|Other Abstract||Anti-N-methyl-D-aspartate receptor encephalitis (anti-NMDARE) is mainly mediated by antibodies and B cell receptor (BCR) specifically binding to the NR1 subunit of NMDA receptor in the central nervous system, these antibodies cause neurological damage to the central nervous system by affecting the NMDA receptors. Therefore, the antibody or BCR specifically targeting NR1 antigen in brain is one of the key objects in the molecular mechanism study of this disease. Immune repertoire (IR) is an important method to study the characteristics and pathogenesis of B-cellmediated autoimmune diseases by obtaining BCR or antibody gene sequences through sequencing. At present, there are only three reports of NR1-positive BCR from foreign patients with anti-NMDARE. They only found a small number of NR1-positive BCR/antibody genes and their methods were less efficient, so it is urgent to find a more efficient method for the study of NR1-positive B cell IR. In addition, their study did not provide additional clues about the differences between patients with anti-NMDARE and healthy people from the perspective of IR. On the other hand, epidemiology and histological results have shown that teratoma is one of the triggers of this encephalitis, but there is no further molecular biology evidence of the correlation between teratoma and anti-NMDARE. Comparing B cell IR characteristics of patients with teratoma and patients with anti-NMDARE can explore the relationship between them at the level of gene. Therefore, we carried out the following three aspects of research to explore the characteristics of these diseases from the perspective of B cell IR.
First of all, we established a NR1-positive BCR gene capture method and NR1-positive BCR gene data set. We selected B cells in cerebral spinal fluid (CSF) that could bind to NR1 antigen by flow cytometry, and then we captured their BCR genes by single cell IR amplification and sequencing. Finally, we got gene sequences of more than 100 NR1-positive B cells from 12 patients with anti-NMDARE, and after sequence alignment, we finally obtained 44 kinds of heavy chains and 38 kinds of light chains. Compared with the three published literature mentioned above, the NR1-positive BCR gene capture method we adopted is more efficient, and we also found more NR1-positive BCR sequences.
Secondly, we explored the overall characteristics of patients' NR1-positive BCR gene data set by bioinformatic methods. We found that there were common clones among patients with anti-NMDARE, and these NR1-positive BCR showed obvious V/J gene usage preference. In addition, the specificity of the obtained NR1-positive BCR gene data set was verified by using BCR data of other immune-mediated disorders of the central system and healthy people from the public databases, which laid a foundation for the data set to be used as potential markers of anti-NMDARE.
Thirdly, we analyzed the B cell IR characteristics in the peripheral blood of the other 7 patients with anti-NMDARE, 4 patients with teratoma (without anti-NMDARE) and 4 healthy people using traditional immune repertoire high-throughput sequencing and bioinformatic methods. We found that the diversity of B cell IR in peripheral blood of these two disease groups were lower than that of the healthy group; there were significant differences in the preference of V/J gene usage, mutation rate and so on among these three groups; some patients with anti-NMDARE and some patients with teratoma had relative high similarity in B cell IR. Combined with the results of flow cytometry that NR1-positive B cells were found in both the teratoma group and the encephalitis group, we deduced that the similarity of B cell IR between these two groups may be related to NR1 antigen. Our study not only reflected the differences in peripheral blood B cell IR between patients with anti-NMDARE and healthy people, but also provided clues for further study on teratoma as a trigger of this encephalitis.
Based on the results of the first study and second study in this paper, we reached the following conclusions: different patients with anti-NMDARE had the same epitope, and there was obvious oligonoclonal hyperplasia in their brain; through the results of the third study, we reached the following conclusions: there were differences between healthy people and patients with anti-NMDARE in the characteristics of peripheral blood B cell immune repertoire; patients only with teratoma whose anti-NMDAR antibodies test were negative in serum can have exposed NR1 antigen in their bodies.|