VH2+ Antigen-Experienced B Cells in the Cerebrospinal Fluid Are Expanded and Enriched in Pediatric Anti-NMDA Receptor Encephalitis

Abstract

Pediatric and adult autoimmune encephalitis (AE) are often associated with Abs to the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Very little is known regarding the cerebrospinal fluid humoral immune profile and Ab genetics associated with pediatric anti-NMDAR-AE. Using a combination of cellular, molecular, and immunogenetics tools, we collected cerebrospinal fluid from pediatric subjects and generated 1) flow cytometry data to calculate the frequency of B cell subtypes in the cerebrospinal fluid of pediatric subjects with anti-NMDAR-AE and controls, 2) a panel of recombinant human Abs from a pediatric case of anti-NMDAR-AE that was refractory to treatment, and 3) a detailed analysis of the Ab genes that bound the NR1 subunit of the NMDAR. Ag-experienced B cells including memory cells, plasmablasts, and Ab-secreting cells were expanded in the pediatric anti-NMDAR-AE cohort, but not in the controls. These Ag-experienced B cells in the cerebrospinal fluid of a pediatric case of NMDAR-AE that was refractory to treatment had expanded use of variable H chain family 2 (VH2) genes with high somatic hypermutation that all bound to the NR1 subunit of the NMDAR. A CDR3 motif was identified in this refractory case that likely drove early stage activation and expansion of naive B cells to Ab-secreting cells, facilitating autoimmunity associated with pediatric anti-NMDAR-AE through the production of Abs that bind NR1. These features of humoral immune responses in the cerebrospinal fluid of pediatric anti-NMDAR-AE patients may be relevant for clinical diagnosis and treatment.

FIGURE 1.. B cell profile in the CSF of pediatric Autoimmune Encephalitis.

Cerebrospinal fluid (CSF) was collected from pediatric patients with autoimmune encephalomyelitis (AE). Shown are B cell subset frequencies by flow cytometry for (A) pediatric patients who tested negative for antibodies against the NMDAR (OND), (B) pediatric patients who tested positive for antibodies against the NMDA receptor and met diagnostic criteria for NMDAR-AE (NMDAR-AE) and (C) one pediatric NMDAR-AE case refractory to treatment (NMDAR-AE CASE). (D) Ratios of B cell subsets in each group. In D, abbreviations are as follows: AntigenE, Antigen Experienced; ASC, antibody secreting cell; MEM, memory; PB, plasmablast.

FIGURE 2.. Antibody variable heavy chain use dysregulated in pediatric NMDAR-AE.

(A) Single B cells from the cerebrospinal fluid of a pediatric NMDAR-AE patient were queried for their antibody variable heavy chain usage and compared to the expected frequency in healthy controls. (B) Mutational frequencies within each variable antibody heavy chain family. (C) Replacement frequencies within each variable antibody heavy chain family. (D) CDR:FR ratio of replacement frequency within each variable antibody heavy chain family.

FIGURE 3.. Binding of recombinant human antibodies to the NR1 subunit of the NMDAR.

(A) Antibodies expressed by individual B cells from the CSF of a pediatric NMDAR-AE patient refractory to treatment were expressed and tested for binding to the NR1 subunit of the NMDAR using a commercially available kit as described in the methods. Blue: DAPI staining. Green: NMDAR. Scale bar: 20 μm. (B) Frequency of positively-stained cells.

FIGURE 4.. Clonal Expansion of VH2+ antigen experienced B cells in the CSF of a pediatric NMDAR-AE patient refractory to treatment.

(A) The CDR3 amino acid sequences of AG06, AG08 and AG09 are in the boxes with differences indicated in color. (B) Clonal relationship of AG06, AG08 and AG09 using a Hamming distance of 2. Replacement mutations are indicated by region, codon number and the amino acid replacement. The gray box indicates a common clone member that was not detected in the repertoire. Germline V, D and J genes are indicated at the root of the clone.

FIGURE 5:. Comparison of rhAbs to germline configuration.

(A) Antibodies expressed by individual B cells from the CSF of a pediatric NMDAR-AE patient refractory to treatment were reverted to their germline configuration and tested for binding to the NR1 subunit of the NMDAR using a commercially available kit as described in the methods. Blue: DAPI staining. Green: NMDAR. Scale bar: 20 μm. (B) Frequency of positively-stained cells. (C) Mean fluorescence intensity of cells. The horizontal dotted line represents the MFI of control cells as the threshold of detection.