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B Cell Receptor Signaling and Protein Kinase D2 Support Regulatory B Cell Function in Pancreatic Cancer.

ABSTRACT: B cells can act as potent suppressors of anti-tumor T cell immunity, presenting a mechanism of resistance to immunotherapy. In pancreatic ductal adenocarcinoma, B cells can display a T cell-suppressive or regulatory phenotype centered on the expression of the cytokine Interleukin 35 (IL-35). While B cell-mediated immunosuppression presents a barrier to anti-tumorigenic T cell function, it is not clear how regulatory B cell function could be targeted, and the signals that promote this suppressive phenotype in B cells are not well understood. Here we use a novel IL-35 reporter model to understand which signaling pathways are important for immunosuppressive properties in B cells. In vitro analysis of IL-35 reporter B cells revealed a synergy between the BCR and TLR4 signaling pathways is sufficient to induce IL-35 expression. However, in vivo, B cell receptor activation, as opposed to MyD88 signaling in B cells, is central to B cell-mediated suppression and promotion of pancreatic cancer growth. Further analysis identified protein kinase D2 (PKD2) as being a key downstream regulator of IL-35 expression in B cells. Regulatory B cells with an inactivating mutation in PKD2 failed to produce IL-35 or fully suppress effector T cell function in vitro. Furthermore, inhibition of PKD in B cells decreased tumor growth and promoted effector T cell function upon adoptive transfer into B cell-deficient mice. Collectively, these data provide insight into how regulatory B cell function is promoted in pancreatic cancer and identify potential therapeutic targets to restrain this function.

SUBMITTER: Michaud D

PROVIDER: S-EPMC8761795 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Glucagon-like peptide 1 (GLP-1) enhances insulin secretion and protects ?-cell mass. Diabetes therapies targeting the GLP-1 receptor (GLP-1R), expressed in numerous tissues, have diminished dose-response in patients with type 2 diabetes compared with healthy human controls. The aim of this study was to determine the mechanistic causes underlying the reduced efficacy of GLP-1R ligands.Using primary mouse islets and the ?-cell line MIN6, outcomes downstream of the GLP-1R were analyzed: Insulin secretion; phosphorylation of the cAMP-response element binding protein (CREB); cAMP responses. Signaling systems were studied by immunoblotting and qRT-PCR, and PKA activity was assayed. Cell surface localization of the GLP-1R was studied by confocal microscopy using a fluorescein-tagged exendin-4 and GFP-tagged GLP-1R.Rodent ?-cells chronically exposed to high glucose had diminished responses to GLP-1R agonists including: diminished insulin secretory response; reduced phosphorylation of (CREB); impaired cAMP response, attributable to chronically increased cAMP levels. GLP-1R signaling systems were affected by hyperglycemia with increased expression of mRNAs encoding the inducible cAMP early repressor (ICER) and adenylyl cyclase 8, reduced PKA activity due to increased expression of the PKA-RI? subunit, reduced GLP-1R mRNA expression and loss of GLP-1R from the cell surface. To specifically examine the loss of GLP-1R from the plasma membrane a GLP-1R-GFP fusion protein was employed to visualize subcellular localization. Under low glucose conditions or when PKA activity was inhibited, GLP-1R-GFP was found at the plasma membrane. Conversely high glucose, expression of a constitutively active PKA subunit, or exposure to exendin-4 or forskolin led to GLP-1R-GFP internalization. Mutation of serine residue 301 of the GLP-1R abolished the glucose-dependent loss of the receptor from the plasma membrane. This was associated with a loss of an interaction between the receptor and the small ubiquitin-related modifier (SUMO), an interaction that was found to be necessary for internalization of the receptor.These data show that glucose acting, at least in part, via PKA leads to the loss of the GLP-1R from the cell surface and an impairment of GLP-1R signaling, which may underlie the reduced clinical efficacy of GLP-1R based therapies in individuals with poorly controlled hyperglycemia.

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B Cell Receptor Signaling and Protein Kinase D2 Support Regulatory B Cell Function in Pancreatic Cancer.

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