Research paper
Ex-vivo α-Galactosylceramide activation of NKT cells in humans and macaques

https://doi.org/10.1016/j.jim.2012.05.019Get rights and content

Abstract

NKT cells are key mediators of antiviral and anticancer immunity. Experiments in mice have demonstrated that activation of NKT cells in vivo induces the expression of multiple effector molecules critical to successful immunity. Human clinical trials have shown similar responses, although in vivo activation of NKT cells in humans or primate models are far more limited in number and scope. Measuring ex vivo activation of NKT cells by the CD1d-restricted glycolipid ligand α-Galactosylceramide (α-GalCer) through cytokine expression profiles is a useful marker of NKT cell function, but for reasons that are unclear, this approach does not appear to work as well in humans and non-human primate macaque models in comparison to mice. We performed a series of experiments on human and macaque (Macaca nemestrina) fresh whole blood samples to define optimal conditions to detect NKT cell cytokine (TNF, IFNγ, IL-2) and degranulation marker (CD107a) expression by flow cytometry. We found that conditions previously described for mouse splenocyte NKT cell activation were suboptimal on human or macaque blood NKT cells. In contrast, a 6 h incubation with brefeldin A added for the last 4 h, in a 96-well plate based assay, and using an α-GalCer concentration of 1 μg/ml were optimal methods to stimulate NKT cells in fresh blood from both humans and macaques. Unexpectedly, we noted that blood NKT cells from macaques infected with SIV were more readily activated by α-GalCer than NKT cells from uninfected macaques, suggesting that SIV infection may have primed the NKT cells. In conclusion, we describe optimized methods for the ex vivo antigen-specific activation of human and macaque blood NKT cells. These assays should be useful in monitoring NKT cells in disease and in immunotherapy studies.

Introduction

NKT cells are lymphocytes of the innate immune system that are important in viral and antitumor immunity through their ability to be rapidly activated and express a wide range of effector molecules (Lindqvist et al., 2009, Motohashi et al., 2009, Brigl and Brenner, 2010, Fujii et al., 2010). NKT cells are activated via an antigen-specific T cell receptor (TCR). Improved methodologies to assess the activation of NKT cells ex vivo should assist dissecting the importance of these immune cells in humans and non-human primates.

Type I or semi-invariant NKT cells express an invariant TCR-α chain (Vα14-Jα18 in mice, Vα24-Jα18 in humans) and are restricted by the MHC-class Ib molecule, CD1d, expressed on APCs presenting lipid based antigens such as glycolipid antigens (Exley et al., 1997). Lipid based antigens are presented to NKT cells by direct ligation onto surface CD1d molecules or internalized into endosomes where loading onto CD1d occurs and subsequent membrane surface presentation on a lipid raft (Venkataswamy and Porcelli, 2010). NKT cells respond to a broad variety of lipid-based antigens including self and foreign glycolipid and phospholipid antigens (Venkataswamy and Porcelli, 2010). α-galactosylceramide (α-GalCer) is a synthetic glycosphingolipid derived from the marine sponge, Agelas mauritianus, and is commonly used in mice and human NKT studies as a potent activator of NKT cells in vivo or in vitro (Kawano et al., 1997). α-GalCer has immunomodulatory effects in cancer immunotherapy (Giaccone et al., 2002, Chang et al., 2005, Uchida et al., 2008, Kunii et al., 2009, Motohashi et al., 2009, Schneiders et al., 2011, Yamasaki et al., 2011, Vivier et al., 2012), autoimmunity (Novak and Lehuen, 2011, Hong et al., 2001, Wu and Van Kaer, 2009), bacterial (Brigl and Brenner, 2010, Emoto et al., 2010), and viral infections (Guillonneau et al., 2009, Lindqvist et al., 2009, Schneiders et al., 2011).

Upon activation with α-GalCer, NKT cells produce large amounts of Th1, Th2 and Th17 cytokines such as IFNγ, TNF, IL-2, IL-4, IL-10, IL-13, IL-17, IL-21 and IL-22 (Godfrey et al., 2010). The cytokines produced by NKT cells trigger the activation of other cells of the immune system such as NK cells, T and B cells and DCs (Fujii et al., 2003, Cerundolo et al., 2009). The detection of the expression of these cytokines by intracellular cytokine staining and flow cytometry permits an analysis of NKT cell functional status.

Studies on α-GalCer activation of mice NKT cells often involve in vivo activation, where α-GalCer induced cytokine production by NKT cells, and indirectly downstream by NK cells, is measured in serum (Sullivan et al., 2010), or directly ex-vivo following α-GalCer administration in vivo (Wilson et al., 2003, Uldrich et al., 2005). In the latter case mice are administered α-GalCer, organs such as spleen, liver or lymph node are harvested a few hours after challenge and the relevant cells placed in culture with a protein transport inhibitor, without any further activation. Alternatively, the global mitogen-induced activation of NKT cells ex vivo is often assessed by PMA/ionomycin stimulation. Such mitogenic stimulation, while providing information on the total functional potential of NKT cells, may not reflect the in vivo capacity of NKT cells when interacting with antigen presented by CD1d. Furthermore, activation with PMA/ionomycin poses difficulties in enumerating intracellular cytokine production from NKT cell subsets as the CD4 surface marker is downregulated upon mitogenic stimulation (O'Neil-Andersen and Lawrence, 2002). Many other studies have assessed activation of mouse NKT cells ex vivo with α-GalCer in combination with protein transport inhibitors such as monensin to detect intracellular cytokines produced in vitro (Uldrich et al., 2005, Patel et al., 2011).

It is important to translate advances in understanding NKT cell biology in mice towards more directly relevant non-human primate models and humans. For example, there is considerable interest in harnessing the antiviral activity of NKT cells in the setting of chronic viral infections such as HIV infection of humans or SIV infection of macaques (Fernandez et al., 2009, Snyder-Cappione et al., 2009). However, such studies require ex vivo/in vitro analysis of NKT cells, yet at present the literature is lacking in clearly defined optimized methods for acute ex-vivo activation of NKT cells with α-GalCer either in humans or macaques. The relative ability of fresh blood NKT cells to respond to acute ex vivo activation with cognate ligands such as α-GalCer has not been studied in HIV infection, or SIV infection, compared to naïve SIV uninfected macaques. Such assays would be useful in immunotherapy or vaccine clinical or pre-clinical trials designed to harness NKT cell based immunity. In this study we assessed ex vivo NKT function upon stimulation of human and macaque blood with α-GalCer under a variety of experimental conditions to optimize methods for NKT cell activation.

Section snippets

Healthy human subjects and animals

For all studies on optimized NKT cell activation methods (Fig. 2, Fig. 3, Fig. 4, Fig. 5) we studied subjects and animals as detailed below. Healthy HIV uninfected human volunteers (n = 5 aged 23–48 years; four females, one male) with varying frequencies of peripheral NKT cells (range of 0.03%-0.23%) were recruited for this study. We also studied a total of 9 juvenile pigtail macaques (Macaca nemestrina) aged 2 to 6 years. Five macaques were infected with SIVmac251 intravaginally and were 21–28 

Activation of NKT cells in macaques using standard conditions studied in mice

Our goal was to develop reliable conditions to assess antigen-specific NKT cell activation in macaque or human blood samples. To assess the ability of primate NKT cells to respond to α-GalCer-induced activation ex vivo, we first assayed the effectiveness of the same experimental conditions used in mouse lymphoid cells (Uldrich et al., 2005, Patel et al., 2011) on the ex vivo activation of pigtail macaque or human peripheral blood NKT cells. We gated on NKT cells as previously described (

Discussion

The ability of NKT cells to rapidly respond with production of a variety of cytokines and other effector functions is key to their potent antiviral and anti-tumor properties. The study of antigen-driven NKT cell activation in macaque and human whole blood samples is hampered by relatively weak cytokine responses, compared to similar assays using mouse NKT cells. We refined and optimized methods to study activation of human or macaque NKT cells with the prototypic ligand, α-Galactosylceramide.

Acknowledgments

We thank Wendy Winnall, Konstantinos Kyparissoudis, Angela Chan, Sheilajen Alcantara, Pradeep Sidana, and Megan Schepers for expert assistance and advice. This research was supported by Australian National Health and Medical Research (NHMRC) awards 510448, 629000 and 454569. GC is supported by a Cancer Research Institute postgraduate scholarship. DIG is supported by an NHMRC research fellowship 454309; SJK is supported by an NHMRC research fellowship 508937.

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