P815 cells were coated with 5 g/ml anti-NKG2D mAb together with titrated amounts of anti-CD3 mAb, and incubated with CD4? NKT cells. of target cells via NKG2D engagement independently of CD1d, and that NKG2D also functions as a co-stimulatory receptor in these cells. NKG2D thus plays both a direct and a co-stimulatory role in the activation of NKT cells. described in physique 1 (data not shown), and responded to GalCer-pulsed monocytes with IFN production and degranulation as assessed by the CD107a assay [27, 28] (Fig. 2B). NKT cell lines generated in this way were subsequently used in functional experiments. Open in a separate window Physique 2 Purified and expanded NKT cells maintain expression of NKG2D, which triggers degranulation independently of CD1d. (A) Generation of a highly purified (98C100%) NKT cell line by growth with IL-2 and GalCer followed by positive magnetic bead selection for V24. (B) IFN production assessed by intracellular staining and CD107a degranulation assessed by surface staining of a highly purified NKT cell line in response to stimulation with GalCer-pulsed monocytes. (C) Expression of granzyme B and perforin in NKT cells, as assessed by intracellular staining, coincide with NKG2D expression. (D) NKT cell degranulation in response to mAb-coated P815 cells assessed by surface expression of CD107a after 6 h stimulation. Data are from four impartial experiments. *, p 0.05 as determined by the paired t-test. NKG2D is an activating receptor that recognizes ligands induced by cellular stress, infection and transformation [26]. Considering the differential expression of NKG2D in CD4+ and CD4? NKT cells [21], we hypothesized that this receptor may direct effector cell responses in CD4? NKT cells. Expression of the cytolytic effector molecules perforin and granzyme B largely overlapped with NKG2D expression in NKT cells (Fig. 2C), suggesting a role for this receptor in cytolytic activity. We next employed the P815 redirected stimulation assay, in Naxagolide which the P815 cell line binds Fc portions of mouse mAbs to provide a triggering ligand for activating receptors on responder cells. P815 cells coated with unloaded CD1d DimerX recombinant Naxagolide reagent, as expected, did not provide a triggering signal to NKT cells, whereas GalCer-loaded CD1d DimerX did induce degranulation as assessed by the CD107a assay (Fig. 2D). Interestingly, anti-NKG2D mAb-coated P815 cells brought on CD107a degranulation in the CD4? subset of NKT cells in the absence of CD1d. In contrast, the engagement of 2B4, which showed an expression pattern similar to that of NKG2D, did not trigger granule exocytosis in NKT cells. In addition, we were unable to detect IFN production by NKT cells in response to either NKG2D or 2B4 stimulation under comparable experimental conditions (data not shown). Together, these data indicate that NKG2D+ NKT cells are armed effector cells that can degranulate independently of TCR stimulation in response to NKG2D engagement. NKG2D localize at the target cell contact We next investigated the expression and localization of NKG2D in NKT cells by using confocal immunofluorescence microscopy. NKT cells were mixed and incubated for 15 min with the classical NK cell target cell line Rabbit Polyclonal to LAT K562, which lacks CD1d but is usually rich in the NKG2D ligands MICA and MICB, and with some expression of ULBP2 and 4 (Fig. 3A). NKT cells were observed either forming conjugates with K562 cells, or alternatively not in contact with or loosely attached to these cells Naxagolide after a 20 min co-incubation (Fig. 3B). In NKT Naxagolide cells contacting K562 cells, NKG2D was often predominantly localized at the site of target cell contact (Fig. 3C), a pattern consistent with the formation of an immunological synapse. Co-staining for CD3 was used to distinguish NKT cells from K562 cells and also revealed an even distribution of CD3 surface expression, which to some extent was co-localized with NKG2D staining at the target cell interface (Fig. 3D). Open in a separate windows Physique 3 NKG2D expression and polarization upon target cell contact. (A) Cell surface expression of NKG2D ligands and CD1d on K562 cells. (B) Light contrast image of three NKT cells, NKT1, NKT2 and NKT3, together with one K562 target cell after a 15 min co-incubation. (C) Confocal image of NKG2D (red) Naxagolide expression in these cells, and polarization of NKG2D in NKT1 in contact with K562 cells (arrow). (D) NKG2D (red), CD3 (green), and DAPI (blue) merged. (E) Quantification of NKG2D+ and NKG2D? NKT cell conjugate formation with K562 target cells..