Elsevier

Cellular Immunology

Volume 277, Issues 1–2, May–June 2012, Pages 33-43
Cellular Immunology

Costimulatory activation of murine invariant natural killer T cells by toll-like receptor agonists

https://doi.org/10.1016/j.cellimm.2012.06.002Get rights and content

Abstract

Invariant NKT (iNKT) cells are glycolipid-reactive lymphocytes with anti-microbial properties. Toll-like receptor (TLR)-primed antigen-presenting cells are known to activate iNKT cells, however, the expression and function of TLRs in iNKT cells remain largely unknown. Here, we show that TCR-activation of murine iNKT cells by α-GalactosylCeramide (α-GalCer) or anti-CD3 antibodies can result in increased expression of TLR genes. TLR3, 5 and 9-mediated costimulation of TCR-preactivated iNKT cells resulted in enhancement of iNKT cell activation, as determined by their cytokine production. Expression of TLR3 and 9 at protein level was also confirmed in TCR-activated iNKT cells. Furthermore, TCR-preactivation followed by TLR9-costimulation of iNKT cells increased their ability to induce maturation of dendritic cells. Thus, our findings show that iNKT cells can up-regulate their TLR expression upon TCR activation and a subsequent TLR-signaling in these cells can lead to their enhanced activation, suggesting a new possible mode of iNKT cell activation.

Highlights

► Stimulation of invariant natural killer T cells results in up-regulation of Toll-like receptors. ► TLR agonist treatment of iNKT cells activates these cells. ► Treatment with TLR ligands increases iNKT cell ability to induce DC maturation.

Introduction

Invariant NKT (iNKT) cells are an important subset of αβ TCR+ T lymphocytes that express an invariant TCR and recognize glycolipid antigens. Alpha-GalactosylCeramide (α-GalCer), a glycolipid antigen, is a potent activator of iNKT cells. Upon activation, iNKT cells can produce large amounts of T helper (Th)1-type and/or Th2-type cytokines. The importance of iNKT cells in the regulation of immune responses, in preventing autoimmune diseases and in immunity against microbial pathogens has been well documented [1], [2], [3], [4].

iNKT cells can be activated either by direct TCR engagement or indirectly through signals received from antigen-presenting cells (APC) that have encountered pathogens [3]. Pattern recognition receptors, such as toll-like receptors (TLR), were earlier thought to be expressed only by APC; however, it has now become evident that TLRs are also expressed by various T cell subsets [5], [6]. Several studies have shown direct costimulatory effects of TLR agonists in T cells leading to their proliferation, activation and enhancement of effector functions, in the absence of APC [7], [8], [9]. An important observation from these studies is that the TLR costimulation in naïve T cells may require preactivation through their TCR as a prerequisite [10], [11], [12]. This notion is supported by findings that suggested memory T cells do not require TCR-stimulation as a prerequisite for responding to direct TLR ligation [7], [10], [13].

The expression and function of TLRs in iNKT cells are poorly understood. Shimizu et al. (2002) reported the expression of TLR2, 4, 5 and 9 genes in purified (∼97%) murine liver NK1.1+TCRβ+ NKT cells [14], [15]. Subsequently, TLR3 and 4 protein expression in hepatic CD1d tetramer positive iNKT cells was reported [16], [17]. More recently, Moreno et al. (2009) reported expression of TLR1 to 9, except for TLR8, at the transcript level in purified (93–100%) Vα24+Vβ11+ human iNKT cells [18]. Previous research has investigated the ‘indirect’ effects of TLR primed APC-derived signals in murine and human iNKT cell activation [18], [19], [20]. For example, Nagarajan et al. (2007) showed increased activation of iNKT cells in response to LPS which was dependant on Dendritic Cells (DC)-derived IL-12 and IL-18 [19]. Tyznik et al. (2008) showed that co-culturing of TLR7/9 primed DCs with splenic iNKT cells can result in enhanced IFN-γ production by iNKT cells. These studies along with a few others [18], [21], [22] collectively indicated the necessity of APC in transmitting TLR-derived signals to iNKT cells leading to enhanced iNKT cell activation. However, despite the expression of certain TLRs in iNKT cells, there is little evidence for the direct TLR-mediated stimulatory or costimulatory effects in the activation of iNKT cells.

In the present study, we used iNKT cell lines primarily to avoid potential contamination of other cells, although present in few numbers, particularly APC that have abundant TLR expression. We investigated the expression of TLR genes at both transcript and protein levels in murine iNKT cell lines, in the presence or absence of TCR ligation by either α-GalCer or anti-CD3 antibodies. Further, TLR costimulatory effects were investigated in iNKT cells that were stimulated through their TCR. Finally, we examined the role of iNKT cells costimulated using TLR ligands in the maturation of DCs.

Section snippets

Cell lines

Vα14-Jα18+ mouse iNKT cell line DN32.D3 [23] was generously provided by Dr. Albert Bendelac (The University of Chicago, Chicago, IL). The rationale for using iNKT cell lines was to exclude the possibility of contamination with other cells, such as APCs, which are known to have abundant expression of a wide range of TLRs and could interfere with our objective of investigating direct effects of TLR ligands on iNKT cells. DN32.D3 cells can be activated with α-GalCer in the absence of APC or

Expression of TLR genes in DN32.D3 iNKT cells

Although many cell types including various T cell subsets, such as conventional αβ, γδ, and regulatory T cells are known to express a diverse array of TLRs, either constitutively or post-activation, expression of TLR genes in iNKT cells is poorly understood. We determined TLR expression in unstimulated as well as in α-GalCer or anti-CD3 antibody stimulated DN32.D3 iNKT cells.

TLR3, 4, 5, 7 and 9 genes except TLR2 were found constitutively expressed in DN32.D3 iNKT cells, as detected by real-time

Discussion

Growing evidence in recent years has demonstrated that in some species, including rodents, humans and cattle, TLRs are expressed by different T cell subsets [6]. However, to date, there have been only a few reports describing the expression of TLRs in iNKT cells [15], [16], [17], [18], [34]. Despite the expression of these TLRs by iNKT cells, possible direct costimulatory effects of TLR agonists in these cells have not been adequately addressed. Therefore, using two iNKT cell lines (DN32.D3 and

Disclosure

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. S. Ole Odemuyi for help in the FACS data analysis. Dr. R.R. Kulkarni was a recipient of an Ontario Ministry of Research and Innovation post-doctoral fellowship.

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    1

    Present address: Department of Pediatrics and Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA 30322, USA.

    2

    Present address: Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.

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