Background/Purpose: Previous studies point to qualitative and quantitative Treg insufficiencies underlying aberrant T-cell activation in SLE. GATA-3 is critical in lineage commitment and functions of murine Treg, whereas IL-21 tips the balance from Treg towards Th17 differentiation through STAT3 phosphorylation in mice. IL-21 and IL-21 receptor polymorphisms confer risk for SLE. However, roles of GATA-3 in lupus Treg biology and mechanisms by which IL-21 regulates human Treg development remain undefined.

Methods: CD3⁺ T cells were isolated from 9 pairs of matched SLE and healthy control (HC) subjects. A part of the CD3⁺ T cells were stained with CD4, CD25, FOXP3, and GATA-3. The remainder was cultured in RPMI culture media with 10% FCS in the presence of plate-bound anti-CD3, soluble anti-CD28 with/without 100 nM rapamycin. After 3 days in culture, IL-21 secretion in the supernatants was quantified by LUMINEX assay. Naïve CD4⁺ T cells were isolated from 5 pairs of matched SLE and HC subjects and cultured for 3 days in the presence of anti-CD3/CD28, TGF-β, and IL-2 with/without IL-21. A portion of cells were stained with CD25, FOXP3, and GATA-3 to determine the frequency of CD4⁺CD25⁺FOXP3^hi Tregs and GATA-3 expression in the CD4⁺CD25⁺FOXP3⁺ Tregs by flow cytometry. Cell lysates were saved for immunoblotting with antibodies against STAT3, pSTAT3^Y705, Akt, and pAkt^Ser473. Signal intensity was normalized to actin. Statistical analysis was made using Student’s t-test.

Results: GATA-3 expression in Tregs was reduced in SLE as compared with HC (relative mean fluorescence intensity: 0.81±0.09, p=0.032) in freshly isolated CD3⁺ T cells, whereas IL-21 secretion appears to be increased in culture supernatants of SLE T cells (SLE: 415.37±207.62 pg/ml, HC: 147.38±45.28 pg/ml, p=0.10). Under Treg-polarizing conditions, IL-21 suppressed CD4⁺CD25⁺FOXP3⁺ Treg differentiation in SLE (frequency of CD4⁺CD25⁺FOXP3^hi cells; control: 57.06±3.35%, IL-21-treated cells: 34.80±2.13%, p=0.0002) and HC (control: 45.30±6.66%, IL-21-treated cells: 30.43±2.84%, p=0.03). IL-21 suppressed GATA-3 expression in CD4⁺CD25⁺FOXP3⁺ Tregs in SLE (control 58.31±7.93%, IL-21-treated cells 26.57±5.40%, p=0.001) and HC (control: 59.53±5.32%, IL-21-treated cells: 38.47±8.51%, p=0.045). Mechanistically, IL-21 phosphorylated STAT3 (relative intensity: 1.48±0.19, p=0.033) as well as Akt (relative intensity: 1.48±0.20, p=0.037) in SLE CD4⁺T cells cultured under Treg-skewing conditions. Rapamycin suppressed IL-21 secretion by SLE T cells stimulated with anti-CD3/CD28 (control: 415.37±207.62 pg/ml, rapamycin: 8.23±3.01 pg/ml, p=0.047).

Conclusion: The data indicate that IL-21 phosphorylates STAT3 and Akt and inhibits Treg differentiation in SLE, possibly via the suppression of GATA-3. Suppression of T-cell IL-21 secretion emerges as one of the mechanisms by which rapamycin promotes Treg expansion in SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/il-21-abrogates-cd4cd25foxp3-treg-differentiation-in-part-by-suppressing-treg-gata-3-expression-in-sle/