Interleukin 10-producing regulatory B-cells (Breg-cells) suppress autoimmune diseases while aberrant elevation of Breg-cells prevents sterilizing immunity, promotes carcinogenesis and cancer metastasis by converting resting CD4+ T-cells to regulatory T-cells (Tregs). It is therefore of interest to discover factors that induce Breg-cells. Here we show that IL-35 induces Breg-cells in-vivo and promotes their conversion to a unique Breg subset that produces IL-35 (IL-35+Breg). Treatment of mice with IL-35 conferred protection from uveitis and mice lacking IL-35 or defective in IL-35-signaling produced less Breg-cells and developed severe uveitis. Ex-vivo generated Breg-cells also suppressed uveitis by inhibiting pathogenic Th17/Th1 while promoting Tregs expansion. We further show that IL-35 induced the conversion of human B-cells into Breg-cells and suppressed uveitis by activating STAT1/STAT3 through IL-35-Receptor comprising IL-12Rβ2/IL-27Rα subunits. Discovery that IL-35 converts human B-cells into Breg-cells, allows ex-vivo production of autologous Breg-cells for immunotherapy and investigating Breg/IL-35+Breg cells roles in autoimmune diseases and cancer.

HighlightsCLRs play an essential role in immunity to fungi and mycobacteria.CLRs are involved in the regulation of homeostasis, autoimmunity and allergy.CLRs recognise and trigger cellular responses to dead and cancerous cells.

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.