Elsevier

Molecular Immunology

Volume 49, Issues 1–2, October–November 2011, Pages 253-259
Molecular Immunology

IDO expression by human B lymphocytes in response to T lymphocyte stimuli and TLR engagement is biologically inactive

https://doi.org/10.1016/j.molimm.2011.08.017Get rights and content

Abstract

The immune system must be under tight control to avoid undesired responses. The enzyme indoleamine 2,3-dioxygenase (IDO) can exert necessary regulating effects by catabolizing tryptophan, leading to the suppression of immune responses in different settings, such as pregnancy and tumor growth. IDO's immuno-suppressive actions are mediated by tryptophan starvation and the accumulation of toxic tryptophan metabolites, resulting in T cell anergy, inhibition of clonal expansion or apoptosis. IDO activity in human macrophages and dendritic cells has been observed after interaction with T lymphocytes, and is triggered by interferon-gamma (IFN-γ) as well as CD40-ligand (CD40L). However, it is unclear whether IDO activity is present in B lymphocytes, which have been identified as having suppressive properties involved in anti-tumor immunity inhibition. In this study, we investigated whether IDO expression is induced in human B cells after exposure to T lymphocyte stimuli and TLR ligands. We report IDO1 and IDO2 mRNA up-regulation by exogenous stimulation with CD40L and IFN-γ. IDO is also upregulated by imiquimod, a TLR 7/8 agonist. In addition, IDO protein is detected after treatment with these exogenous factors or with supernatant from activated CD4+ T cells. We, however, report weak or absent enzymatic activity from these IDO-expressing cells, as assessed by tryptophan consumption. We conclude that IDO may not be a counter-regulatory mechanism utilized by B lymphocytes to down-regulate immune responses, although its expression is inducible.

Highlights

IDO mRNA and protein are expressed in human B lymphocytes when activated with CD40L and IFN-γ. ► TLR7/8 synergizes with CD40L + IFN-γ stimulation to enhanced IDO mRNA. ► The IDO expressed in human B lymphocytes is enzymatically inactive when tryptophan catabolism is measured by HPLC.

Introduction

Counter-regulatory mechanisms are necessary in the immune system to avoid inappropriate or excessive responses (reviewed in Kyewski and Klein, 2006) and promote contraction phases. By limiting the intensity and extent of immune responses, these inhibitory mechanisms prevent further damage to the host. Such functions can be fulfilled by the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Degradation of the essential amino acid tryptophan by IDO, depleting tryptophan from the intracellular pool or local microenvironment, was first described as a defense mechanism against intracellular pathogens (Pfefferkorn, 1984, Taylor and Feng, 1991). Its role was thereafter extended to fetal protection against maternal T cells, when Munn et al. (1998) demonstrated that administration of the IDO inhibitor 1-methyl-tryptophan (1MT) to pregnant mice led to fetal rejection. This pivotal study ascribed a new immunosuppressive function to the enzyme, and prompted an intense search for its contribution in tumor escape (Friberg et al., 2002, Uyttenhove et al., 2003). In fact, enzymatically active IDO and the recently identified IDO2 proteins (Ball et al., 2007, Metz et al., 2007) lead to tryptophan starvation and downstream metabolite accumulation. This culminates in multiple effects on immune cells, such as T lymphocyte proliferative arrest (Munn et al., 1999, Frumento et al., 2002, Terness et al., 2002), anergy (Munn et al., 2004a), apoptosis (Fallarino et al., 2002a), conversion of naive CD4+ T cells to regulatory T cells (Tregs) (Fallarino et al., 2006), and activation of mature CD4+CD25+Foxp3+ Tregs (Sharma et al., 2007).

Active IDO is induced in human macrophages and monocyte-derived dendritic cells (DC) upon interaction with T lymphocytes, and this induction is dependent not only on interferon-gamma (IFN-γ) production, but also on CD40-ligand (CD40L) up-regulation on these T cells (Munn et al., 1999, Hwu et al., 2000). Moreover, a subtype of IDO+ plasmacytoid DC, co-expressing a B-lineage marker, can be found in tumor-draining lymph nodes of tumor-bearing mice or patients, creating a suppressive environment and culminating in a profound anergic state of anti-tumor T lymphocytes (Munn et al., 2004a).

In the course of an effective humoral response, B lymphocytes interact closely with T lymphocytes, and signals are exchanged through cell-cell contacts involving major histocompatibility complex/T cell receptor (MHC/TCR) and CD40/CD40L interactions, and cytokines such as IFN-γ are released. Based on what is known about IDO induction in macrophages and DC, this counter-regulatory mechanism also possibly arises in B lymphocytes after T lymphocyte encounter. B cell-deficient mice have been shown to mount a more effective anti-tumor response in comparison to wild type mice, suggesting that B cells hamper anti-tumor immunity (Inoue et al., 2006, Shah et al., 2005). Such suppressive activity has been associated with interleukin (IL)-10 production by B cells after CD40 ligation, leading to decreased IFN-γ production by T lymphocytes, at least in vitro (Inoue et al., 2006). However, it is not clear whether B lymphocyte inhibitory effects depend on the same mechanism of action, to prevent anti-tumor responses effectiveness in vivo (Rosenblatt et al., 2007). Some other mechanism orchestrated by B cells might participate in the inhibition of anti-tumor responses in vivo.

A recent study demonstrated the existence of a murine PDCA-expressing B lymphocyte subpopulation, in which IFN-α and IDO1/IDO2 are induced at the mRNA level upon stimulation with cytotoxic T lymphocyte antigen-4-immunoglobulin (CTLA4-Ig). However, neither protein expression nor enzymatic activity was evaluated (Vinay et al., 2010). Also, a B-lymphoid population with DC attributes (CD19+, CD11c+) was able to induce Treg activation by IDO expression upon CpG oligonucleotide stimulation (Johnson et al., 2010). In the present work, we investigated whether IDO expression could be up-regulated in human B lymphocytes in response to T cell signals. In addition, we sought to determine whether this induction would beget a functional enzyme capable of metabolizing tryptophan. We found that IDO was produced by human B cells at the mRNA and protein levels after stimulation with IFN-γ, CD40L and imiquimod (TLR 7/8 ligand), but in an inactive form, indicated by the lack of tryptophan consumption and kynurenine accumulation.

Section snippets

Healthy donors and cells

Heparinized blood obtained by leukapheresis from healthy donors was centrifuged on lymphocyte separation medium (Wisent, St-Bruno, Québec, Canada) to isolate peripheral blood mononuclear cells (PBMC). Donors were recruited by the Division of Hematology and Immunodeficiency Service of Royal Victoria Hospital. The study was approved by the local ethics committee, and informed consent was obtained from each donor.

All cell lines were procured from the American Type Culture Collection (Manassas,

IDO mRNA expression in B lymphocytes in response to exogenous cytokines

To determine whether stimuli from activated T cells could up-regulate IDO expression in B lymphocytes, we treated isolated human B cells with either soluble trimeric CD40L or IFN-γ or both. We evaluated IDO expression in B lymphocytes from multiple donors, and observed that single or combined treatment induced IDO, as detected by standard RT-PCR (Fig. 1A). Quantitative PCR allowed us to determine that IDO mRNA was expressed at comparable levels in CD40L- and IFN-γ-activated B cells (Fig. 1B).

Discussion

Cellular cross-talk between B and T lymphocytes is central to humoral responses. After antigen recognition by B cell receptors, followed by processing and presentation of this antigen, B lymphocytes must receive signals from CD4+ helper T cells specific to the same antigen to start antibody production. These include contact-mediated signals through MHC/TCR, B7/CD28 and CD40/CD40L, but also cytokines secreted by CD4+ T cells (reviewed in Mills and Cambier, 2003). It has been known for over a

Conflicts of interest

The authors have no financial conflicts of interest.

Acknowledgments

This work was supported by operating grants from the Natural Sciences and Engineering Research Council [grant number 340955-2010]; the Canadian Institutes of Health Research [grant number MOP-89727]; and by a fellowship from Fonds de la recherche en santé du Québec to [RL].

Healthy donor samples were provided by Dr. Jean-Pierre Routy and Dr. Mohamed-Rachid Boulassel from the Division of Hematology and Immunodeficiency Service of Royal Victoria Hospital. The editorial assistance of Ovid Da Silva

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