Quantification of autoantibodies using a luminescent profiling method in autoimmune interstitial lung disease

Abstract

Autoantibodies are important for the diagnosis of autoimmune interstitial lung disease (ILD). Standard immunoassays have limitations, including their qualitative nature and/or a narrow dynamic range of detection, hindering the usefulness of autoantibodies as biomarkers of disease activity. Here, the luciferase immunoprecipitation system (LIPS) was evaluated for measuring myositis-specific and other lung-related autoantibodies in 25 subjects with idiopathic inflammatory myopathies (IIM), 26 with Sjögren's disease (SjD), and 10 healthy volunteers. LIPS detected a broad dynamic range of autoantibodies, to MDA5, Jo-1, PL12, KS, U1-70K, and Ro52, and matched seropositivity status with established immunoassays. Robust anti-MDA5 autoantibodies in four IIM-ILD patients had a median value of 1,134,000 LU (IQR 473,000-2,317,000), which was 500 times higher than in 21 seronegative IIM patients. Markedly elevated anti-Jo-1 autoantibodies in five IIM-ILD patients demonstrated a median value of 1,177,000 LU (IQR: 604,000-2,520,000), which was 1000-fold higher than in seronegative patients. Robust anti-Ro52 and other anti-tRNA-synthetase autoantibodies were detected in a subset of IIM-ILD subjects. In SjD, only anti-U1-70K and KS autoantibodies were identified in ILD patients with a prevalence of 30% and 20%, respectively. In longitudinal samples of five IIM-ILD patients, anti-Jo-1 autoantibody levels paralleled clinical improvement of lung function. LIPS can accurately quantify autoantibody levels as biomarkers for treatment response in patients with autoimmune ILD.

Keywords: Sjögren’s disease; idiopathic inflammatory myopathies; interstitial lung disease; myositis-associated autoantibody; myositis-specific autoantibody.

Figure 1

Autoantibodies against a panel of aminoacyl-tRNA synthetases. Autoantibody levels by LIPS were determined against six aminoacyl-tRNA synthetases including (A) Jo-1, (B) PL7, (C) PL12, (D) Zo, (E) KS, and (F) Ha. Each symbol represents a sample from subjects who were healthy volunteers (NV) or diagnosed with SjD (Sjogren’s disease) and IIM (idiopathic inflammatory myopathy). SjD and IIM subjects with interstitial lung disease are shown by the open circles. Autoantibody levels are plotted in light units on a log₁₀ scale, and the dashed lines represent the cut-off level for determining seropositive autoantibodies for each antigen, as described in the Methods.

Figure 2

Detection of autoantibodies against the N- and C-terminal protein fragments of the MDA5 (IFIH1) autoantigen. Autoantibody levels against the (A) N-terminal (MDA5-N) and (B) C-terminal half of MDA5 protein (MDA5-C) were measured by LIPS in the cohort. Each symbol represents a sample from subjects who were healthy volunteers (NV) or diagnosed with SjD (Sjogren’s disease) and IIM (idiopathic inflammatory myopathy). Autoantibody levels are plotted in light units on a log₁₀ scale, and the dashed lines represent the cut-off level for determining seropositive autoantibodies, as described in the Methods.

Figure 3

Autoantibodies against six known pulmonary-associated autoantigens by LIPS assay. Autoantibody levels against six known autoantigens including (A) Ro52, (B) Ro60, (C) TRIM38, (D) CENP-A, (E) U1-70K, and (F) IFN-omega determined by LIPS. Each symbol represents a sample from individual healthy subjects (NV) or patients with SjD and IIM. Autoantibody levels are plotted in light units on a log₁₀ scale, and the dashed lines represent the cut-off level for determining seropositive autoantibodies for each antigen as described in the Methods.

Figure 4

Heatmap analysis of autoantibodies in the IIM and SjD cases. Heatmap analysis shows the seropositivity observed in (A) IIM patients with and without ILD and (B) in the SjD cases with and without ILD. Autoantibody levels against 13 of the target proteins are shown where there is seropositivity in at least one subject. Only the results with N-terminal fragment for MDA5 are shown. Color coding denotes relative autoantibody levels in standard deviations above the baseline cut-off value. Autoantibody levels in the patients ranged from low levels (pink) to extremely high autoantibody levels (black). The clear boxes represent seronegative responses with the autoantigens in each subject. Physician global activity score (PGA) is shown for the IIM subjects on the left side of the panel.

Figure 5

Jo-1 autoantibody trajectory following rituximab immunosuppressive treatment in IIM-ILD patients. (A) Jo-1 autoantibody levels (LU) are shown over time in five IIM-ILD patients. Three of the patients (Pt 1-3) received rituximab therapy, in which the time of rituximab administration is shown by the arrow. The cut-off for Jo-1 seropositivity is shown by the dotted line. Patient #5 was seronegative for Jo-1 autoantibodies. (B) The corresponding lung function of forced vital capacity (FVC) percent changes (C) Diffusion capacity of the lungs for carbon monoxide (DLCO) changes over time (D) Creatine kinase (CK) levels and (E) Physician global activity (PGA) score for each of the serial samples from the patients shown in panel (A).