Background/Purpose: Studies to date have primarily focused on stimulators that are overexpressed and activate B cells in SLE subjects.  Factors that can maintain B cells in a resting state and may be under-expressed in SLE are largely unknown.  We utilized a combined single-cell B cell transcriptome and high dimensional flow cytometry analysis approach for a coordinated understanding of factors that are both over- and under-expressed in B-cell developmental checkpoints in SLE.

Methods: The expression of IL-4R and interferon beta (IFNβ) in subsets of B cells was analyzed using high dimensional flow cytometry analysis (n=47 patients who met the ACR classification criteria for SLE). Total B cells from 3 autoantibody (anti-DNA,^,anti-Sm, and anti-histone)⁺ African American (AA) SLE patients, 1 autoAb(−) Europeran American (EA) SLE patient and 1 AA healthy control were purified using the StemCell Human B cell isolation kit.  A high-throughput scRNA-seq was carried out using a droplet-based 10x Chromium approach. The effects of IL-4 on IFNβ-induced IRF7 as well as on IFNɣ + a defined stimulatory media (DSM)(anti-Ig+BAFF+IL-21+TLR7+IL-2)-induced double negative 2 (DN2: CD21⁻CD27⁻IgD⁻CD11c⁺T-bet⁺) B cells were analyzed using an in vitro culture approach.

Results: At the protein level, within naïve (IgD⁺CD27⁻) B cells, higher expression of IFNβ and lower expression of IL-4R correlated with the percent of activated naïve CD21^loIgD⁺ and CD21^loIgD⁻ DN B cells.  There was higher percent of IFNβ⁺IL-4R^lo naïve B cells in AA compared to EA SLE patients.  However, in EA, but not AA, patients, higher percent of IFNβ⁺IL-4R^lo naïve B cells was found in anti-Sm⁺ and renal disease⁺ patients. scRNA-seq analysis shows that the molecular signature of B cells from autoAb(+) patients is type I IFN stimulated genes (ISGs), including IRF7, ISG15, ISG20, MX1, and STAT1.  The molecular signature of B cells from autoAb(-) patients is IL4R, BACH2, S1PR1, and FCER2.  The high ISG and low IL4R was evident during the early transitional stage 1 B cells and persisted in pre-switched IGHD+ B cells.  Stimulation of B cells with DSM+IFNɣ promoted DN2 development whereas addition of IL-4 inhibited DN2 and preserved B cells at the resting naïve stage (IgD⁺CD11c⁻T-bet⁻). IL-4 also inhibited IFNβ-induced IRF7.

Conclusion: Down-regulation of IL-4R in naïve B cells is a major dysregulation in SLE since it may predispose SLE B cells to both type I IFN and type II IFN stimulation.  Further, lower IL-4R together with upregulation of endogenous IFNβ in naïve B cells was an important signature for activated naïve B cells.  Such signature was associated with development of DN2 B cells and, in EA patients, development of anti-Sm autoantibody. Modulation of IL-4R signaling may be developed into a novel therapy for SLE.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/lower-il-4r-in-igd-naive-b-cells-is-a-pre-disposing-factor-for-development-of-t-bet-dn2-b-cells-in-systemic-lupus-erythematosus/