A stromal platform for robust expansion of functional IL-10–producing B cells for immune regulation
Ryo Kawakami, Keisuke Imabayashi, Akemi Baba, Yuichi Saito, Kazuhiko Kawata, Yutaro Yada, Airi Shibata, Rinka Ito, Ryo Kurasawa, Ryota Higuchi, Sungyeon Park, Hiroaki Niiro, Shinya Tanaka, Yoshihiro Baba
Ryo Kawakami, Keisuke Imabayashi, Akemi Baba, Yuichi Saito, Kazuhiko Kawata, Yutaro Yada, Airi Shibata, Rinka Ito, Ryo Kurasawa, Ryota Higuchi, Sungyeon Park, Hiroaki Niiro, Shinya Tanaka, Yoshihiro Baba
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Research Article Immunology

A stromal platform for robust expansion of functional IL-10–producing B cells for immune regulation

Abstract

IL-10–producing B cells exert immunosuppressive effects, yet their low abundance and poor in vitro viability have limited their therapeutic application. Here, we developed a stromal coculture system using MS5 cells engineered to express human CD40L, BAFF, and IFN-β1 (MS5-3F, for “3 factors”), which enables robust induction and greater than 1000-fold expansion of human IL-10–producing B cells. The expanded cells showed phenotypic and transcriptional profiles characteristic of unswitched (IgM+) plasmablasts and potently suppressed CD4+ T cell proliferation in an IL-10–dependent manner. MS5-3F–expanded B cells also increased the frequency of regulatory T cells in vitro, an effect that was not abrogated by IL-10/IL-10R blockade, suggesting contributions from additional mechanisms. IL-10 production originated predominantly from naive B cells, rather than memory B cells. Furthermore, B cells from patients with systemic lupus erythematosus, despite impaired IL-10 production under conventional conditions, were efficiently differentiated into IL-10–producing B cells using this system. The expanded cells showed minimal IgG-secreting output. Our platform offers a scalable strategy for generating human regulatory B cells, laying the foundation for B cell–based immunotherapies.

Authors

Ryo Kawakami, Keisuke Imabayashi, Akemi Baba, Yuichi Saito, Kazuhiko Kawata, Yutaro Yada, Airi Shibata, Rinka Ito, Ryo Kurasawa, Ryota Higuchi, Sungyeon Park, Hiroaki Niiro, Shinya Tanaka, Yoshihiro Baba

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Figure 1

Establishment of a stromal coculture system for human IL-10–producing B cell induction and expansion.

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Establishment of a stromal coculture system for human IL-10–producing B ...
(A) Schematic overview of the coculture system. MS5 stromal cells were genetically engineered to express human IFN-β1 (MS5-IFNβ1), CD40L and BAFF (MS5-CD40L/BAFF), or all 3 factors (MS5-3F), to support the differentiation of peripheral blood B cells into IL-10–producing B cells. (B) Representative flow cytometry histograms of human CD40L and BAFF expressed on MS5 lines. See also Supplemental Figure 1. (C) Quantification of IFN-β1 production by MS5 lines. (D) Fold expansion of total B cells cocultured with different MS5 lines in the presence of CpG ODN. Cell numbers were assessed every 4 days and normalized to the initial input to calculate fold change. See also Supplemental Figure 2A. (E and F) Induction and expansion of IL-10–producing B cells cocultured with MS5-CD40L or MS5-3F. (E) Representative flow cytometry plots of IL-10–producing B cells on days 0 and 12 of coculture, assessed using IL-10 secretion assay. (F) Fold expansion and temporal changes in frequencies of IL-10+ cells. (G and H) Comparison of B cell expansion and IL-10 production between MS5-3F coculture and conventional liquid culture conditions. (G) Fold expansion of total B cells. (H) IL-10 secretion in culture supernatants on day 4 assessed by ELISA. (I) Expansion of B cells from multiple healthy donors in MS5-3F coculture. (J) Summarized graph of percentage IL-10 in B cells from different donors on day 12 of MS5-3F coculture. See also Supplemental Figure 2E. Data from 3 independent experiments were combined (C, D, and F: n = 3; G–J: n = 9). Data are presented as mean ± SEM. P values are from 1-way ANOVA with Tukey’s post hoc test (C, H, and J) or 2-way (D and F) ANOVA with Bonferroni’s post hoc test. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS, not significant.