TLR4 Directly Activates Invariant NKT Cells Figure 3. TLR4-mediated cytokine modulation by iNKT cells is triggered by MyD88 and TRIF, and depends on endocytosis and formation of the endosome compartment. (A, D, E) Sorted iNKT cells were incubated with anti-CD3+ CD28 mAbs or anti-CD3+ CD28 mAbs + LPS in RPMI media for 24 h. The concentration of IL-4 and IFN-c was measured in culture supernatant by ELISA. (A) To block TLR4 signals, iNKT cells were pre-treated with Myd88 or TRIF inhibitor, or control (ctl) peptide for 1 h. iNKT cells (NKT or N), preincubated with 100 mM/ml of dynamin inhibitor (DI) (B), 100 nM/ml of bafiliomycin A1 (Bafli) for 30 min or 100 mM/ml of chloroquine (Chlo) for 2 h (C), were incubated with irradiated splenocytes (SPC or S) in the presence of a-GalCer. (D) iNKT cells were incubated with agarose bead-bound or unbound LPS in the presence of anti-CD3 and CD28 mAbs. (E) To inhibit surface TLR4 and/or CD14, iNKT cells were preincubated with anti-TLR4 mAb (25 mg/ml) or anti-CD14 mAb (50 mg/ml) for 30 min at 4uC before anti-CD3+ CD28 mAb stimulation. Data are representative of three independent observations. (n = 3 in each group; *p,0.05, **p,0.01, ***p,0.001). doi:10.1371/journal.pone.0045348.g003 deficient iNKT cells was attributable to functional defects in TLR4-deficient iNKT cells. These data suggest that endogenous ligand-mediated signaling through TLR4 in iNKT cells con- tributes to their activation, resulting in the promotion of antibody- induced joint inflammation. Discussion Data regarding the expression patterns of TLR4 in NKT cells are contradictory [33,34,35]. Nagarajan et al. suggested that neither TLR4 nor CD14 were expressed by iNKT cells [35]. In contrast, TLR4 expression was demonstrated in sorted NK1.1+TCR-b+ NKT cells by RT-PCR [33] and in the cytoplasm of iNKT cells using flow cytometry [34]. However, cell surface expression and cytosolic localization of TLR4 in iNKT cells was not fully characterized. Our experiments clearly demonstrate that iNKT cells expressed TLR4 on the cell surface and in the early endosome. Several lines of reasoning might explain the contra- dictory observations of TLR4 expression patterns in iNKT cells. TLR4 expression is constitutively detected in na¨ıve murine T cells and down-regulated in activated murine T cells [25,26], indicating that the level of TLR4 expression depends on activation status. However, constitutive expression of TLR4 in iNKT cells was not reduced after TCR-mediated activation (data not shown). Therefore, it is unlikely that the different activation status of iNKT cells in these experiments accounts for the contradictory expression pattern of TLR4 in iNKT cells. Moreover, it is also less likely that positive TLR4 expression pattern in NKT cells of our experiments might be attributable to contamination of other cell types in sorted NKT cells because purity of sorted NKT cells was extremely high (Fig. S1B). Alternatively, the sensitivity of the equipment, such as the flow cytometer, technical limitations, and/ or the use of different reagents might affect detection of TLR4 expression by iNKT cells. Although TLR4 has been reported to be expressed on the surface of most cell types [12,13,14,16], it is also localized in the PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e45348