Activation and clonal expansion of CD8+T cells in patients with anti-NMDAR encephalitis revealed by single-cell RNA sequencing analysis

Background Anti-N-methyl-D-aspartate receptor encephalitis (NMDAR-E) is a rare and severe form of antibody-mediated autoimmune encephalitis. While the roles of B cells and NMDAR antibodies in NMDAR-E have been extensively studied, the contribution of T cell subsets to the development of the disease remains unclear. Methods We utilized single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) to examine a comprehensive collection of 45,088 individual immune cells sourced from both NMDAR-E patients and control subjects. Besides, we incorporated 14,536 immune cells obtained from a publicly available database. Additionally, cytometry analysis was conducted on samples from 60 NMDAR-E patients (with 89 samples) and 44 individuals in the control group. To investigate the influence of CD8+T cells on B cells, we performed in vitro co-culture experiments. Results We observed the activation of effector memory CD8+T cells in the CSF and peripheral blood (PB) of NMDAR-E patients, accompanied by an expansion of TCR clones. These clonal CD8+T cells exhibited upregulated gene expression associated with cytotoxicity and cell trafficking. Furthermore, we detected elevated levels of KIR+CD8+T cells in the CSF and PB of NMDAR-E patients. Cell communication analysis revealed that these inhibitory KIR+CD8+T cells could interact with B cells through MHC-I molecules. Surprisingly, CD8+T cells isolated from the PB of NMDAR-E patients induced autologous B cell death, resulting in a higher rate of B cell apoptosis compared to the control group. Conclusion Activated CD8+T cells with TCR clones in NMDAR-E patients may contribute to the pathogenesis of NMDAR-E. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by grants from National Science Foundation of China (Grant No. 81571272 and 82071459); The 1.3.5 project for disciplines of excellence and Brain Science project of West China Hospital, Sichuan University (Grant No: ZYGD20011 and ZYJC21001). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The research conducted in this study received ethical approval from the Institutional Review Board (IRB) of West China Hospital, under the protocol number ChiCTR1900027074. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The raw sequence data reported in this article are currently being uploaded to the Genome Sequence Archive (GSA: PRJCA014916) at the National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences. Once the upload is complete, these data will be publicly accessible at . Currently, interested parties can request access to the relevant data by contacting the corresponding author. The data sets supporting the conclusions of this article are included within the article and its accompanying files. ### Abbreviations NMDAR : Anti-N-methyl-D-aspartate receptor CBA : cell-based assay CNS : central nervous system CSF : cerebrospinal fluid PBMCs : Peripheral blood mononuclear cells PB : peripheral blood scTCR-Seq : single-cell TCR sequencing scRNA-seq : Single cell RNA sequencing Temra : CD45RA+ effector memory T cells Tem : effector memory T Tcm : central memory T Tn : naïve T HC : Healthy control IIH : Idiopathic intracranial hypertension Bm : memory B cells Bn : naïve B cells ASCs : Antibody-secreting cells DN : CD27-IgD-double negative B cells t-SNE : t-distributed neighbor embedding MHC : Major histocompatibility complex DEGs : Differentially expressed genes KIRs : killer-cell immunoglobulin-like receptors KEGG : Kyoto Encyclopedia of Genes and Genomes pathway * ### Abbreviations NMDAR : Anti-N-methyl-D-aspartate receptor CBA : cell-based assay CNS : central nervous system CSF : cerebrospinal fluid PBMCs : Peripheral blood mononuclear cells PB : peripheral blood scTCR-Seq : single-cell TCR sequencing scRNA-seq : Single cell RNA sequencing Temra : CD45RA+ effector memory T cells Tem : effector memory T Tcm : central memory T Tn : naïve T HC : Healthy control IIH : Idiopathic intracranial hypertension Bm : memory B cells Bn : naïve B cells ASCs : Antibody-secreting cells DN : CD27-IgD-double negative B cells t-SNE : t-distributed neighbor embedding MHC : Major histocompatibility complex DEGs : Differentially expressed genes KIRs : killer-cell immunoglobulin-like receptors KEGG : Kyoto Encyclopedia of Genes and Genomes pathway