Background/Purpose:  Studies indicate quantitative and qualitative Treg insufficiencies underlying the dysregulated immune response in SLE. However, it is unknown what mechanisms drive the Treg dysfunction in SLE. GATA-3 is critical in differentiation and functions of murine Treg. IL-21 is upregulated in SLE and tips the balance from Treg to Th17 differentiation in mice. We sought to identify a mechanism that would link IL-21 and GATA-3 to understand the altered differentiation and function of SLE Tregs.

Methods:  CD3⁺ T cells were isolated from matched SLE and healthy control (HC) subjects. The cells were cultured in the presence of anti-CD3/CD28 with or without rapamycin, after which IL-21 was measured in the supernatant by LUMINEX assay. Naïve CD4⁺ T cells from matched SLE and HC subjects were cultured for 3 days in the presence of anti-CD3/CD28, TGF-β, and IL-2 with or without IL-21. Frequency of CD4⁺CD25⁺FOXP3⁺ Tregs and expression of GATA-3 and CTLA-4 in the Treg were determined by flow cytometry. Total STAT3 and its phosphorylation at tyrosine 705 (pSTAT3^Y705) were examined by immunoblotting. Next, CD4⁺CD25^– responder T cells (Tresp cells) and autologous CD4⁺CD25⁺ Tregs were magnetically isolated. Treg function was determined in the presence or absence of IL-21 by assessing the % suppression of proliferation of CFSE-stained Tresp cells cultured for 5 days in the presence of anti-CD3 and irradiated autologous PBMCs. FOXP3, GATA-3, and CTLA-4 expression in the Treg were determined upon coculture. In some experiments, Tregs were expanded in vitro for 4 weeks with or without rapamycin before coculture. Statistical analyses were done using Student’s t-test.

Results:  IL-21 inhibited SLE Treg differentiation (% CD4⁺CD25⁺FOXP3⁺ cells with and without IL-21: 40.78±3.26%, 64.75±3.16%; p=0.0002). Suppressive function of SLE Tregs was diminished (SLE: 30.25±4.48%, HC: 46.63±4.90%, p=0.03) and further inhibited by IL-21 (18.99±4.95%, p=0.042), while IL-21 did not promote the Tresp cell proliferation. pSTAT3 was upregulated in SLE CD4⁺ T cells, which was further promoted by IL-21 under Treg-polarizing conditions. GATA-3 (Relative MFI: 0.81±0.09, p=0.032) and CTLA-4 were downregulated in freshly isolated CD4⁺CD25⁺FOXP3⁺ cells in SLE (% CTLA-4⁺ cells, SLE: 5.11±0.75%, HC: 6.96±0.79%; p=0.034). IL-21 inhibited GATA-3 expression in CD4⁺CD25⁺FOXP3⁺ cells in SLE (% GATA-3⁺ cells with and without IL-21: 35.29±5.87%, 67.72±7.37%; p=0.0002). IL-21 suppressed CTLA-4 expression in SLE Tregs (% FOXP3⁺CTLA-4⁺cells with and without IL-21: 8.62±1.35%, 29.46±5.31%; p=0.001). In turn, rapamycin blocked T-cell IL-21 secretion, while it promoted FOXP3, GATA-3, and CTLA-4 expression and suppressive function of SLE Tregs.

Conclusion:  IL-21inhibits Treg differentiation and function in SLE potentially by downregulating GATA-3 and CTLA-4, whereas rapamycin promotes Treg differentiation and function in part by suppressing IL-21.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-21-inhibits-treg-differentiation-and-function-in-sle-by-modulating-gata-3-and-ctla-4/