Costimulatory activation of murine invariant natural killer T cells by toll-like receptor agonists
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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Innate Immunity and Inflammation
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2016, Clinical ImmunologyCitation Excerpt :This potent adjuvant effect is due in part to the fact that the activation of iNKT cells by α-GalCer also rapidly induces the full maturation of DCs in vivo and thereby acts as an adjuvant for both CD4+ and CD8+ T-cell immunity [7,10]. The Toll-like receptor (TLR) pathway has also been shown to participate in the interaction between iNKT cells and DCs [11]. Activation of the TLR pathway induces transcription of several components of the inflammatory response, such as factor NF-κB, interferon-regulatory factors (IRFs) and MAP kinases, which lead to the production of pro-inflammatory cytokines such as TNFα, IL-12, IL-6, and IL-1 by DCs [12–15].
Toll-like receptor regulation of effector T lymphocyte function
2013, Trends in ImmunologyCitation Excerpt :Multiple reports have since demonstrated varying roles of TLRs in regulating NKT cell function. Mouse invariant NKT (iNKT) cell lines were found to constitutively express TLR3, TLR4, TLR5, TLR7, and TLR9 [103]. Engagement of these TLRs alone did not result in appreciable activation effects; however, preactivation through the TCR followed by TLR ligation enhanced activation.
Invariant natural killer T cells as sensors and managers of inflammation
2013, Trends in ImmunologyCitation Excerpt :TIM-1 recognition of apoptotic cells induces iNKT cell IL-4 and IFN-γ secretion and this mechanism is suggested to contribute to the development of allergic airway hyper-reactivity in mice [64]. ( vii) Although iNKT cells fail to express TLRs under noninflammatory conditions, a recent study has provided evidence that TCR engagement on iNKT cells can induce expression of several TLRs, and that subsequent stimulation with TLR agonists leads to enhanced iNKT cell activation [65]. ( viii) A recent study has shown that hepatic iNKT cells can be directly activated to secrete IL-10 in response to neurotransmitters in an experimental model of stroke [66].
- 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.