IL4‐induced gene 1 is secreted at the immune synapse and modulates TCR activation independently of its enzymatic activity

Abstract

Amino-acid catabolizing enzymes produced by mononuclear phagocytes play a central role in regulating the immune response. The mammalian phenylalanine-catabolizing enzyme IL4-induced gene 1 (IL4I1) inhibits effector T lymphocyte proliferation and facilitates regulatory T-cell development. IL4I1 expression by macrophages of various human tumors may affect patient prognosis as it facilitates tumor escape from the T-cell response in murine models. Its enzymatic activity appears to participate in its effects, but some actions of IL4I1 remain unclear. Here, we show that the presence of IL4I1 during T-cell activation decreases early signaling events downstream of TCR stimulation, resulting in global T-cell inhibition which is more pronounced when there is CD28 costimulation. Surprisingly, the enzymatic activity of IL4I1 is not involved. Focal secretion of IL4I1 into the immune synaptic cleft and its binding to CD3 + lymphocytes could be important in IL4I1 immunosuppressive mechanism of action. Keywords: CD28 stimulation r Immunosuppressive enzymes r T cells r TCR signalingAdditional supporting information may be found in the online version of this article at the publisher's web-site IntroductionClonotypic TCR engagement by antigen presenting cells via the MHC/peptide complex is the central event of T-cell activation.Correspondence: Dr. Flavia Castellano and Dr. Valérie Molinier-Frenkel e-mail: flavia.castellano@inserm.fr; valerie.frenkel@inserm.fr TCR signaling consists of a molecular cascade propagated from the cytosol to the nucleus via a multilayered signal-transduction network. This network involves proximal kinases, such as Lck and ZAP70, and a molecular platform of adaptor proteins, such as LAT * These authors contributed equally to this work.C 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2018. 48: 106-119 Molecular immunology and signaling 107 Figure 1. IL4I1 decreases early TCR signaling. (A & B) CD3+ T lymphocytes obtained by negative sorting were stimulated with an anti-CD3 mAb alone (A) or together with an anti-CD28 mAb (B) in conditioned media from HEK cells that produce IL4I1 (IL4I1) or those that do not (Ctrl) for 0 (unstimulated), 1, or 2 min. Cell lysates were analyzed by western blotting with an anti-phospho-ZAP70 mAb (Y319). One representative experiment is shown in the upper panels. Relative quantification of the bands (ratio phospho-ZAP70 to total ZAP70) is shown in the lower panels (A and B: average of 3 and 5 independent experiments, respectively). (C) Western blot analysis of phosphorylated ZAP70 (Y319), ERK p42 (T202/Y204), and p44 (T185/Y187) after activation of T cells as in B, with 100 ng purified recombinant IL4I1 (rIL4I1) or without (Ctrl) for 0 (unstimulated), 2, or 5 min. One representative experiment of 3 independent experiments is shown. Numbers under each blot represent the OD ratios of the respective phosphorylated protein over total Western blot analysis of phosphorylated PLC-γ (Y783) after T-cell activation as in A and B, respectively, in c...

Emerging Role of IL-4–Induced Gene 1 as a Prognostic Biomarker Affecting the Local T-Cell Response in Human Cutaneous Melanoma

“…These observations suggest that IL4I1 can affect both the proliferation and function of this subset. This is supported by several of our previous results: (i) IL4I1 inhibits in vitro human T-cell proliferation by down-regulating TCR signaling (Aubatin et al, 2017;Boulland et al, 2007), (ii) the in vivo escape of B16 melanoma overexpressing IL4I1 is accompanied by reduced expansion of cytotoxic anti-tumor CD8 þ T cells (Lasoudris et al, 2011), and (iii) genetic inactivation of IL4I1 in the mouse Ret model of spontaneous melanoma leads to an increased infiltration of cytotoxic CD8 þ T cells in primary tumors (Bod et al, 2017).…”

“…IL4I1 is a secreted L-amino acid oxidase that mainly catabolizes L-phenylalanine to produce hydrogen peroxide, ammonia, and phenylpyruvate. In the past 10 years, IL4I1 has been shown to be expressed by tumor-associated macrophages (TAMs) of numerous cancer types and to regulate T-cell properties both in vitro and in vivo (Aubatin et al, 2017;Boulland et al, 2007;Cousin et al, 2015;Lasoudris et al, 2011;Santarlasci et al, 2012).…”

Inflammatory Dendritic Cells, Regulated by IL-4 Receptor Alpha Signaling, Control Replication, and Dissemination of Leishmania major in Mice

“…Recent studies have demonstrated other mechanisms of alternative activation of macrophages. For example, interleukin-4-indiced gene 1 (IL-4I1) has been defined as a strong modulator of alternative macrophage activation (Yue et al, 2015), and is also expressed on DCs (Aubatin et al, 2018).…”

An Overview of l-Amino Acid Oxidase Functions from Bacteria to Mammals: Focus on the Immunoregulatory Phenylalanine Oxidase IL4I1

“…Our group has shown that mouse and human IL4I1 are glycosylated and secreted LAAOs that preferentially degrade phenylalanine and, to a lesser extent, arginine [ 11 , 16 ]. IL4I1 limits T lymphocyte activation and proliferation [ 16 , 58 ], in part via the production of H 2 O 2 ( Figure 2 ). A recent interaction of IL4I1 with T lymphocytes has been shown, which could participate in its inhibitory functions, either by concentrating the enzyme at the T cell surface or by mediating a negative intracellular signal [ 58 ].…”

“…IL4I1 limits T lymphocyte activation and proliferation [ 16 , 58 ], in part via the production of H 2 O 2 ( Figure 2 ). A recent interaction of IL4I1 with T lymphocytes has been shown, which could participate in its inhibitory functions, either by concentrating the enzyme at the T cell surface or by mediating a negative intracellular signal [ 58 ].…”