1070 Hagberg N, et al. Ann Rheum Dis 2018;77:1070–1077. doi:10.1136/annrheumdis-2017-212794 Basic and translational research EXTENDED REPORT The STAT4 SLE risk allele rs7574865[T] is associated with increased IL-12-induced IFN-γ production in T cells from patients with SLE Niklas Hagberg, 1 Martin Joelsson, 1 Dag Leonard, 1 Sarah Reid, 1 Maija-Leena Eloranta, 1 John Mo, 2 Magnus K Nilsson, 2 Ann-Christine Syvänen, 3 Yenan T Bryceson, 4,5 Lars Rönnblom 1 ABSTRACT Objectives Genetic variants in the transcription factor STAT4 are associated with increased susceptibility to systemic lupus erythematosus (SLE) and a more severe disease phenotype. This study aimed to clarify how the SLE-associated intronic STAT4 risk allele rs7574865[T] affects the function of immune cells in SLE. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 52 genotyped patients with SLE. Phosphorylation of STAT4 (pSTAT4) and STAT1 (pSTAT1) in response to interferon (IFN)-α, IFN-γ or interleukin (IL)-12, total levels of STAT4, STAT1 and T-bet, and frequency of IFN-γ + cells on IL-12 stimulation were determined by fow cytometry in subsets of immune cells before and after preactivation of cells with phytohaemagglutinin (PHA) and IL-2. Cellular responses and phenotypes were correlated to STAT4 risk allele carriership. Janus kinase inhibitors (JAKi) selective for TYK2 (TYK2i) or JAK2 (JAK2i) were evaluated for inhibition of IL-12 or IFN-γ-induced activation of SLE PBMCs. Results In resting PBMCs, the STAT4 risk allele was neither associated with total levels of STAT4 or STAT1, nor cytokine-induced pSTAT4 or pSTAT1. Following PHA/IL-2 activation, CD8 + T cells from STAT4 risk allele carriers displayed increased levels of STAT4 resulting in increased pSTAT4 in response to IL-12 and IFN-α, and an augmented IL-12-induced IFN-γ production in CD8 + and CD4 + T cells. The TYK2i and the JAK2i effciently blocked IL-12 and IFN-γ-induced activation of PBMCs from STAT4 risk patients, respectively. Conclusions T cells from patients with SLE carrying the STAT4 risk allele rs7574865[T] display an augmented response to IL-12 and IFN-α. This subset of patients may beneft from JAKi treatment. Systemic lupus erythematosus (SLE) is an auto- immune rheumatic disease with a strong genetic component. 1 Genetic association studies have identified >80 loci associated with an increased susceptibility to SLE. 2 One of the strongest SLE risk loci outside the HLA region is signal trans- ducer and activator of transcription (STAT)4, with the most significantly associated single-nucleotide polymorphisms (SNPs) in STAT4 being located in the third intron of the gene. 3–5 However, there is a large linkage disequilibrium (LD) block extending towards the 3′ end of the gene and the causal SNP has not been inferred. 6 Since STAT1 is located adjacent to STAT4, it is possible that the risk SNPs found in STAT4 have a regulatory effect on STAT1. Both STAT4 and STAT1 encode transcription factors of importance to the immune system. STAT4, mainly expressed in T cells and NK cells, is required for the inflammatory response following IL-12 receptor (IL-12R) stimulation. 7–9 STAT4 is also involved in the non-canonical signalling pathway of the type I interferon (IFN) receptor (IFNAR). 10 11 On IL-12R engagement, STAT4 is phosphorylated by JAK2 and TYK2, leading to dimerisation and translocation to the nucleus, where it binds DNA and induces the expression of a large number of proinflammatory genes, including IFNG. 12 STAT1 is widely expressed and part of the canonical signalling pathways for the type I IFN receptor (IFNAR) and the IFN-γ receptor (IFNGR). 13 Increased expression and activation of STAT1 has been reported in patients with lupus, which support the important role of the IFN system in SLE. 14 The SLE-associated STAT4 SNPs are linked to a more severe disease phenotype with an earlier onset of disease, and an increased risk for stroke and nephritis with severe renal insufficiency. 4 15–17 Despite the strong association of STAT4 risk SNPs with rheumatic diseases and clinical subphenotypes, very little is known about the molecular mecha- nisms whereby these risk gene variants contribute to autoimmune disease. Previous studies have reported increased expression of STAT4 mRNA in osteoblasts 4 and peripheral blood mononuclear cells (PBMCs) from healthy individuals 18 and patients with SLE 19 carrying the STAT4 risk allele. Furthermore, patients with SLE carrying the STAT4 risk allele have an increased expression of IFN-induced genes in peripheral blood cells despite having a decreased IFN-α activity in sera. 20 In the present study, we set out to define how the STAT4 risk variant affects cellular functions in different types of immune cells in SLE. Using primary cells from genotyped patients with SLE, we examined STAT4-dependent and STAT1-dependent cellular functions on the single- cell level in unstimulated and in vitro stimulated cells and correlated these to rs7574865, which is one of the strongest SLE-associated SNPs in STAT4. Finally, Janus kinase (JAK) inhibitors were evaluated in vitro for their capacity to restore augmented cellular responses in PBMCs from patients with SLE carrying the STAT4 risk allele. To cite: Hagberg N, Joelsson M, Leonard D, et al. Ann Rheum Dis 2018;77:1070–1077. Handling editor Josef S Smolen ► Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ annrheumdis-2017-212794). 1 Department of Medical Sciences, Rheumatology and Science for Life Laboratories, Uppsala University, Uppsala, Sweden 2 Respiratory, Infammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden 3 Department of Medical Sciences, Molecular Medicine and Science for Life Laboratories, Uppsala University, Uppsala, Sweden 4 Department of Medicine, Center for Hematology and Regenerative Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 5 Department of Clinical Sciences, Broegelmann Research Laboratory, University of Bergen, Bergen, Norway Correspondence to Dr Niklas Hagberg, Department of Medical Sciences, Section of Rheumatology, Uppsala University, Rudbeck laboratory, Uppsala S-75185, Sweden; niklas.hagberg@medsci.uu.se Received 4 December 2017 Revised 30 January 2018 Accepted 3 February 2018 Published Online First 23 February 2018 on June 18, 2020 by guest. 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