels. IL-2 upregulates while IL-21 constrains GATA-3 and CTLA-4 expression selectively in CD4⁺CD25⁺FOXP3⁺ Tregs (% GATA-3⁺ cells with or without IL-21, HC: 47.78±6.12%, 71.20±4.14%; p=0.0075, SLE: 33.49±5.28%, 66.85±6.29%; p=8.15×10^-5, % CTLA-4⁺ cells with or without IL-21, HC: 36.18±2.31%, 43.92±3.87%; p=0.016, SLE: 39.82±4.38%, 52.11±5.66%; p=0.0058). mTORC1 blockade by three-day rapamycin treatment reverses TGF-β deficiency, while dual blockade of mTORC1 and mTORC2 by four-week rapamycin treatment induces autophagy and restores GATA-3 and CTLA-4 expression specifically in CD4⁺CD25⁺FOXP3⁺ cells (GATA-3⁺ cells with or without rapamycin, 25.48±5.73%, 10.78±3.31%; p=0.037, CTLA-4⁺ cells, 23.11±9.08%, 7.51±5.88%; p=0.035), and Treg function (% suppression with or without rapamycin, 57.77±9.62%, 45.41±8.83%; p=0.04).

Conclusion: The data identify IL-21-driven mTOR activation as a pharmacologically targetable checkpoint of deficient autophagy and diminished expression of TGF-β, GATA-3, and CTLA-4 which underlie Treg dysfunction in SLE.

« Back to 2017 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-21-mtor-axis-blocks-the-differentiation-and-function-of-sle-tregs-via-suppression-of-autophagy/