Figure 2.

Structural and functional properties of disease-associated OT: NR1-autoantibody isotype detection, flow cytometry, cell culture and multiplex histology. (A) NR1-antibody isotype levels in matched fluids from four OTs from three patients [indicated by symbols and lines; Patient 2 (P2) had initial and recurrent OTs analysed]. Patient 7 NR1-IgG reproduced here for comparison.¹⁴ (B) B cell populations in the recurrent OT (top row) and peripheral blood (PBMCs) of Patient 2. Flow cytometry plots show ASCs (CD19⁺CD27⁺CD38⁺; left), naïve (CD27⁻IgD⁺) and memory (CD27⁺IgD⁻) B cells (middle) and memory B cell receptor isotypes (IgA versus IgG staining; with IgM staining indicated by colour; right). Prior gates shown in Supplementary Fig. 1 (scatter plot lymphocyte gate, doublet exclusion, CD45⁺CD3⁻). (C) Frequency of NR1-IgG detection in cell culture supernatants (percentage of wells) from three sets of OT explants and dissociated cells (right) using activation (black bars) or maintenance (grey bars) conditions. (D) Distributions of lymphocytes, accessory vascular structures, and neuroglial tissue from NMDAR-antibody encephalitis-associated OT tissue (Patient 8). A low power fluorescence image shows a region of co-localized neuronal (MAP2) and glial (GFAP) markers towards the outer aspect of the section (red box). (E–G) The region of interest is then examined with neuroglial/vascular/dendritic cell markers (E), NR1 (F) and CD19 (G). NR1 staining is most dense in the lymphocytic region (grey box) but can also be seen in the neuroglial region. The CD19⁺ region (grey box and H–K), dendritic cells (purple box, L), and vascular structures (blue and yellow boxes, M and N, respectively) are then presented. (H–K) Within the lymphoid aggregate, CD19⁺ cells are densely aggregated with occasional CD38⁺; representative rarer CD138⁺ cells from outside the region are shown in the green inset (H). Within the centre of the CD19⁺ region (H, brown box) there were BCL6⁺ cells, with some cells also positive for AID (I). This region also shows CXCR5 expression (J) alongside a CD21⁺ meshwork (K). (L) A cluster of LAMP3⁺ cells consistent with dendritic cells. (M) A PNAd⁺ vascular structure consistent with a high endothelial venule. (N) A podoplanin⁺ vascular structure consistent with a lymphatic vessel. BCL6 = B cell lymphoma 6 protein; CXCR5 = C-X-C chemokine receptor type 5; DAPI = 4′,6-diamidino-2-phenylindole; GFAP = glial fibrillary acidic protein; LAMP3 = lysosome-associated membrane glycoprotein 3; MBC = memory B cell; P = patient; PNAd = peripheral node addressin.