Notch1-Activated B Cells Have an Immunomodulatory Function Enhancing Th2 and Treg Immune Response Via IL-33-ST2 Pathway

Notch1 signaling pathway is involved in T-cell fate decision and development, but it is also known to be activated in B cells upon anti-IgM or LPS stimulation. In addition to its physiological upregulation in B cells, Notch1 signaling is often aberrantly activated in several lymphoid malignancies of B-cell origin, such as classical Hodgkin lymphoma, mantle cell lymphoma and chronic lymphocytic leukemia. However, functional roles of Notch1 in B cells have not been well elucidated to date. Here we report a novel immunomodulatory role of Notch1-activated B cells that alters T-cell immune response in an IL-33-dependent manner.

Functional analysis of Notch1 in mature B cells had been hampered by its substitutability for Notch2, which is involved in early B-cell fate decision towards marginal zone B cells (Zhang et. al. J Immunol 2013). To eliminate such irrelevant effect of Notch1 on early B-cell differentiation, we generated a mouse model in which Notch1 intracellular domain (NICD), a constitutively active form of Notch1, began to be expressed in mature B cells after AICDA promoter-dependent Cre expression in germinal centers (Stop^Floxed-NICD Tg mice×Aicda-Cre mice, hereby designated as NICD Tg mice). In this mouse model, NICD transgene was expressed in about 5% of total splenic B cells, with normal B cell maturation and differentiation. Alternatively, subsets of splenic CD4⁺ T cells were significantly altered, with increase in Th2 and Treg cells and decrease in Th1 and Th17 cells. IFN-γ production by CD8⁺ T cells was also significantly reduced.

Consequently, NICD Tg mice were susceptible to fungal infections, and more importantly, they began to die of spontaneous malignant neoplasms such as sarcoma and lymphoma at 9 months of age. The tumor development was further increased when TP53 gene was heterozygously deleted in NICD Tg mice. None of the tumors having developed in NICD Tg mice expressed the NICD transgene, suggesting that these tumors did not develop as a result of direct oncogenic effect of NICD. As serum levels of IFN-γ and TNF-α were significantly lower in NICD Tg mice than in control mice, it was rather suggested that these tumors had developed under a condition of suppressed anti-tumor immunity.

To elucidate the mechanism of immunomodulatory activity of Notch1-activated B cells, we performed a comparative gene expression analysis using B cells from NICD Tg and control mice. Among several candidate genes whose expression levels were increased in Notch1-activated B cells, we focused on elevated IL-33 as a potential cause for the immunomodulation. Upregulation of IL-33 protein in Notch1-activated B cells was validated by intracellular cytokine flow cytometry.

IL-33 is a cytokine that is expressed in nuclei of broad types of cells in their resting state. However, we found that it was also present in the cytoplasm of Notch1-activated B cells, suggesting that IL-33 is actively produced in these cells. To confirm whether extracellular release of IL-33 from B cells was enhanced through Notch1, we cultured splenic B cells from wild-type mice with LPS stimulation in the presence of L cells with or without Notch1 ligand Delta-like 1 (Dll1) expression. We found that IL-33 secretion from B cells was increased twofold in the presence of Dll1-positive compared to Dll1-negative L cells. As expected, the Dll1-mediated increase in IL-33 levels was successfully blocked by DAPT, a Notch signaling inhibitor.

To determine whether the IL-33 secreted from Notch1-activated B cells was responsible for the functional modulation of T cells, we cultured wild-type CD4⁺ T cells with B cells from NICD Tg or control mice, and measured cytokine levels produced by T cells. As a result, IL-4, IL-13 and IL-10 secretion was markedly increased when T cells were cocultured with Notch1-activated B cells. Strikingly, the increase in these Th2- and Treg-associated cytokine levels was completely canceled by addition of a blocking antibody against the IL-33 receptor ST2.

In summary, we have shown that Notch1-activated B cells have a novel immunomodulatory function to alter T-cell immunity towards Th2 and Treg immune response via IL-33 secretion, thereby suppressing cellular immunity. This immunomodulatory mechanism may potentially be utilized by Notch1-activated B-cell neoplasms to escape anti-tumor immunity, and we propose that the Notch1-IL-33-ST2 axis can be a promising target for immunotherapy of lymphoid malignancies.