Background/Purpose: Myositis-specific autoantibodies (MSAs) play a crucial role in diagnosing and classifying idiopathic inflammatory myopathies (IIMs). However, approximately 30% of seronegative IIM patients may be unrecognized or misdiagnosed due to the absence of reliable biomarkers. This study aims to identify potential autoantibodies in seronegative IIM.

Methods: We enrolled 42 seronegative IIM patients, 149 MSA-positive IIM cases, 83 healthy controls (HCs), and 83 autoimmune disease controls (DCs). Initial high-throughput autoantibody screening was performed using the HuProtTM human proteome microarray in a discovery cohort. Subsequently, candidate antigens were expressed, purified, and validated via western blotting in an expanded cohort. An autoantigen-induced murine model was then utilized to explore immunological properties.

Results: Through systematic screening and validation, anti-hepatoma-derived growth factor-like protein 1(HDGFL1) was identified as a novel autoantibody, detected in 33% (14/42) of seronegative IIM patients but absent in HCs (0/83, P< 0.0001). Within the seronegative IIM group, anti-HDGFL1 antibody-positive patients demonstrated significantly higher frequencies of rash (71.4% vs 25.0%, P=0.004) and arthritis (28.6% vs 3.6%, P=0.035) compared to anti-HDGFL1 antibody-negative patients. HDGFL1-immunized mice developed specific autoantibodies, alongside elevated levels of serum creatine kinase and aspartate aminotransferase compared to the control group. Histopathological examination revealed pronounced inflammatory cell infiltration in skeletal muscle, characterized by increased CD3+ T cells and CD68+ macrophages. Flow cytometric analysis indicated enhanced splenic T cell proliferation in the HDGFL1-immunized group compared to controls.

Conclusion: Anti-HDGFL1 autoantibody was identified as a novel biomarker for the diagnosis of seronegative IIM, offering new insights into immunopathogenesis.

Figure 1 Discovery of HDGFL1 as an autoantigen candidate. (A) Comparative signal intensity profiles of top 50 candidate autoantigens between MSA-negative patients and healthy controls (HC) in the discovery phase. Proteins are ranked by descending fold change (FC) relative to HC. Gene symbols used to refer to protein products. (B) Representative fluorescence images of HDGFL1 autoantigens identified from the sera of MSA-negative IIM and HC on HuProt protein microarrays.

Figure 2 Immune response to HDGFL1. (A) Schematic representation of the immunization protocol. C57BL/6 mice received intradermal injections of complete Freund’s adjuvant emulsions with or without HDGFL1 protein at the dorsal region, followed by booster immunization at day 14 and final evaluation at day 28. (B) Serum levels of HDGFL1-specific autoantibodies detected by ELISA in immunized vs adjuvant-only control groups. (C-D) Comparative serum analysis of muscle damage biomarkers: creatine kinase (CK, C) and aspartate aminotransferase (AST, D) levels between experimental groups. (E) Representative histopathological features (H&E staining, upper panel a-b) and immune cell infiltration patterns (IHC analysis, lower panel c-f) in muscle tissues (100× magnification). (F-G) Quantitative comparison of CD68⁺ macrophage infiltration (F) and CD3⁺ T lymphocyte infiltration (G) in muscle tissues between groups. Data presented as percentage of positive staining area.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-anti-hdgfl1-as-a-novel-autoantibody-in-seronegative-idiopathic-inflammatory-myopathies/