1097: Intrinsic STAT1 Deficiency Underlies Proinflammatory Imprint of Naive CD4+ T Cells in Spondyloarthritis

Bilade CHERQAOUI¹, Frederic Cremazy¹, Marc Lauraine¹, Ghazal Shammas², Roula Said-Nahal³, Hendrick Mambu Mambueni⁴, Felicie Costantino², Marine Fourmont², Simon Glatigny², Luiza Maria Araujo² and Maxime Breban², ¹INSERM U1173, UFR Simone Veil, Versailles-Saint Quentin / Paris-Saclay University, Montigny-le-Bretonneux, France, ²1INSERM U1173, UFR Simone Veil, Versailles-Saint Quentin / Paris-Saclay University, Montigny-le-Bretonneux, France, ³Rheumatology Division - Ambroise Pare Hospital, APHP, Boulogne-Billancourt, France, ⁴Genomic Platform of Faculty of Health Simone Veil - UVSQ university, Montigny-le-Bretonneux, France

Background/Purpose: Spondyloarthritis (SpA) development is associated with type 3 immune response activation. In HLA-B27/human β2-microglobulin transgenic rat model (B27-rat), this might at least be related to enhanced differentiation of naive CD4+ T cells into T helper-17 (Th17). Here, we aimed to decipher the molecular mechanisms that might lead to altered naive CD4+ T cells differentiation in SpA.

Methods: Naive CD4+ T cells were sorted by flow cytometry from mesenteric lymph nodes (mLN) of B27-rat, control healthy nontransgenic (NTG) littermates or healthy transgenic B7-rats, and from peripheral blood of patients with axial SpA (N=28, including 7 with juvenile onset), healthy controls (N=17) or patients with rheumatoid arthritis (N=8). The transcriptome and epigenome were characterized ex vivo rat naive CD4+ T cells, by using RNA-seq and ChIP-seq of 2 activating histone marks (H3K4me3, H3K27ac). In addition, selected genes expression was studied by q-RT-PCR. Differentiation of rat naive CD4+ T cells was analyzed 3 to 6 days after stimulation with anti-CD3/anti-CD28 alone or in the presence of rat IFNγ or a selective STAT1 transcriptional activator (2-(1,8-Naphthyridin-2-ly)phenol).

Results: Under anti-CD3/anti-CD28 neutral stimulation B27-rat (but not B7-rat) naive CD4+ T cells were skewed to develop a Th17 proinflammatory profile, even before disease onset, as compared to NTG rats. Both transcriptomic (Figure 1) and epigenomic studies consistently revealed pro-Th17 and decreased interferon signatures in ex vivo-sorted B27-rat CD4+ naive T cells. Imbalance between increased STAT3 and decreased STAT1 was predicted to be a critical upstream regulator of this molecular signature (Table 1). At the protein level, STAT3 increased upon disease establishment, whereas STAT1 decrease was an early and persistent characteristic of B27-rat naive CD4+ T cells, occurring before disease onset. Moreover, Stat1 and STAT1-related genes mRNA expression was precociously deficient in thymic precursors of B27-rat naive CD4+ T cells, even in newborn. Furthermore, STAT1 mRNA expression was also significantly decreased in naive CD4+ T cell from SpA patients, as compared to healthy controls and rheumatoid arthritis patients (p < 0.05 and p < 0.01, respectively). Finally, in vitro stimulation of B27-rat naive CD4+ T cells with either IFNγ or a selective STAT1 transcriptional activator abolished IL-17A overexpression, confirming that STAT1 deficiency might be an upstream determinant of Th17 differentiation bias in SpA.

Conclusion: STAT1 deficiency appears a precocious signature in naive CD4+ T cells from B27-rat, preceding disease onset. Similar observation was evidenced in SpA patients. This phenomenon was associated with heightened Th17 signature that paralleled disease development in B27-rat, was determined at the epigenomic level and predicted to be orchestrated by STAT3 hyperactivity. Thus imbalance between STAT1 and STAT3 pathways appears a fundamental mechanism explaining Th17 expansion in B27-rat and SpA patients.
RNA-seq performed on mLN naive CD4+ T cells from 3 wk-old B27-rats (n = 7) and controls (n = 5), or 3-mo old B27-rats (n = 7) and controls (n = 7).
Expression heatmap showing the log2 fold-change expression of IFN/Th1 and Th17 pathways-related genes, in naive CD4+ T cells, as compared to controls, before (premorbid) of after (adult) disease onset. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

RNA-seq performed on mLN naive CD4+ T cells from 3 wk-old B27-rats (n = 7) and controls (n = 5), or 3-mo old B27-rats (n = 7) and controls (n = 7).
Upstream transcription factors (TF) predicted to explain differentially enriched genes profile in naive CD4+ T cells from B27-rats, as compared to controls; -log (adj p value) calculated by Fisher’s exact test; z-score refers to the activation (red), inhibition (blue) or undefined (white) state of the downstream regulated genes in B27-rats.
Disclosures: B. CHERQAOUI, None; F. Cremazy, None; M. Lauraine, None; G. Shammas, None; R. Said-Nahal, None; H. Mambu Mambueni, None; F. Costantino, None; M. Fourmont, None; S. Glatigny, None; L. Araujo, None; M. Breban, None.