Autoimmune encephalitis (AE) is an uncommon neurological disorder mediated by autoimmune mechanisms and is an important cause of rapidly progressive cognitive dysfunction, refractory epilepsy, and psychiatric abnormalities (1, 2). While most patients have been shown to respond well to immunotherapy, some patients continue to have intractable seizures and varying degrees of cognitive impairment, which have a serious effect on their quality of life (3, 4).

With the development of detection technology, an increasing number of AE antibodies have been reported (5–7). AE-associated antibodies can be divided into two categories: antibodies against cell-surface-targeting antigens and antibodies against intracellular-targeting antigens. The cell surface-targeted antigen antibodies comprise primarily the anti-N-methyl-D-aspartate receptor (NMDAR) antibodies, anti-g-aminobutyric acid B receptor (GABABR) antibodies, anti-glioma inactivated 1 protein (LGI1) antibodies, anti-contactin-associated protein-like 2 (CASPR2) antibodies, and anti-AMPAR antibodies. Intracellular antibodies primarily include the anti-Hu, Yo, Ri, Ma2, CV2/CRMP5, amphiphysin, and glutamic acid decarboxylase (GAD) antibodies. The clinical manifestations and prognoses of the various AE subtypes differ (6, 8). Given of the diversity of the antibody subtypes and the clinical manifestations of AE as well as the disorder’s insidious onset in some patients (e.g., minor cognitive dysfunction), early diagnosis and treatment of AE remain difficult. Therefore, an in-depth study of the clinical features, treatment, and prognosis of the different antibody subtypes associated with AE is of great significance.

In recent years, investigators have shown the presence of co-existent neuronal antibodies in patients with AE (9–11), and only a few case series or scattered cases of antibody overlap have been reported in the literature, with some patients having an extremely poor prognosis. However, the mechanisms by which antibody overlap exists and its clinical significance are unclear. To improve our understanding of the clinical significance, treatment, and prognosis of AE antibody superposition, we conducted a retrospective analysis of the clinical characteristics, treatment, and prognostic details of 22 patients with AE who were treated at multiple clinical centers in China.

We described the clinical characteristics, treatment, and prognosis of 22 patients with coexisting neuronal antibodies, and observed that the clinical symptoms and laboratory findings of patients with coexisting antibodies were more diverse and atypical at onset compared with single-antibody positive AE, leading to increased difficulty in early diagnosis and treatment. Our study provided new evidence for the diagnosis and prognosis of multiple antibody-positive patients with AE. To our knowledge, this was the first case series report on multiple-antibody coexistence in patients with AE from multiple clinical centers in the East of China.

The coexistence of multiple antibodies in AE is uncommon. Currently, studies in China and abroad are predominantly small-sample case series and single-case reports (10, 12–14). In 2018, Fan et al., in a study comprising 42 patients with MOG-Ab disease and 491 patients with neuromyelitis optica spectrum disorder (NMOSD), reported that 11.9% of patients with MOG-Ab disease and 0.6% of patients with NMOSD had overlapping NMDAR-Ab (10). A study involving 189 patients with AE in Southwest China in 2019 showed that 8 (4.23%) patients had two co-existing neuronal antibodies, of whom 50% (4/8) of patients with anti-NMDAR antibody-positivity were observed to have co-existing antibodies of other types (11). In our cohort, there were only 22 (7.97%) patients with multiple antibodies, of whom 63.63% (14/22) of patients with anti-NMDAR antibody-positivity were observed as having other types of co-existing antibodies.

Our results were similar to those of previous reports. In addition to anti-NMDAR encephalitis, anti-GABABR encephalitis was more often associated with other anti-neuronal antibodies. In a study of 20 cases of anti-GABABR encephalitis by Höftberger et al. in 2013 (15), seven cases (35%) had additional antibodies, including three cases with SOX1, two amphiphysin, one GAD65, one NMDAR, and one Ri (ANNA2). In our study, 75% (3/4) of anti-GABABR antibody-positive patients had coexisting paraneoplastic antibodies such as Hu and Yo. In general, the positive phenomenon of multiple anti-neuronal antibodies in AE is rare, and it also suggests that multiple antibodies may coexist in the presence of NMDAR and GABABR antibody-related AE.

The presence of coexisting anti-neuronal antibodies may lead to diversification of the clinical characteristics of AE and the presence of atypical clinical and radiographic features (10, 16–18). For example, anti-NMDAR encephalitis may co-exist with a demyelinating disease in the presence of anti-MOG or anti-GFAP antibodies. In our study, patients with anti-AMPAR encephalitis were noted to also have stiff-person syndrome (anti-GAD) or sensory neuropathy (anti-amphiphysin). As reported by Höftberger in 2013, in a study of anti-GBABR encephalitis (15), patients with coexisting additional NMDAR antibodies showed prominent psychiatric symptoms, whereas patients with coexisting GAD65 antibodies developed refractory seizures. In our study, seizures, memory deficit, and mental behavioral disorders were more common in the single-antibody cohort at onset, whereas the clinical presentation of the multi-antibody cohort was more diverse. For example, Patient 14—who had four antibodies (NMDAR, AMPA1, AMPA2, and Ma2)—experienced vertigo, sleep disturbances, memory impairment, seizures, and absence of sweating on the left side of the neck and face; these symptoms were diverse and atypical. Patient 13 with NMDAR-Ab and MOG-Ab predominantly showed limb weakness and visual impairment and had multiple demyelinating lesions in the white matter of the left cerebral hemisphere and the optic nerve. Therefore, the potential for the coexistence of non-specific clinical symptoms associated with each anti-neural antibody, also suggests the importance and necessity of antibody testing, while it should be noted that whether there is a certain correlation between antibody overlap and phenotype overlap still needs more studies to clarify.

The clinical symptoms and laboratory findings of multiple antibody-associated AE may overlap with those of other autoimmune diseases and are more diverse and atypical than those of a single antibody-positive AE (4, 19–21). The results of tests for autoimmune antibodies are not readily available at some institutions and may take days or even weeks to become available. This makes the early diagnosis and treatment of multiple AE more difficult, particularly in some AE patients who are negative for anti-neuronal antibodies and whose initiation of immunotherapy is more dependent on clinical manifestations and ancillary findings during the course of the disease. Therefore, new biomarkers should be explored to assist in early diagnosis (22–24).

AE imaging has also received considerable attention in recent years. MRI findings in the brain have been more commonly reported in previous studies. The brain MRI in patients with AE involves most commonly the temporal lobe or hippocampus unilaterally or bilaterally and may sometimes accumulate in the thalamus, occipital lobe, and brainstem with or without enhancement. Previously, an ¹⁸F-FDG PET/CT scan was primarily used to assess whether the etiology of encephalitis was an occult malignancy (18, 25, 26). However, recent studies have begun to note the positive role of cerebral ¹⁸F-FDG PET/CT scans in the evaluation of patients with AE (27, 28). A retrospective study in 2017 assessed the positivity rate of MRI versus ¹⁸F-FDG PET/CT scans, which included 23 patients who were proven to have antibody-positive anti-NMDAR encephalitis, showed that a PET/CT scan more often showed abnormal findings compared with a brain MRI scan (16). In our study, the predominant finding from the brain ¹⁸F-FDG PET/CT imaging was lobar hypometabolism, with hypometabolism being observed most commonly in the parietal and occipital lobes. Another study involving 24 patients with anti-NMDAR encephalitis in the acute phase in 2021 showed the characteristics of patients with anti-NMDAR encephalitis of different etiologies (18). Those findings showed that patients in the cryptogenic and paraneoplastic subgroups exhibited hypermetabolism in the frontotemporal and basal ganglia and hypometabolism in the occipital lobe. Other patients, secondary to viral encephalitis, exhibited significantly lower metabolism in the bilateral occipital and temporal lobes on the side of viral encephalitis and part of the basal ganglia. This suggests that the brain ¹⁸F-FDG PET/CT scan may be more likely to be abnormal than the MRI scan of the brain and that patients with AE triggered by different factors present distinct cerebral metabolic patterns. In the preliminary exploratory stage, cumulative evidence supported the value of the characteristic presentation of ¹⁸F-FDG PET/CT scanning as an early diagnostic indicator of AE.

Patients with combined multiple anti-neuronal antibody autoimmunity, regardless of the clinical manifestations, have a more complex and diverse disease than those with AE with a single antibody; however, they are still treated primarily with immunotherapy (7, 29, 30). First-line immunotherapy included steroids, intravenous immunoglobulin, and plasma exchange, and second-line immunotherapy included options such as rituximab, mycophenolate mofetil, and cyclophosphamide. Early tumor resection or other antitumor therapy should be performed in the presence of a combined tumor (4, 30–32). The co-existence of different antibody stacks leads to differences in immunotherapy response and long-term prognosis. AE patients with multiple anti-neuronal antibodies, especially the paraneoplastic antibodies (e.g., Hu, Yo, and Ri), often have other tumors and have a poor response to immunotherapy and a poor prognosis. In a study of 17 cases of anti-GABABR encephalitis, 8 patients died in the last follow-up; seven of these eight patients died as a result of tumor progression or complications of chemotherapy (31). Among them, five patients had additional antibodies: three SOX1, one Amphiphysin, and one Ri (ANNA2). The only patient without a tumor had refractory status epilepticus, in association with antibodies to GAD65, and was the cause of death. They also observed that patients with small-cell lung cancer had a shorter survival time than patients without tumors.

In our study, 20 patients were treated with steroids and intravenous immunoglobulin, and most patients showed an improvement after first-line immunotherapy. Three patients died of tumor progression or complications of chemotherapy, and all of them had small-cell lung cancer. It is important to note that 22.73% of patients had tumors in the multi-antibody cohort, while 9.09% of patients with single antibody positive had concomitant tumors. Furthermore, in the multi-antibody cohort, four (100%) patients with GABABR antibody (+) had tumors (small-cell lung cancer being more common), whereas 36.36% of patients with anti-GABABR encephalitis had tumors in the single-antibody positive group. This suggests that when these AE-associated antibodies coexist in superposition, they are highly suggestive of tumors. Given the poor prognosis of AE combined with tumors, especially with small-cell lung cancer, early tumor screening is of great significance. Particularly in AE patients with combined GABABR and paraneoplastic antibodies, regular tumor screening and long-term follow-up are necessary if the tumor is not detected early in the course of the disease.

This study has certain limitations. Although a more varied demographic background is implied by this being a multicenter study, to some extent this study reflects the characteristics of patients with multi-antibody AE, specifically in eastern China; the geographical limitations of the regional design limit the variability of the demographic characteristics, thus limiting our ability to generalize the findings. The current study is retrospective and has limitations in terms of completeness of patient data and monitoring of dynamic changes in the disease. In addition, another limitation of this study is that most patients did not retest autoimmune encephalitis related antibodies during and after treatment, so the possibility of false positives for some antibodies cannot be excluded. On the one hand, we need to objectively assess the clinical phenomenon and significance of the coexistence of multiple antibodies, on the other hand, dynamic detection of antibodies is required to exclude false positives. Thus, large-sample, multi-center, prospective, and longitudinal studies are needed.

The coexistence of multiple anti-neuronal antibodies is rare, and the superposition of antibody types may lead to superimposed or atypical clinical features, making early diagnosis difficult. Antibody superimpositions respond differently to immunotherapy, and differences in long-term prognosis exist. The coexistence of multiple anti-neuronal antibodies, especially when combined with paraneoplastic antibodies, suggests the possibility of a combined potential tumor range. The clinical significance and mechanism of the coexistence of multiple anti-neuronal antibodies still need to be further studied.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by the Ethics Committee of Qilu Hospital, Cheeloo College of Medicine, Shandong University. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

X-WL conceived the study and supervised this work. SQ conceived the study, organized and statistical data, and drafted the manuscript. S-CZ collected and organized datas. Z-HW, LW, R-RZ, H-YL, YJ, L-LL, M-LW, and A-HW assisted in collecting datas. All authors reviewed and approved the final manuscript. All authors contributed to the article and approved the submitted version.

The National Natural Science Foundation (No. 81873786) supported this work.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

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