B cells secrete GABA, which provokes a pro-tumor immune microenvironment

Abstract

In a recent publication in Nature, Zhang et al. report that foreign antigen stimulation elicits bountiful changes in lymphatic metabolite production—changes that include B cells secreting GABA, which reprograms macrophages and limits T cell cytotoxicity. This signifies a new mechanism by which B cells regulate immune suppression and facilitate tumor progression.

Advances in the field of cancer immunometabolism have revealed that metabolic crosstalk impacts anti-tumor immune potential. A recent study by Zhang et al. published in Nature reveals lymphatic B cell secretion of γ-aminobutyric acid (GABA) and GABA positive B cells as suppressors of cytotoxic T cells (Zhang et al., 2021). The current knowledge of GABA activity connects this metabolite to diverse functions in the body, including cellular protection, development, and differentiation. GABA is made from glutamine, glutamate, and glucose by the glutamic acid decarboxylase enzymes GAD65 and GAD67. GABA can bind to GABA-A, B, and C receptors with differing function. Although it is commonly appreciated as a neurotransmitter, recent studies show that GABA is secreted by a variety of cell types, including cancer cells, dendritic cells, T cells, natural killer (NK) cells, macrophages, and with this discovery from the Fagarasan lab, B cells.

Human prognostic data and mouse models suggest that there are pro- and anti-tumor B cell subsets (Sharonov et al., 2020; Hollern et al., 2019; Roghanian et al., 2016). Anti-tumor B cell subsets, such as those found within tertiary lymphoid structures, activate T helper cells to effector and memory cell types through antigen presentation and molecular stimulation. T helper cells, in turn, support B cell activation with cytokines and ligands to foster B cell differentiation to memory and antibody-secreting cells. Among the protumor B cells, B regulatory cells and regulatory plasma cells (a subset of antibody-secreting B cells) express immunosuppressive cytokines (IL-10, IL-15, IL-35, and PDL-1) which dampen T cell cytotoxicity and support an anti-inflammatory microenvironment. Revealing a new mechanism of immune suppression, Zhang et al. uncover B cells that produce and secrete GABA, which fosters anti-inflammatory macrophage phenotypes and limits T cell cytotoxicity.

Following vaccination with ovalbumin (a model foreign antigen), Zhang and colleagues identified numerous changes in metabolite production within the antigen experienced lymph node (Figure 1A). These changes included a prominent increase in GABA concentration. Genetic models further showed that lymphatic elevation of GABA required an intact B cell compartment. In an eloquent validation experiment, the authors used labeled glutamine and cell sorting to verify that B220⁺ B cells in the lymph node were major producers of GABA following antigen stimulation.

Figure 1. Zhang et al. highlight the importance of B cell metabolites in immune cell regulation and suppression of anti-tumor immunity.

(A) Upon antigen encounter, lymphatic B220⁺ B cells secrete γ-aminobutyric acid (GABA) through the enzyme GAD67.

(B) GABA binds to GABA-A receptors on other immune cells, thus provoking tumor growth, reducing cytotoxic CD8⁺ T cell activity, and upregulating the IL-10 ligand and receptor expression in macrophages, thereby polarizing macrophages to an anti-inflammatory phenotype.

GABA dampens T cell cytotoxicity (Tian et al., 1999), and it is well established that B cells engage in immunosuppression in the MC38 colorectal cancer (CRC) mouse model (Zhang et al., 2013). This prompted the authors to test whether B cell GABA secretion impacts tumor progression and immune suppression of T cells. In the MC38 mouse model, enhanced T cell cytotoxicity and control of tumor growth was achieved with genetic deletion of B cells. However, when B cell knockout mice were given a slow-release GABA pellet, MC38 tumors grew faster, and cytotoxic T cell activity was suppressed. This implied that B cell GABA production suppressed anti-tumor immunity. More evidence of this mechanism came by translational experiments using a GABA-A receptor antagonist picrotoxin. Picrotoxin limited tumor growth and enhanced T cell cytotoxicity in wild-type mice with MC38 CRC. Solidifying the conclusion of these findings, genetic knockout of GAD67 in B cells led to reduced GABA levels and superior control of tumor progression coinciding with enhancement of T cell activation. These data support a new suppressive function for B cells in which secreted GABA suppresses the anti-tumor immune response and T cell cytotoxicity (Figure 1B).

B regulatory cells suppress T cells and polarize macrophages to a protumor cell state via IL-10 (Schwartz, Zhang and Rosenblatt, 2016). Thus, the authors next tested whether B cell-secreted GABA might function with IL-10 to control the polarization of macrophages in the tumor. GABA signaling increased expression of IL-10 receptors and reprogrammed macrophages to an anti-inflammatory phenotype. Adoptive transfer of macrophages co-cultured with B cells or treated in vitro with IL-10 led to enhanced tumor growth and suppression of CD8⁺ T cell activity in vivo beyond the effects of singular IL-10 treatment. Together, these results suggest a cooperative B cell-macrophage interaction and that GABA modulates IL-10 signaling. They also indicate that GABA amplifies anti-inflammatory responses by targeting multiple immune cells.

During vaccination, B cells have been demonstrated to molecularly stimulate and present antigens to CD4⁺ T cells (Whitmire et al., 2009), thereby eliciting CD8⁺ and CD4⁺ T cell memory and effector function. Adding to the heterogenous functions of B cells in antigen-driven responses, Zhang et al. find that a major component of the lymphatic B cell metabolite response to ovalbumin serves to suppress T cells. Because their measurements were based on B220⁺ B cells, it is unclear which conventional B cell subsets are engaged in GABA secretion, although the nature of the described B cell-macrophage interactions is reminiscent of B regulatory cells. Because B cells also express GABA-A receptors, it could be possible that the measured GABA in macrophages consequentially reinforces B regulatory phenotypes and high levels of B cell GABA production. Future molecular phenotyping and positioning of lymphatic GABA-producing B cells should help resolve questions about these GABA-secreting B cells that suppress T cells from those that support their activity. Additional clarification is needed to determine whether the B cell subset(s) that secrete GABA are located within the tumor or if they are specific to the lymph node. Such results could be accomplished through the use of glutamine tracing and flow cytometry approaches established by Zhang et al. to determine whether these mechanisms influence the CD8⁺ tumor infiltrates by B cell dynamics in the tumor-draining lymph node or within the tumor.

Across solid tumor types, patient prognosis data indicate that tumor-infiltrating B cells frequently facilitate longer patient survival (Sharonov et al., 2020). However, there are also frequent instances in which immunosuppressive B cells are found in mouse and human tumors. Thus, despite a disconnect between prognostically favorable B cells in the majority of human CRCs (Edin et al., 2019) and the pro-tumor B cell function in the MC38 CRC mouse model (Zhang et al., 2013), the work of Zhang et al. holds broad implications for improving the therapeutic treatment of cancer. In cancers that cultivate suppressive GABA⁺ B cell activity, targeting metabolite signaling may enhance therapy. Indeed, the authors’ use of picrotoxin provides a rationale for targeting metabolite signaling to enhance the efficacy of immune-checkpoint and CAR-T therapies. Theoretically, future studies might also learn how to leverage GABA selectively in order to limit specific immune related adverse events. Indicating even broader application, Zhang et al. show that exogenous GABA can dampen T cell responses in the absence of B cells. This adds potential insight as to how alternative sources of GABA such as the stroma, microbiome, and cancer cells themselves might influence the immune microenvironment and tumor progression. GABA has been observed to have intrinsic effects on tumor cell invasion and migration. As a result, the immunosuppressive GABA signaling in the lymph node may also contribute to metastatic spread and colonization. Therefore, Zhang et al.’s work should prompt expanded inquiry into the cell and molecular regulation of GABA secretion and its impact on cells with GABA receptors. More broadly, this work reinforces the need to decipher the metabolic crosstalk within the tumor microenvironment, as emerging studies continue to advance definitions of immune cell heterogeneity, regulation, and function while also revealing novel modes for therapeutic control of tumor progression through immunomodulation and metabolites.