Mechanisms of disease perpetuation and pathogenesis in autoantibody-mediated epilepsies with a focus on LGI1- and CASPR2-related syndromes
Keywords: Encephalitis
Autoimmune Encephalitis
LGI1-autoantibodies
Bone marrow-derived long-lived plasma cells
Germinal centre reactions
CASPR2-autoantibodies
Autoimmune Neurology
Neuroimmunology

Background and Aims: This thesis explores the clinical and immunological aspects of LGI1- and CASPR2-autoantibody syndromes – rare, immunotherapy-responsive conditions mediated by autoantibodies against neuronal cell surface antigens. Two paradigms of autoantibody production exist: ongoing germinal centre (GC) reactions, and long-lived plasma cells (LLPCs). We aimed to expand the clinical phenotypes, and explore the underlying immunobiology using patient biological samples. We aimed to examine the concept of GC reactions through study of serum IgM antibodies, CXCL13 levels, and patient serum binding patterns to the two major LGI1 domains (LRR and EPTP), and to directly investigate the potential role of the bone marrow (BM) compartment by asking whether BM-derived antigen-specific LLPCs contribute to LGI1-autoantibody production.
Methodology: Detailed clinical phenotyping was undertaken for 107 LGI1-autoantibody patients. The CASPR2 IgM live cell-based assay (CBA) was developed, optimised, and tested in 120 sera including n=51 from 25 patients with CASPR2-IgG. LGI1 IgMs were tested by live CBA in 120 IgG-depleted sera (n=70 from patients with LGI1-IgG). End-titrations against full-length LGI1, and LRR and EPTP were quantified by live CBA for 102 sera and 11 CSF samples from 38 LGI1-autoantibody patients. A CXCL13 assay was developed and serum CXCL13 levels were determined in 543 samples from 398 subjects, including 161 sera from 52 LGI1 autoantibody patients. LLPCs, defined by flow cytometry as live, CD3-CD14-CD20-CD19-/lowCD138+ cells, were purified from fresh BM aspirate from n=2 patients with LGI1-autoantibody encephalitis. BM LLPC oligocultures were maintained in optimized culture conditions (IL-6, CXCL12, APRIL, and BAFF). After 7 days, culture supernatants were tested for LGI1-IgG by live CBA.
Results: Potential triggers including prodromal infection and vaccination were identified in ~20% of patients, at a median time of 3.5 weeks prior to symptom onset. 11 distinct subtypes of seizures were identified, and patients manifested up to 5 seizure semiologies. When compared to a pharmacoresistant epilepsy cohort (n=218), the proportion of patients with ≥ 3 seizure types was higher in the LGI1-autoantibody patient group (p=0.0039**). Relapses were common, (39/107; 36%), and 36% of these had multiple relapses. The most common precipitant of relapse was steroid weaning or cessation (80%). FBDS significantly associated with relapse, present in 77% relapsing versus 46% non-relapsing patients (p=0.002**). Novel clinical features including hoarding and obsessive-compulsive behaviours were found in 35%, and unusual manual stereotypies which we have termed ‘complex motor behaviours’ in 5%, which may represent either a novel seizure semiology or the sleep disorder agrypnia excitata.
IgMs were uncommon, detected in 16% of CASPR2- and 17% of LGI1-autoantibody patients. In LGI1-antibody patients, 7/8 (88%) positive samples were from relapsing patients, suggesting that IgM may be a marker of relapse. Almost all (97%) patients had serum autoantibodies to both LGI1 domains, and within individual samples EPTP and LRR end-titres showed strong correlations (Spearman r = 0.9257, p<0.0001****). Patients showed constant EPTP:LRR ratios across disease course, suggesting ongoing production of autoantibodies to both LGI1 domains, representing putative ongoing GC activity. LGI1-antibody patients had the highest serum CXCL13 levels (mean 386 pg/ml; LGI1 versus pharmacoresistant epilepsy, seizure-free epilepsy, and HCs p<0.0001****; LGI1 versus CASPR2 p=0.0015**). Serum CXCL13 levels were higher in LGI1-antibody patients with relapses compared to non-relapsing patients (p<0.0001****). LGI1-IgG autoantibodies were detected in the BM LLPC culture supernatant from one of the patients at a frequency of 1:4920 of total cultured BM LLPCs.
Conclusions: Herein, the clinical features of the largest UK cohort of patients with LGI1-autoantibodies are described, expanding the current clinical phenotypes with seizure and relapse characteristics, and novel descriptions including hoarding and ‘complex motor behaviours’. We show that whilst IgMs are rare, LGI1-IgM may be a marker of relapse, and other GC activity markers including CXCL13 may be useful biomarkers in LGI1-autoantibody patients. We demonstrate the presence of LGI1-specific BM-derived LLPCs in a patient with autoimmune encephalitis by direct sampling of their BM. These proof-of-concept data demonstrate that the BM is a site for long-term autoantibody production and suggest a role for BM-derived LLPCs in autoimmune encephalitis. Collectively, these results suggest that both GC reactions and LLPC-driven mechanisms of autoantibody production appear to operate synergistically in patients with LGI1 autoantibodies. These paradigms may be applied to other autoantibody-mediated conditions. Overall, these findings expand the clinical phenotypes and provide novel insights into disease immunobiology which may inform use of more targeted immunotherapies to improve clinical outcomes in these patients.