GPR174 signals via Gαs to control a CD86-containing gene expression program in B cells

GPR174 is abundantly expressed in B and T lymphocytes and has a role in restraining T cell responses, but the function of GPR174 in B cells is less clear. Here we report that upon in vitro culture B cells undergo a spontaneous GPR174-dependent activation process that is associated with marked changes in gene expression, including up-regulation of Cd86, Nr4a1, Ccr7, and phosphodiesterases. B cells lacking Gαs show a block in induction of the GPR174-dependent program. Spontaneous up-regulation of CD86 in cultured B cells is dependent on protein kinase A. Both GPR174- and Gαs-deficient B cells show enhanced survival in culture. In vivo, GPR174 contributes to NUR77 expression in follicular B cells and is needed for establishing a marginal zone compartment of normal size. Treatment of mice with lysophosphatidylserine (lysoPS), a GPR174 ligand, is sufficient to promote CD86 up-regulation by follicular B cells. These findings demonstrate that GPR174 can signal via Gαs to modulate B cell gene expression and show this can occur in vivo in response to lysoPS. Additionally, the findings illuminate a pathway that might be targeted to improve systems for the in vitro study of B cell responses.

G-protein–coupled receptors (GPCRs) comprise a large family of seven-transmembrane signaling proteins, many of which have roles in the development and function of the mammalian immune system. The outcome of GPCR signaling is determined via coupling to various G proteins. Signaling via Gαi-coupled GPCRs promotes cell migration, whereas GPCR coupling to Gα12/13 proteins often leads to migration inhibition. In contrast, Gαs signaling exerts its effect through cAMP production and leads to complex and often suppressive effects on immune cell activation and proliferation. GPCR GPR174 is one of several related X-linked receptors that are highly expressed on lymphocytes (1–3). Several genome-wide association studies (GWAS) have linked GPR174-associated single-nucleotide polymorphisms (SNPs) to autoimmune disorders including Graves’ and Addison’s diseases (4–6).GPR174 was found to be a receptor for lysophosphatidylserine (lysoPS) and was suggested to signal via Gα12/13-containing heterotrimeric G proteins (1). Studies on GPR174-deficient mice revealed a role for the receptor in restraining T regulatory (Treg) cell development and function (2) and conventional T cell proliferation and IL-2 production (7, 8). In these reports, signaling was suggested to occur via Gαs-containing heterotrimeric G proteins (7, 8). In another study, GPR174 was reported to support B cell migration to spleen stromal cell culture supernatants and biochemical fractionation led to the suggestion that CCL19 and CCL21, well-defined CCR7 ligands, were ligands for GPR174 (3). In that study, GPR174 was suggested to signal via Gαi and Gα13 (3). Taken together, there is currently a lack of clear understanding regarding the signaling pathway(s) engaged downstream of GPR174.CD86 is a critical costimulatory molecule in antigen-presenting cells including B cells (9, 10). Induction of CD86 by B cell receptor (BCR) signaling is an important feature of the BCR-induced activation program, although CD86 can also be induced in B cells by CD40 and cytokine signaling (11–13). Interestingly, Cd86 was originally characterized as a cAMP-inducible gene in B cells (14–18). However, since the BCR, CD40, and cytokines are not thought to induce CD86 via cAMP, the receptors leading to cAMP-mediated induction of CD86 in B cells have been unclear.The study of B cells in vitro has been critical to numerous advances in the understanding of adaptive immunity and it remains a crucial method for dissecting the B cell response. Although not widely reported on, it is generally understood by B cell biologists that B cells undergo some amount of gene expression change during in vitro culture. Indeed, this aspect of cultured B cells presented a major challenge to efforts by the Alliance for Cell Signaling (19, 20) to dissect the signaling circuits in mouse B cells. The pathways involved in causing these gene expression changes are not understood.Here we report that cultured follicular B cells rapidly up-regulate CD86 independently of stimulation in a GPR174- and Gαs-dependent manner. The GPR174-mediated response was not dependent on exogenous ligand or BCR signaling. RNA-sequencing analysis revealed that B cells underwent changes in expression of 1,000 genes after 4 h of unstimulated culture and many of these changes were GPR174 dependent. This included induction of Cd86, Nr4a1, Ccr7, and phosphodiesterases and down-regulation of immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors. There was a strong overlap in the alterations in gene expression between GPR174- and Gαs-deficient B cells, indicating that GPR174 signals via Gαs in follicular B cells. When B cells were maintained in vitro for 1 to 2 d in the absence of stimulation, GPR174 and Gαs deficiencies were associated with augmented survival. In vivo GPR174 deficiency led to reduced NUR77-GFP reporter expression, and GPR174- and Gαs-deficient mice had a reduced marginal zone (MZ) B cell compartment. Treatment of mice with lysoPS was sufficient to cause increased CD86 expression by follicular B cells. These findings establish GPR174 as a receptor capable of exerting a large influence on B cell gene expression and they provide insight into the B cell properties that can be influenced by the receptor.