Table_3_High Interferon Signature Leads to Increased STAT1/3/5 Phosphorylation in PBMCs From SLE Patients by Single Cell Mass Cytometry.xlsx

The establishment of an “interferon (IFN) signature” to subset SLE patients on disease severity has led to therapeutics targeting IFNα. Here, we investigate IFN signaling in SLE using multiplexed protein arrays and single cell cytometry by time of flight (CyTOF). First, the IFN signature for SLE patients (n=81) from the Stanford Lupus Registry is determined using fluidigm qPCR measuring 44 previously determined IFN-inducible transcripts. IFN-high (IFN-H) patients have increased SLE criteria and renal/CNS/immunologic involvement, and increased autoantibody reactivity against spliceosome-associated antigens. CyTOF analysis is performed on non-stimulated and stimulated (IFNα, IFNγ, IL-21) PBMCs from SLE patients (n=25) and HCs (n=9) in a panel identifying changes in phosphorylation of intracellular signaling proteins (pTOF). Another panel is utilized to detect changes in intracellular cytokine (ICTOF) production in non-stimulated and stimulated (PMA/ionomycin) PBMCs from SLE patients (n=31) and HCs (n=17). Bioinformatic analysis by MetaCyto and OMIQ reveal phenotypic changes in immune cell subsets between IFN-H and IFN-low (IFN-L) patients. Most notably, IFN-H patients exhibit increased STAT1/3/5 phosphorylation downstream of cytokine stimulation and increased phosphorylation of non-canonical STAT proteins. These results suggest that IFN signaling in SLE modulates STAT phosphorylation, potentially uncovering possible targets for future therapeutic approaches.

以“干扰素（IFN）特征”对系统性红斑狼疮（SLE）患者按疾病严重程度进行分型，已催生了靶向IFNα的治疗方案。本研究借助多重蛋白芯片与飞行时间单细胞质谱流式技术（CyTOF），探究SLE患者体内的IFN信号通路。首先，本研究通过Fluidigm qPCR技术，对斯坦福狼疮登记库中81名SLE患者（n=81）的44种已被证实的IFN诱导转录本进行检测，以此确定患者的IFN特征。IFN高表达（IFN-H）患者的SLE临床评分更高，肾脏、中枢神经系统（CNS）及免疫系统受累情况更严重，且针对剪接体相关抗原的自身抗体反应性更强。本研究针对25名SLE患者（n=25）与9名健康对照（HCs，n=9）的外周血单个核细胞（PBMCs），分别在未刺激与IFNα、IFNγ、IL-21刺激条件下，通过检测胞内信号蛋白磷酸化水平的流式面板（pTOF）开展CyTOF分析。另有一组流式面板，用于检测31名SLE患者（n=31）与17名健康对照（n=17）的PBMCs在未刺激与PMA/离子霉素（ionomycin）刺激条件下的胞内细胞因子（ICTOF）分泌变化。通过MetaCyto与OMIQ开展的生物信息学分析显示，IFN-H与IFN低表达（IFN-L）患者的免疫细胞亚群存在表型差异。最为显著的是，IFN-H患者在细胞因子刺激下的STAT1/3/5磷酸化水平升高，且非经典STAT蛋白的磷酸化水平也有所提升。上述结果表明，SLE患者体内的IFN信号通路可调控STAT磷酸化，这或为未来的治疗策略发掘潜在靶点。