Human IL35-producing B cells (I35Bregs) are induced by CpG DNA

Abstract

Purpose : In the mouse, in vitro activation of B cells by LPS induces the expansion of regulatory B cells that produce IL-35 (i35-Bregs) and i35-Bregs play critical roles in suppressing ocular inflammation and amelioration of experimental autoimmune uveitis (EAU). In this study we have investigated whether TLR agonists also induce the expansion of human i35-Breg cells.

Methods : CD4⁺ T or CD19⁺ B cells were isolated from human PBMC of healthy subjects by using CD4- or CD19-magnetic bead sorting. The cells were stimulated with LPS, CD40L, CpG, PGN, anti-CD40 or anti-IgM in presence or absence of hIL-35 for 2 days. The immunophenotype was characterized by FACS using labeled mAbs specific to CD4, CD19, CD20, CD24, CD25, CD27, CD38, CD45RA, CD45RO, CD138, IgM, IgD, IL-10, IL-12p35, EBI3, IL-12Rbeta2. ELISA was used for IL-10 detection. PCR and Western blotting assays were used for cytokine detections. ³[H]-thymidine incorporation was performed with five replicate cultures.

Results : Among the TLR ligands examine (LPS, CpG, PGN), CpG was the most effective inducer of IL-10- and IL-35-producing Bregs. Similar to mouse IL-35, hIL-35 induced autocrine production of IL-35 by promoting the expansion of i35-Bregs and mediated its effects through activation of STAT1 and STAT3. We also show that human i35-Breg cells are CD27^HiCD38^HiCD138⁺ CD19⁺ plasmablast.

Conclusions : The expansion of human i35-Bregs by CpG and discovery that hIL-35 can induce the conversion of B-cells into IL-10- producing B cells (Bregs) or i35-Bregs allows ex-vivo production of large amounts of Bregs and i35-Bregs. These findings may also facilitate the development of adoptive Breg cell immunotherapy for autoimmune diseases.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.