Background/Purpose: Follicular helper T cells (Tfh) cells in lupus help shape the germinal center (GC) response by delivering contact-dependent and soluble signals, including the cytokines IFN-γ and IL-21. In conventional immune responses, synthesis of Tfh-cell cytokines is regulated by the transcription factors STAT4, T-bet, and Bcl6, although their regulation in lupus is not known. To answer this question, we examined the differential expression of these transcription factors in splenic Tfh cells prior to onset of disease, after immune cell activation but before end organ disease onset, and then in later severe disease in lupus-prone B6.Sle1.Yaa mice. We also assess applicability to human lupus.

Methods: Spleens were harvested from 2,4 and 6 months old B6.Sle1.Yaa mice. Transcription factor (T-bet and Bcl6) and cytokine production were assessed by intercellular flow cytometry. Cell were stimulated with PMA and ionomycin, and Tfh (CD4⁺CD44^hiLy6c^loPSGL-1^loCXCR5^hiPD-1^hi) and Th1 (CD4⁺CD44^hiLy6c^hiPSGL-1^hi) cells were stained for IL-21 and IFN-γ. Phosphorylation STAT4 in Tfh and Th1 cells were analyzed by flow cytometry after stimulation by IFN-α or IL-12 for 20 minutes in DMEM medium with 1% serum.

Twenty-eight SLE patients from age 18 to 75 years and twenty age-matched healthy control were enrolled in this study. All SLE patients had fulfilled the revised disease criteria of the American College of Rheumatology 1997. Disease activity was assessed with the SLE Disease Activity Index 2000 (SLEDAI2K). Mononuclear cells from the peripheral blood samples were isolated using Ficoll-Paque density gradient media and rested suspended in DMEM medium with 10% serum overnight. Phosphorylation STAT4 in circulating Follicular helper-like T cells (CD4⁺CD45RA^–CXCR5^hiPD1^hiCCR7^lo) and CD4+ T memory cells (CD4⁺CD45RA^–CXCR5^hiPD1^loCCR7^hi) were analyzed by flow cytometry after stimulation by IFN- α or IL-12 for 20 minutes in DMEM medium with 1% serum.

Results: We determined that GC Tfh cells produced IL-21 and IFN-γ at similar rates during these three stages of disease; however, Tfh-cell STAT4 activity increased while Bcl6 and T-bet expression declined as disease progressed, suggesting a role of STAT4 in driving T-cell dependent GC B-cell maturation responses in murine lupus.

We found that circulating Tfh and circulating CD4+ T memory cells from patients secreted IL-21 and IFN-γ after stimulation. We next examined STAT4 activity these cells, after IFN-α and IL-12 stimulation in vitro, in comparison to cells from healthy controls. STAT4 phosphorylation, as an indicator of STAT4-mediated nuclear translocation, was more robust upon IFN-α than IL-12 stimulation, by contrast to canonical STAT4 phosphorylation driven by IL-12 in Th1 cells. Furthermore, the STAT4 phosphorylation correlated to greater disease activity (SLEDAI2K).

Conclusion: Our study indicates that as disease progresses in mice and humans with lupus, Tfh-cells and circulating CD4+ T memory cells are robustly responsive to STAT4-guided gene expression, which plays an important role in driving pathologic cytokine production in sustaining moderate and late stage disease activity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/stat4-activation-by-type-i-interferons-regulates-pathogenic-il-21-and-ifn-%ce%b3-in-lupus/