Background/Purpose: TCRαβ+CD4-CD8-T cells (double negative T cells, DNT) have been reported to produce inflammatory cytokines such as IL-17, and infiltrate the kidneys of patients with lupus nephritis. We previously reported that inhibition of the transcription regulator STAT3 ameliorates systemic lupus erythematosus (SLE) by reducing DNT accumulation in MRL/lpr lupus –prone mice. However, the underlying mechanism remains unclear.

Methods: MRL/lpr mice were treated with Stattic, a STAT3 inhibitor, or placebo starting at 8 weeks of age for 4-8 weeks. B6/lpr T cell STAT3 knockout (TCKO) mice were bred by crossing B6-CD4 ^Cre Stat3^fl/flmice with B6/lpr mice and followed until disease development. Proteinuria and serum dsDNA antibody levels were monitored. Spleens, kidneys and lymph nodes were collected for flow cytometry and histology at different time points. Peripheral blood T cells from SLE patients and healthy controls were used to evaluate the proportion of DNT, as well as PD-1 and IL-17 expression. DNT cells were sorted from B6/lpr STAT3 TCKO and Stattic-treated MRL/lpr mice, and were used to assay glycolysis stress by Seahorse XFp analyzer. Glycolysis associated genes expressions were analyzed by q-PCR.

Results: Stattic significantly attenuated disease activity in MRL/lpr mice, with decreased serum dsDNA antibody levels (Stattic treated vs placebo: 1,634±211 vs. 18,365±2,132U/ml, p<0.0001), suppressed crescentic glomerulonephritis, reduced spleen size, and diminished skin lesions. Similar results were found in B6/lpr STAT3 TCKO mice. The number of DNT cells were reduced significantly in B6/lpr STAT3 TCKO mice in both kidneys (wild type vs STAT3 TCKO: 31.3±9.6% vs 17.6±6.5%, p=0.0005) and peripheral lymphoid organs (wild type vs STAT3 TCKO: Spleen: 35.4±7.8% vs 26.5±6.1%, p=0.0051; Lymph node: 73.1±11.8 vs. 61.5±10.1, p=0.0168). Moreover, IL-17 production and PD-1 expression were significant decreased in DNT cells from B6/lpr STAT3 TCKO mice. Moreover, p-STAT3 was increased in DNT cells from SLE patients compared with healthy control (17.6±2.1% vs. 6.5±1.3%, p<0.0001). Accordingly, PD-1, as well as IL-17 expression was upregulated in p-STAT3+ DNT as compared to p-STAT3- DNT from SLE patients. XF glycolysis stress assay revealed that DNT from B6/lpr STAT3 TCKO (or Stattic treated MRL/lpr) mice showed lower extracellular acidification rate and glycolysis capacity than matched controls. In vitro treatment with 2-DG could significantly reduced generation and IL-17 production in DNT from B6/lpr STAT3^fl/fl mice, but has less effect on B6/lpr STAT3 TCKO mice. Gene expression analysis shown that HIF1α, GLUT-1, PDK1 mRNA levels were increased in DNT from B6/Lpr STAT3^fl/flmice as compared to B6/lpr STAT3 TCKO mice (4.5 ±2.1 fold, 3.1 ±1.0 fold, and 2.5 ±0.7 fold, respectively). Similar increase was seen in SLE patients derived DNT cells as compared to healthy control DNT.

Conclusion: This is the first study to provide evidence that STAT3 regulates the generation, function and pathogenicity of DNT in systemic lupus erythematosus. Specific targeting of STAT3 in SLE T cells may help restore the balance between pro-inflammatory and regulatory T cells, an imbalance that underlies SLE pathophysiology.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/signal-transducer-and-activator-of-transcription-stat-3-regulates-tcr%ce%b1%ce%b2cd4-cd8-t-cells-in-systemic-lupus-erythematosus-sle/