Low-dose IL-2 enhances the generation of IL-10-producing immunoregulatory B cells

Abstract

Dysfunction of interleukin-10 producing regulatory B cells has been associated with the pathogenesis of autoimmune diseases, but whether regulatory B cells can be therapeutically induced in humans is currently unknown. Here we demonstrate that a subset of activated B cells expresses CD25, and the addition of low-dose recombinant IL-2 to in vitro stimulated peripheral blood and splenic human B cells augments IL-10 secretion. Administration of low dose IL-2, aldesleukin, to patients increases IL-10-producing B cells. Single-cell RNA sequencing of circulating immune cells isolated from low dose IL2-treated patients reveals an increase in plasmablast and plasma cell populations that are enriched for a regulatory B cell gene signature. The transcriptional repressor BACH2 is significantly down-regulated in plasma cells from IL-2-treated patients, BACH2 binds to the IL-10 gene promoter, and Bach2 depletion or genetic deficiency increases B cell IL-10, implicating BACH2 suppression as an important mechanism by which IL-2 may promote an immunoregulatory phenotype in B cells.

Fig. 1. Activated B cells express CD25 and the addition of IL-2 increases IL-10 production by in vitro and vivo.

a Heatmap showing IL2RA and IL2RB transcript expression in IL-10-positive human B cells relative to IL-10-negative B cells in n = 6 subjects. b Flow cytometry plots and quantification of surface expression of CD25 (IL-2Rα) on human blood (b) and splenic (c) B cells. Gated on live single CD19⁺ events. All bar graphs in figures show mean and error bars representing standard error of mean (SEM). Representative of three experimental replicates. d Quantification of IL-10 in culture supernatants from human splenic B cells stimulated with IL-2, CpG and/or CD40L. Quantification of IL-6 (e) and TNFα (f) in culture supernatant from negatively-isolated human splenic B cells stimulated with IL-2 and CpG. Graphs show mean and SEM of triplicates and are representative of three experimental repeats. g Quantification of IL-10 in culture supernatants from negatively isolated human blood B cells stimulated with IL-2, and lower concentrations of CpG. h CD4 T cell TNFα MFI following co-culture with CpG-stimulated B cells generated in the presence or absence of IL-2. Graphs show mean and SEM of duplicates and are representative of three experimental repeats. p-values generated using an unpaired two-tailed parametric t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 2. Analysis of B cells and plasma cells from patients given LD IL-2 treatment.

a Diagrammatic schema summarizing the two treatment arms of the LD aldesleukin study. Patients with recently-diagnosed type 1 diabetes received 0.09 × 10⁶ IU/m² or 0.2–0.47 × 10⁶ IU/m² aldesleukin every two to five days. Proportion (of total B cells) or absolute number of IL-10-positive B cells in patients prior to and following the administration of 0.09 × 10⁶ IU/m² (c) or 0.2–0.47 × 10⁶ IU/m² (d) aldesleukin. Coloured line indicates mean changes following treatment. Representative flow cytometry plot of IL-10 expressing B cells pre- and post- 0.09 × 10⁶ IU/m² (b) or 0.2–0.47 × 10⁶ IU/m² (e) aldesleukin. p-values generated using a paired two-tailed parametric t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.

Fig. 3. In-depth analysis of B cells and plasma cells from patients given LD IL-2 treatment.

a Diagrammatic schema summarising the three treatment groups of the trial. Patients received either saline, or 1.5 × 10⁶ IU, or 2.5 × 10⁶ IU IL-2. b Proportion of IL-10-positive B cells in patients prior to and following the administration saline (left) or 1.5 × 10⁶ IU IL-2 (right). n = 4 and 6 analysed by flow cytometry of untreated and treated groups respectively. p-values generated using a paired two-tailed parametric t-test. *p < 0.05. c Representative flow cytometry plot of IL-10 expressing B cells pre- and post-LD IL-2. d Uniform manifold approximation and projection of four clusters of all B and plasma cells from all samples analysed by scRNAseq: Naïve, non-switched memory and switched memory B cells and plasma cells (left). Dot plot of enriched identifying genes of each cluster (right). e Proportion of each cell type before treatment and following saline or LD IL-2 treatment. LD IL-2 treatment groups combined. p-values generated using an unpaired two-tailed parametric t-test. **p < 0.01 (f) Dotplot showing Breg gene set enrichment (gene signatures from recently published meta-analysis of all human Breg RNAseq studies). The intensity of gene signature expression is indicated by the colour of the dot and proportion of cells expressing this signature is indicated by size of the dot. Source data are provided as a Source Data file.

Fig. 4. BACH2 inhibits IL-10 production in B cells.

a Volcano plots of relative transcription factors expression following LD IL-2 treatment versus following saline treatment in plasma cells. p-values generated using an unpaired two-tailed parametric t-test. b Fold change in IL-10 gene expression comparing murine Bach2^−/− versus Bach2^+/+ B cells from publicly available data. c escarpment plots showing gene set enrichment analysis using Breg gene signature comparing murine Bach2^−/− versus Bach2^+/+ B cells from publicly available data. d Confocal imaging of BACH2 in B cells stimulated with RGFP966, an HDAC3 inhibitor. e Quantification of IL-10 in culture supernatants from human blood B cells stimulated with CpG, IL-2 and/or RGFP966, an HDAC3 inhibitor. p-values generated using an unpaired two-tailed parametric t-test. *p < 0.05, **p < 0.01. f CHIP sequencing data of BACH2 on the Il10 region of DNA from publicly available data. B cells were unstimulated. Bar graphs show mean and SEM of duplicates. Source data are provided as a Source Data file.