Transitional B cells in quiescent SLE: An early checkpoint imprinted by IFN

Abstract

Systemic lupus (SLE) is characterized by a break of B cell tolerance that plays a central role in disease pathophysiology. An early checkpoint defect occurs at the transitional stage leading to the survival of autoreactive B cells and consequently the production of pathogenic autoantibodies. The main purpose of our work was to determine whether transitional B cells, as the most immature naïve B cell subset upstream of pathogenic B cells, display specific features compared to healthy non SLE subjects. Through extensive analysis of transitional B cells from untreated or low treated, mostly Caucasian, SLE patients, we demonstrated that transitional (T1 and T2) B cell frequencies were increased in SLE and positively correlated with disease activity. SLE transitional B cells displayed defects in two closely inter-related molecules (i.e. TLR9 defective responses and CD19 downregulation). RNA sequencing of sorted transitional B cells from untreated patients revealed a predominant overexpression of interferon stimulated genes (ISGs) even out of flares. In addition, early transitional B cells from the bone marrow displayed the highest interferon score, reflecting a B cell interferon burden of central origin. Hence, the IFN signature in transitional B cells is not confined to African American SLE patients and exists in quiescent disease since the medullary stage. These results suggest that in SLE these 3 factors (i.e. IFN imprintment, CD19 downregulation and TLR9 responses impairment) could take part at the early transitional B cell stage in B cell tolerance by-pass, ultimately leading in periphery to the expansion of autoantibodies-secreting cells.

Keywords: CD19; Interferon; Systemic lupus erythematosus; TLR9; Transitional B cells.

Fig. 1

(A) Representative dot plots of transitional B cells and their subsets in one HD and one SLE patient. (B) Frequency of transitional B cells (top left panel) and their subsets (bottom panel) (% of CD19⁺) in HDs compared to SLE patients. Correlation between transitional B cells (% of CD19⁺) and disease activity assessed by the SLEDAI-2K score (top right panel). (C) MFI of CD19 in transitional B cells subsets from HDs and SLE patients. (D) Frequency of CD86⁺CD69⁺ transitional B cells from HDs or SLE patients after no stimulation (NS) or in vitro stimulation with CD40L (CD40 ligand), R848 (TLR7 agonist) or CpG (TLR9 agonist) for 2 days. HDs: healthy donors; MFI: mean of fluorescence intensity; SLE: Systemic lupus erythematosus.

Fig. 2

(A) Flow chart of RNA Sequencing analysis. (B) Venn Diagram representing genes significantly up (in red) or down-regulated (in blue) between quiescent SLE patients, active SLE patients and HDs. A: active; HDs: healthy donors; Q: quiescent; SLE: systemic lupus erythematosus.

Fig. 3

(A) Volcano plot representation of RNA-Seq data comparing transitional B cells from HDs (n=6) versus untreated quiescent SLE patients (n=3) (left panel) and transitional B cells from untreated quiescent versus active patients (n=3) (right panel). (B) Hierarchical representation of interferon stimulated genes (ISGs) expression in transitional B cells from each patient compared to HDs. (C) Modular analysis of RNA-Seq data representing the % of transcripts over/under-expressed (P<0.01) in untreated SLE patients (quiescent, n=3; active, n=3) compared to HDs (n=6). (D) qRT-PCR of ISGs in sorted transitional B cells of quiescent SLE patients compared to HDs. (E) Correlation between transitional B cells (% of CD19⁺ cells) and the interferon score. HDs: healthy donors; ND: not detected; SLE: Systemic lupus erythematosus.

Fig. 4

(A) Gating strategy for BM analysis of pre/proB (CD19⁺CD24⁺⁺CD38⁺⁺IgM⁻IgD⁻), immature (CD19⁺CD24⁺⁺CD38⁺⁺IgM⁺IgD⁻) and early transitional B cells (CD19⁺CD24⁺⁺CD38⁺⁺IgM⁺IgD⁺). (B) qRT-PCR of eight interferon stimulated genes in sorted early transitional B cells from BM of SLE patients compared to control. (C) Representation of the interferon score in BM and peripheral B cell subpopulations in three SLE patients. BM: Bone marrow; ND: not detected; SLE: Systemic lupus erythematosus.

Fig. 5

(A) Representative dot plots of Ki-67⁺ cells (top panel) and Ki-67 MFI (bottom left panel) in gated transitional B cells from one HD and one SLE patient; frequency of Ki-67⁺ transitional B cells from HDs and SLE patients (bottom right panel). (B) Frequency of early apoptotic (DAPI⁻ AnnexinV⁺) transitional B cells in HDs and SLE patients. FMO: Fluorescence Minus One; HDs: healthy donors; MFI: mean of fluorescence intensity; SLE: systemic lupus erythematosus.