B-Cell Compartmental Features and Molecular Basis for Therapy in Autoimmune Disease

Abstract

Background and objectives: To assess the molecular landscape of B-cell subpopulations across different compartments in patients with neuromyelitis optica spectrum disorder (NMOSD).

Methods: We performed B-cell transcriptomic profiles via single-cell RNA sequencing across CSF, blood, and bone marrow in patients with NMOSD.

Results: Across the tissue types tested, 4 major subpopulations of B cells with distinct signatures were identified: naive B cells, memory B cells, age-associated B cells, and antibody-secreting cells (ASCs). NMOSD B cells show proinflammatory activity and increased expression of chemokine receptor genes (CXCR3 and CXCR4). Circulating B cells display an increase of antigen presentation markers (CD40 and CD83), as well as activation signatures (FOS, CD69, and JUN). In contrast, the bone marrow B-cell population contains a large ASC fraction with increased oxidative and metabolic activity reflected by COX genes and ATP synthase genes. Typically, NMOSD B cells become hyperresponsive to type I interferon, which facilitates B-cell maturation and anti-aquaporin-4 autoantibody production. The pool of ASCs in blood and CSF were significantly elevated in NMOSD. Both CD19- and CD19+ ASCs could be ablated by tocilizumab, but not rituximab treatment in NMOSD.

Discussion: B cells are compartmentally fine tuned toward autoreactivity in NMOSD and become hyperreactive to type I interferon. Inhibition of type I interferon pathway may provide a new therapeutic avenue for NMOSD.

Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

Figures

Figure 1. B-Cell Clusters and Distribution Revealed by High-Throughput Single-Cell RNA Sequencing in NMOSD

(A. A.a) Integrated analysis reveals distinct subpopulations of B cells in NMOSD including naive B cells, memory B cells, age-associated B cells, and antibody-secreting cells (ASCs). (A.b-e) Individual plots showing B-cell subpopulations in control blood (A.b), NMOSD blood (A.c), NMOSD CSF (A.d), and NMOSD bone marrow (BM) (A.e). Two clusters of memory B cells were identified: IGHMhighCOCHlowITGB1low and IGHMlowCOCHhigh ITGB1high memory B cells. Four clusters of ASCs were identified: MS4A1high, CD27high, MK67high, and SDC1high ASCs. (B) Heatmap showing unsupervised hierarchical clustering of gene expression pattern in B cells from NMOSD blood, bone marrow, and CSF. Scaled expression of discriminative gene sets for each B cell subpopulation is shown. (C) Plots showing the characteristic expression of indicated molecules in B-cell subpopulations. NMOSD = neuromyelitis optica spectrum disorder.

Figure 2. Distinctive Transcriptome Profiles of B Cells Across Blood, Bone Marrow, and CSF From Patients With NMOSD

(A) Scatter dot plots showing expression of inflammation-related genes in B cells from NMOSD blood (A.a), bone marrow (A.b), and CSF (A.c). Blood from healthy individuals was used as control. Red dots: upregulated genes. Blue dots: downregulated genes. (B) Heatmap showing differential expression pattern of selected inflammation-related genes in B-cell subsets across blood, CSF, and bone marrow. NMOSD blood vs control blood: increased genes include FOS, CD69, JUN, DUSP1, PPP1R15A, IER2, ZFP36, and HLA-DQB1; reduced genes include S1PR4, YBX3, and SNHG7. (C) Bar graphs showing top 10 enriched gene ontology (GO) terms of indicated pathways in B cells from NMOSD bone marrow (C.a) or blood (C.b). Color scheme is based on adjusted p values from high (blue) to low (red). GO pathway analysis shows significant increase of type I interferon pathway in CSF B cells but not in bone marrow B cells. (D) Bar graph showing the differences in indicated pathways between blood B cells and CSF B cells in NMOSD. Shown in t-statistics from low (green) to high (red) resulting from the GSVA score. NMOSD = neuromyelitis optica spectrum disorder.

Figure 3. B-Cell Reactivity to Type I Interferon in Patients With NMOSD

(A) Unsupervised hierarchical clustering of 145 IFN-related genes expressed in blood B cells vs CSF B cells obtained from patients with NMOSD. (B) Counts of B-cell subpopulations with upregulation or downregulation of interferon-related genes in blood, bone marrow, and CSF. Upregulation: green; downregulation: red. (C) Left panel: Z-scores of IFN-related genes in patients with NMOSD vs controls. Right panel: Z-scores of IFN-related genes in main clusters of B cells from blood, CSF, and bone marrow in patients with NMOSD. (D and E) Flow cytometry plots and bar graphs show B-cell subpopulations from NMOSD blood in response to IFN-α2b stimulation with or without an IFN-α2b inhibitor. UM B cells: memory IgM cells (IgM+IgD+CD27+); DN B cells: double-negative B cells (CD27−IgD−); SW B cells: switched memory B cells (IgD−CD27+); ASCs: antibody-secreting cells (CD27highCD38high). n = 8 per group. (F) Effects of IFN-α2b stimulation on production of AQP4-IgG in the supernatant of cultured B cells. B cells were obtained from NMOSD blood and cultured in the presence of sCD40L (100 ng/mL), IL-2 (50 ng/mL), IL-21 (50 ng/mL), IL-6 (5 ng/mL), and TNF-α (5 ng/mL) for 10 days, with or without IFN-α2b (5,000 U/mL). Data for AQP4-IgGs are expressed as median fluorescence intensity (ΔMFI) values. n = 6 per group. ASC = antibody-secreting cell; NMOSD = neuromyelitis optica spectrum disorder. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4. Trajectory Trace of B Cells and Their Functional Response to Type 1 Interferon in NMOSD

(A) Expression of indicated markers in naive B cells from NMOSD blood. (B) Trajectory analysis shows early development signature membrane metalloendopeptidase (MME) expressed mainly in transitional B cells. (C) Heatmap of naive B cells in NMOSD blood, bone marrow (BM), and CSF showing 3 clusters: CCR7− transitional B cells; CCR7int-naive B cells; and CCR7high-naive B cells. (D) Interferon response among 3 clusters of naive B cells. Intermediate and late naive B cells displayed strong type I interferon response. (E and F) Indicated groups of cultured blood B cells were treated with vehicle, IFN-α2b, or IFN-α2b + IFN-α receptor inhibitor for 3 days. Flow cytometry analysis revealed increased proportion of transitional B cells (CD24highCD38high) after IFN-α treatment. Inhibition of IFN-α receptor abolished the effects of IFN-α2b treatment, n = 8 per group. NMOSD = neuromyelitis optica spectrum disorder. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Characterization of Antibody-Secreting Cells (ASCs) and Their Responsiveness to Rituximab or Tocilizumab in NMOSD

(A) Plots showing different expression of inflammatory signatures in CSF ASCs vs blood ASCs in NMOSD. (B) GSVA scores of indicated pathways expressed in NMOSD ASCs vs healthy controls. Shown in t-statistics from low (green) to high (red) that results from the GSVA score comparison tests comparing ASCs from NMOSD blood vs control blood. (C) UMAP analysis showing 4 subpopulations of ASCs that highly expressed MS4A1, SDC1, MK67, or CD27, respectively. (D) Differential expression of MS4A1 (D.a), CD19 (D.b), CD24 (D.c), CD27 (D.e), MKI67 (D.e), and SDC1 (D.f) in 4 subpopulations of ASCs. (E) Expression of immunoglobulin constant region genes, IGHA, IGHD, IGHG, and IGHM, in subpopulations of ASCs. (F) Plots showing transitioning B-cell clones in patients with NMOSD vs controls (F.a, controls; F.b, NMOSD). (G and H) Trajectory analysis of ASCs reveals 4 subpopulations of ASCs based on the expression level of CD19 and CD138. CD19−CD138+ plasma cells were obviously terminally differentiated. (I and J) Changes of peripheral blood CD19+ and CD19− ASCs before and after rituximab (I) or tocilizumab (J) treatment. The counting of 2 subsets was expressed as the percentage of peripheral blood mononuclear cells (PBMCs). n = 10 patients with NMOSD per group. *p < 0.05. NMOSD = neuromyelitis optica spectrum disorder.