Elsevier

Cytokine

Volume 65, Issue 1, January 2014, Pages 33-41
Cytokine

Expression regulation of co-inhibitory molecules on human natural killer cells in response to cytokine stimulations

https://doi.org/10.1016/j.cyto.2013.09.016Get rights and content

Highlights

  • Co-inhibitors express differently in response to varied cytokine stimulations.

  • IL-12 is a powerful inducer of LAG-3.

  • TGF-β is a powerful suppressor of PD-1.

  • Modulation of co-inhibitors may be a potential approach for cancer immunotherapy.

Abstract

Co-inhibitory molecules have become the key targets in cancer immunotherapy with the strategy of blocking immune checkpoints to reverse the pathogenic regulation of T cells. However, their expression regulations in NK cells, the most important innate immune cells against tumor, remain largely undefined. In this study, we showed that the expressions of co-inhibitors on NK cells, including LAG-3, PD-1, and TIGIT, are differently regulated by cytokines IL-10, IL-12, IL-15, IFN-α, and TGF-β. Among the tested cytokines, IL-12 is the most powerful inducer of LAG-3, and TGF-β is the strongest suppresser of PD-1. Notably, the expression of these co-inhibitors responds to the time course of stimulus progressively. Together, these findings illustrated that the co-inhibitors on NK cells express differently in response to cytokine stimulations of IL-10, IL-12, IL-15, IFN-α, and TGF-β, providing an initial information on the expression regulation of co-inhibitors in human NK cells.

Introduction

Natural killer (NK) cell is one of the three major lymphocytes arisen from bone marrow progenitors, distincts from T and B cells due to larger size and cytoplasmic granules. Unlike T and B cells, NK cell does not express receptors requiring somatic gene rearrangement, carries out its effector functions without prior sensitization to antigens, and kills target cells by releasing cytotoxic granules such as granzymes and perforins [1]. NK cells are the first line of defense in innate immunity against pathogens and tumors, and play key roles in immunodefense, immunosurveillance, and immunohomeostasis [2]. The cytotoxicity of NK cells is strictly regulated by the complex repertoires of activating and inhibitory receptors to ensure proper activation against pathogens while preventing inadvertent attacks to normal cells [3].

Activation of NK cells is solely dependent on the balance between activating signals and inhibitory signals induced by their receptors. When NK cells are in contact with normal cells, the binding between major histocompatibility complex (MHC) class I molecules on the normal cells and their recognized inhibitory receptors on NK cells induces inhibitory signals to override any activation signals and thus keeping NK cells in calm [4]. However, when NK cells are engaged with abnormal or stressed cells, with either altered or lost MHC class I molecules, NK cells would be fully activated due to the absence or weakening of MHC class I-induced inhibitory signals [4]. Though the concept of negative regulation by co-inhibitory molecules was proposed earlier, it has not become the forefront of immunological research until recently, with the identification of numerous co-inhibitory molecules including lymphocyte activation gene-3 (LAG-3), programmed death-1 (PD-1), and T cell immunoglobulin and ITIM domain (TIGIT), and their critical functions in immunotherapy [5].

Lymphocyte activation gene-3 (LAG-3), also termed as CD223, is a member of immunoglobulin superfamily, owns a structure resembling that of CD4, with four extracellular IgG domains, and binds to MHC class II molecule [6]. LAG-3 expresses on plasmacytoid dendritic cells, B cells, NK cells, NKT cells, γδT cells, activated T cells, regulatory T cells, and tumor-infiltrating lymphocytes. It has a unique cytoplasmic tail with two special motifs: KIEELE motif is essential for inhibitory functions; and EP repeat, contributes to LAG-3 localization by binding to LAG-3-associated protein (LAP) [7], [8]. The membrane-bound LAG-3 expresses as a dimer on the cell surface, which can be cleaved by metalloproteases ADAM10 and ADAM17, inducing a soluble form of LAG-3 [9], [10], that may be further artificially modified into a recombinant soluble dimeric form of LAG-3, also known as IMP321 (sLAG-3), which has been intensively tested in clinical trials [11], [12].

Inhibitory molecules of the B7/CD28 family play key roles in immune suppression and immune tolerance. Programmed death-1 (PD-1), also termed as CD279, is a distant member of CD28 family, with a single immunoglobulin superfamily domain and a cytoplasmic domain containing two motifs: an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immnoreceptor tyrosine-based switch motif (ITSM) [13], [14]. PD-1 interacts with its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), inducing inhibitory pathways downstream [15], [16], [17], [18]. On the other hand, these inhibitory signals contribute to the immunosuppressive tumor microenvironment, and therefore, clinical blockade of these pathways has validated its potential as a target for cancer immunotherapy [18], [19].

T cell immunoglobulin and ITIM domain (TIGIT), is a recently identified co-inhibitory molecule expressed on T and NK cells with an immunoglobulin variable (IgV) domain, a transmembrane domain, and an immunoreceptor tyrosine-based inhibitory motif (ITIM) that binds to poliovirus receptor (PVR; CD155), nectin-2 (CD112), and possibly nectin-3 (CD113) [20], [21], [22]. Yu et al. have originally identified TIGIT on T cells with the function of inhibiting T cell activity indirectly through the induction of tolerogenic dendritic cells [20]. However two years later, Joller et al. have proved that TIGIT may inhibit T cell responses directly independent of APCs by using an agonistic anti-TIGIT antibody [23]. On the other hand, in human NK cells, TIGIT inhibits NK cytotoxicity directly by binding to PVR and nectin-2 through its ITIM [21].

Cytokines have been well known for their abilities in signal transduction and communication governing between cells. They may as well modulate the expressions of co-inhibitory molecules on different cells. For example, the immunosuppressive potential of activated Treg may be increased by up-regulating the expression of PD-1 and the secretion of IL-10 and TGF-β [24]. The expression of LAG-3 is strongly up-regulated in CD4+ and CD8+ T cells when primed with IL-12 [25], [26]. The expression of LAG-3 is up-regulated by IL-2, IL-7, and IL-12, but not by IL-4, IL-6, TNF-α, TNF-β, and IFN-γ on activated human T cells [27].

In the present study, we investigated the effects of IL-10, IL-12, IL-15, IFN-α, and TGF-β on regulating the transcription of co-inhibitory molecules, including LAG-3, PD-1, and TIGIT on human NK cells. We used real-time PCR to examine the changes in the mRNA level of co-inhibitory molecules after NK cells were stimulated with different dosages of these cytokines for various time intervals. The expression of co-inhibitory molecules was differently regulated by the tested cytokines and this regulatory effect was dependent on time course of the stimulation. Our work provides initial information on the expression regulation of co-inhibitors on human NK cells and directs new insights into future researches on the understanding of immune networks and the potential interventions in cancer immunotherapy.

Section snippets

NK cell culture

NK-92 cells were cultured in modified α-MEM medium (Thermo Scientific, Waltham, Massachusetts, USA) supplemented with 12.5% fetal bovine serum (ExCell Biology, Shanghai, China), 12.5% horse serum (Thermo Scientific, Waltham, Massachusetts, USA), 100 IU/ml recombinant human IL-2 (Jinsili, Yixing, Jiangsu, China), 100 IU/ml penicillin and 100 μg/ml streptomycin (Beyotime, Nantong, Jiangsu, China), cultures were kept at 37 °C in 5% CO2 humidified atmosphere.

Total RNA extraction and reverse transcription

NK-92 cells were stimulated with recombinant

Expression regulation of co-inhibitory molecules by IL-15 in human NK-92 cells

Interleukin-15 (IL-15) is essential for NK cell development, differentiation, and survival. In order to explore the effects of this essential NK cell developmental cytokine on the gene regulation of co-inhibitory molecules, NK-92 cells were cultured and stimulated with IL-15 at varying dosages and time intervals. In a solely time-dependent study (Fig. 1A), NK-92 cells were stimulated with 10 ng/ml IL-15 and the relative mRNA level of co-inhibitory molecules LAG-3, PD-1, and TIGIT was analyzed

Discussion

The balance between pro-inflammatory and anti-inflammatory is critical in regulating immune responses, which requires co-operations between different immune cells that are controlled by receptor-ligand interactions and cytokines. Co-inhibitory molecules work alone or together to prevent over-activation of immune cells, and their expressions and functions vary in T cells and NK cells. Although LAG-3 was initially discovered on NK cells, intense studies have been conducted on T cells only. LAG-3

Conclusion

Co-inhibitory molecules on human NK cells respond differently to varied cytokine stimulations. LAG-3 is significantly induced by IL-12; PD-1 is significantly induced by IL-12 and IFN-α while suppressed by IL-10 and TGF-β; and TIGIT shows irresponsiveness to IL-12, IL-15, IFN-α, and TGF-β. In addition, the expression regulation of these co-inhibitory molecules is time-dependent. This study provides insights into the regulation of co-inhibitory molecules by different cytokines, indicating that

Acknowledgments

This work was supported in part by funds from the National Basic Research Program of China (2013CB944903 and 2012CB825800), the National Natural Science Foundation of China (Grant No. 91029710, 81071683 and 81272327), the National Hitech Project (863 project, #2012AA020901), and the National Science & Technology Major Projects (2012ZX10002014).

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