747: Different Models, Different Narratives: Autoantibody Binding Divergence Across Pain Conditions

Topic: Clinical Science
Subtopic: Pain category (ICD-11)

Background and Aims: Neuropathic pain (NP), caused by damage or disease of the somatosensory system, is increasingly recognised as involving immune–neuronal interactions. Autoantibodies may contribute to NP by targeting neural structures and altering neuronal function without overt inflammation[1]. This is particularly relevant in idiopathic pain conditions, including idiopathic small fibre neuropathy (SFN), where autoantibodies have been reported[2,3], and Fibromyalgia Syndrome (FMS), in which patient IgG shows increased binding to sensory neurons compared with healthy controls (HC)[4]. This study aimed to screen patient and HC sera for IgG binding to live primary mouse DRG sensory neurons using an automated imaging pipeline, and to human induced pluripotent stem cell-derived sensory neurons (hIPSC-dSN).

Methods: Samples from FMS (n=70), SFN (n=66) cohorts were screened along with age and sex-matched HCs (n=70). Sera screening was performed using live cell-based assays involving primary dorsal root ganglion (DRG) cultures and hIPSC-dSN from both healthy individuals and patients. For primary cultures, Images were then acquired using automated microscopy, which were processed through Cellpose 2.0 for segmentation training to distinguish β3-tubulin positive sensory neurons [5], followed by a quality check of the model by blinded segmenters who generated ground truth labels. Fluorescence intensity of IgG binding to membranes of segmented cells was quantified using ImageJ software, allowing for measurements at the single-cell level to assess cellular responses. Internal positive and negative controls were used to normalise membrane binding intensity. Screening on hIPSC-dSN was assessed blindly by eye on day 40+ maturation using three control lines.

Results: Our imaging analysis on primary mouse culture revealed variable levels of binding positivity in all cohorts and no significant difference in IgG membrane binding intensity between the SFN cohort (mean= 0.1351 ± 0.02563) and HC (0.1525 ± 0.03072). However, FMS cohort (mean = 0.5265 ± 0.1372) exhibited significantly higher membrane binding intensity (p= 0.0008) compared to healthy controls, suggesting a differential role of autoantibodies in this condition. On the other hand, both the HC and SFN cohorts showed no detectable binding across all three (hIPSC-dSN) cell lines, whereas a single FMS sample demonstrated consistent positive binding in all three lines.

Conclusions: A clear divergence was observed between the two model systems. In primary mouse DRG cultures, IgG binding was detectable across all cohorts, with significantly elevated binding in the FMS cohort compared to both SFN and HC, indicating broader and more heterogeneous autoreactivity. In contrast, screening on hIPSC-dSN demonstrated a marked reduction in binding frequency, highlighting key differences between the two models. These findings underscore the importance of model selection and the need for complementary screening approaches to account for the limitations of each system, particularly in the context of autoantibody discovery.

References: 1. Xu M, Bennett DLH, Querol LA, Wu LJ, Irani SR, Watson JC, et al. Pain and the immune system: emerging concepts of IgG-mediated autoimmune pain and immunotherapies. Journal of neurology, neurosurgery, and psychiatry [Internet]. 2020;91(2). Available from: https://pubmed.ncbi.nlm.nih.gov/30224548/

2. Chan ACY, Wong HY, Chong YF, Lai PS, Teoh HL, Ng AYY, et al. Novel Autoantibodies in Idiopathic Small Fiber Neuropathy. 2022;:66–77. Available from: https://europepmc.org/article/PMC/9300200

3. Farah A, Daifallah O, Singh E, Dawes JM. Autoantibodies as drivers of neuropathic pain. Trends in molecular medicine [Internet]. 2026;32(2). Available from: https://pubmed.ncbi.nlm.nih.gov/40744832/

4. Goebel A, Krock E, Gentry C, Israel MR, Jurczak A, Urbina CM, et al. Passive transfer of fibromyalgia symptoms from patients to mice. The Journal of clinical investigation [Internet]. 2021;131(13). Available from: https://pubmed.ncbi.nlm.nih.gov/34196305/

5. Pachitariu, M. & Stringer, C. Cellpose 2.0: how to train your own model. Nature Methods 2022 19:12 19, 1634–1641 (2022).

Presenting Author: Omar Daifallah
Submitting Author: Omar Alzahrany

Senior Author: John M Dawes

IASP 2026 Global Year theme of Neuropathic Pain: Yes

• Benjamin Wastie - Nuffield Department of Clinical Neurosciences, University of Oxford
• Marva Chan: MSc - Nuffield Department of Clinical Neurosciences, University of Oxford
• Peck Miles: BSc - Nuffield Department of Clinical Neurosciences, University of Oxford
• Faisal Feroz: BSc - Nuffield Department of Clinical Neurosciences, University of Oxford
• Jishi John: NA - Nuffield Department of Clinical Neurosciences, University of Oxford
• Eoin Kelleher: MD, PhD - Nuffield Department of Clinical Neurosciences, University of Oxford
• Yuki Terajima: MD - Nuffield Department of Clinical Neurosciences, University of Oxford
• Mandy Tseng: PhD - Nuffield Department of Clinical Neurosciences, University of Oxford
• Adham Farah: PhD - Nuffield Department of Clinical Neurosciences, University of Oxford
• Victoria Tan: MSc - University of Oxford
• Andreas Themistocleous: MD - Nuffield Department of Clinical Neurosciences, University of Oxford
• Anushka Soni: MD, PhD - Nuffield Department of Clinical Neurosciences, University of Oxford
• David L Bennett: MD, PhD - Nuffield Department of Clinical Neurosciences, University of Oxford
• John M Dawes: PhD - Nuffield Department of Clinical Neurosciences, University of Oxford