Activated αβ T and reduced mucosa-associated invariant T cells in LGI1- and CASPR2-encephalitis

Abstract

Anti-leucine-rich glioma inactivated-1 (LGI1) and anti-contactin-associated-protein-2 (CASPR2) autoimmune encephalitis (AIE) are common and characterized by pathogenic antibodies targeting neuronal autoantigens. However, the drivers of the antibody-secreting cells and involvement of T cells remain unresolved. We performed single-cell RNA sequencing of fresh CSF and parallel blood samples of 15 patients with LGI1-AIE (n = 9) and CASPR2-AIE (n = 6) compared with control patients [multiple sclerosis (n = 15) and idiopathic intracranial hypertension (n = 18)]. We validated our observations in independent cohorts using flow cytometry of CSF and blood. We confirmed autoantibody specificity using recombinant human monoclonal antibodies. In comparison to idiopathic intracranial hypertension and multiple sclerosis controls, we observed clonal CSF-specific antibody-secreting cell expansion in LGI1/CASPR2-AIE despite mostly normal CSF findings. Antibody-secreting cells were dominantly plasmablasts and transcribed IgG4 and IgG1/2 heavy chains. Expanded clones showed signs of affinity maturation and bound the respective neuronal autoantigen. Within CD4 and CD8 T-cell clusters, CD4 and CD8 central memory T cells were activated, clonally restricted and expanded. T-cell clones were often shared between CSF and blood. We also observed a shift of natural killer cells and loss of mucosa-associated invariant T (MAIT) cells in the CSF of LGI1-AIE and the blood of LGI1- and CASPR2-AIE compared with idiopathic intracranial hypertension and multiple sclerosis controls. MAIT-like T cells were detected in autopsied brains of LGI1- and CASPR2-AIE patients, and mice lacking MAIT cells displayed an increased antibody seroconversion and higher titres following active LGI1/CASPR2 immunization. Our data: (i) confirm the intrathecal antigen-specific plasma cell expansion in LGI1- and CASPR2-AIE in a large cohort of untreated AIE patients; (ii) suggest that activated and expanded central memory CD4 and CD8 T cells in the CSF participate in disease pathogenesis; and (iii) implicate invariant T-cell receptor-expressing lymphocytes in the brain, CSF and blood in disease pathogenesis.

Keywords: autoimmune encephalitis; contactin-associated protein 2; flow cytometry; leucin-rich glioma inactivated 1; mucosa-associated invariant T cells; single-cell transcriptomics.

Figure 1

Single-cell transcriptomics and flow cytometry identified expansion of plasma cells as a hallmark of LGI1-/CASPR2-AIE. (A) Sankey diagram showing overlap between LGI1-AIE, CASPR2-AIE, non-inflammatory-disease controls (IIH in cohort 1, functional disorder in cohort 2 and healthy controls in cohort 3) across the four sample cohorts. IHC = immunohistochemistry analysis of formalin-fixed, paraffin-embedded (FFPE) autopsy brain tissue. (B) Uniform manifold approximation and projection (UMAP) plot depicting the cell-type clusters of CSF cells. The second cell cluster included only PBMCs and is therefore shown only in Supplementary Fig. 1D. (C–F) Comparison of the relative cell-type abundance between LGI1 and IIH (C), CASPR2 and IIH (D), LGI1 and MS (E) and CASPR2 and MS (F). (G) Flow cytometry validation: relative percentage of plasma cells (%CD3⁻CD19⁺CD138⁺) quantified as percentages of all lymphocytes in CSF cells of the second cohort. Statistical significance was determined by the Kruskal–Wallis test with Dunn’s post hoc test and adjusted with the Benjamini–Hochberg method. FACS = fluorescence-activated cell sorting; FND = functional neurological disorders; IIH = idiopathic intracranial hypertension; MAIT = mucosal-associated invariant T cell; MS = multiple sclerosis.

Figure 2

B-lineage cells in CSF are preferentially plasmablasts, clonally expanded, and express IgG1/2 and IgG4 heavy chains in LGI1-/CASPR2-AIE. Single-cell transcriptomes of all B-cell clusters (from Fig. 1B) in CSF and PBMCs from LGI1, CASPR2, IIH and MS patients were subclustered and analysed. (A) UMAP plot with B-lineage subclusters of CSF cells from AIE patients. Cluster 4 contains only PBMCs and is therefore not shown here. Expressions of selected marker genes are shown in Supplementary Fig. 3B and C. Cluster 8 is composed of ‘contaminating’ T cells. (B) UMAP plot illustrating the distribution of B-cell clone sizes in CSF from AIE patients. (C) UMAP depicting the immunoglobulin subtype transcribed by CSF cells from AIE patients. (D) Comparison of the frequency of transcribed immunoglobulin heavy chains (IGHG1 or IGHG2 versus IGHG4) in antibody-secreting B lymphocytes (ASCs) from CSF in all AIE patients. Only samples with >30 cells were considered. Significance was tested with a Mann–Whitney U-test. (E and F) Visual representation of B-cell receptor (BCR) clones (and clonotypes connected by lines) in the CSF (blue) and PBMCs (red) of two representative patients with LGI1-AIE (E) and CASPR2-AIE (F). Each dot indicates the amount of identical BCRs belonging to one clone. The area of each dot is proportional to the clone size. Clonotypes (connected by lines) were defined as identical VDJ gene segments, identical CDR3 length and a CDR3 nucleotide sequence with two or fewer mismatches. Asterisks mark clones that were verified experimentally as autoantigen specific. (G) Circos plot highlighting BCR clones composed of mixtures of plasmablasts, plasma cells and memory B cells. The line thickness indicates the number of cells. Clones consisting of only one cell type are marked in grey. AIE = autoimmune encephalitis; PBMC = peripheral blood mononuclear cell; UMAP = uniform manifold approximation and projection; VDJ = variable–diversity–joining.

Figure 3

T-cell repertoires in CSF of LGI1-/CASPR2-AIE patients show differential changes preferentially in the CD4 TCM clusters, clonal expansion of activated CD4 TCM and CD8 TCM clusters, and blood–CSF-spanning T-cell clones. (A) UMAP plot based on the T-cell subclustering (of all T-cell clusters from Fig. 1B) of all PBMCs and CSF cells. (B and C) Unbiased, cluster-free analysis of differential T-cell abundance was analysed in a pairwise fashion using the DAseq tool and visualized in shades of red for increases and shades of blue for decreases; comparison of LGI1 and IIH CSF T cells (B) and CASPR2 and IIH CSF T cells (C). (D) T-cell clone sizes in CSF from AIE patients projected onto the UMAP plot. (E and F) Visual representation of T-cell receptor clones in CSF (blue) and PBMC (red) of two representative patients with LGI1-AIE (E) and CASPR2-AIE (F). In the network, each dot indicates one clone (identical VDJ genes and CDR3 regions). The area is proportional to the clone size. The pie charts indicate the percentage of CSF cells and PBMCs within each clone. AIE = autoimmune encephalitis; IIH = idiopathic intracranial hypertension; MAIT = mucosal-associated invariant T cell; NK = natural killer cell; PBMC = peripheral blood mononuclear cell; TCM = central memory T cell; UMAP = uniform manifold approximation and projection; VDJ = variable–diversity–joining.

Figure 4

Patients with LGI1-/CASPR2-AIE show trans-compartment loss of innate-like MAIT cells and shifts in the NK cell subpopulations. (A) UMAP showing the subclustering of the mixed T-cell cluster (cluster 10, Fig. 3). The expression levels of marker genes for each cell type shown are visualized in Supplementary Fig. 8A. (B) Heat map comparing selected cell-type abundances (of the subclustering shown in Fig. 4A) between AIE patients, IIH and MS controls across CSF and PBMC compartments. Colours indicate an increase (red) or decrease (blue), and asterisks indicate significance (Supplementary Fig. 8B and C). (C–E) Flow cytometry validation: cell proportions of dnTc (CD3⁺CD4⁻CD8⁻) (C), CD56dim NK cells (CD3⁻CD56^dim) (D) CD56bright NK cells (E), in the CSF (cohort 2). (F) Cell proportion of MAIT cells in PBMCs in another independent flow cytometry cohort (cohort 3). Frequency respective to parent gate. Gating schemes are shown in Supplementary Figs 2 (cohort 2) and 9 (cohort 3). AIE = autoimmune encephalitis; FND = functional neurological disorders; IIH = idiopathic intracranial hypertension; MAIT = mucosal-associated invariant T cell; MS = multiple sclerosis; NK = natural killer cell; PBMC = peripheral blood mononuclear cell.

Figure 5

MAIT cells are present in the brain of patients with CASPR2-AIE and LGI1-AIE, and their absence leads to reduced presence of autoantibodies in a murine immunization model. (A) Multiplexed immunofluorescence (mIF) staining of post-mortem CASPR2-AIE brain tissue, showing CD3 (red), CD8 (green), DAPI (white), CD4 (yellow) and CD161 (blue). Scale bars = 1 mm in overview; 20 μm in subpanels. (B and C) Box plots representing CD3⁺CD161⁺CD4-gdTc-CD8^+/− T cells as a percentage of CD3⁺ cells in the parenchyma (B) and meninges (C) of three human CASPR2- and three LGI1-AIE patients as shown in multiplex immunohistochemistry. (D and E) Murine immunization model using full-length LGI1/CASPR2 protein immunization (AIE) versus sham immunizations (control) of MAIT-deficient mice (MR1) and littermates (C57BL6). Serum was analysed on Day 28 for LGI1/CASPR2 antibody positivity using a cell-based assay, and the end point titre was determined. (D) Bar plot depicting percentage of antibody-positive mice. (E) Box plot showing antibody titre with respect to their disease group. Statistical significance was determined by Fisher’s exact test in D and by Wilcoxon rank test in E. AIE = autoimmune encephalitis; IHC = immunohistochemistry; MAIT = mucosal-associated invariant T cell.