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The Journal of Immunology, 2007, 178: 3418-3426.
Copyright © 2007 by The American Association of Immunologists, Inc.

The Activating NKG2D Ligand MHC Class I-Related Chain A Transfers from Target Cells to NK Cells in a Manner That Allows Functional Consequences1

Fiona E. McCann2,3, Philipp Eissmann2, Björn Önfelt4, Rufina Leung and Daniel M. Davis5

Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recently, it has become apparent that surface proteins commonly transfer between immune cells in contact. Inhibitory receptors and ligands exchange between cells during NK cell surveillance and we report here that NK cells also acquire activating ligands from target cells. Specifically, the stress-inducible activating ligand for NKG2D, MHC class I-related chain A (MICA), transferred to NK cells upon conjugation with MICA-expressing target cells. Acquisition of MICA from target cells was dependent on cell contact and occurred after accumulation of MICA at the immunological synapse. Moreover, transfer of MICA was facilitated by specific molecular recognition via NKG2D and augmented by Src kinase signaling. Importantly, MICA associated with its new host NK cell membrane in an orientation that allowed engagement with NKG2D in trans and indeed could down-regulate NKG2D in subsequent homotypic interactions with other NK cells. MICA captured from target cells could subsequently transfer between NK cells and, more importantly, NK cell degranulation was triggered in such NK cell-NK cell interactions. Thus, NK cells can influence other NK cells with proteins acquired from target cells and our data specifically suggest that NK cells could lyse other NK cells upon recognition of activating ligands acquired from target cells. This mechanism could constitute an important function for immunoregulation of NK cell activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The activity of NK cells is controlled by a balance of signals from activating and inhibitory receptors (1). Inhibitory NK cell receptors predominantly recognize cognate MHC class I protein and provide self-tolerance toward healthy cells (2). Cells with absent or reduced expression of MHC class I protein, as often observed after transformation or viral infection, are unable to trigger sufficient inhibitory signals and become susceptible to NK cell attack. Furthermore, up-regulated expression of ligands for activating NK cell receptors can render cells sensitive to NK cell attack (3, 4). One such activating receptor is the C-type lectin-like receptor NKG2D.

Human NKG2D is expressed on NK cells and a variety of T cell subsets (5). Ligands for NKG2D in humans are MHC class I-related chain A (MICA)6 and chain B, the UL-16 binding proteins (ULBP)-1, ULBP-2, and ULBP-3 and the retinoic acid early transcript 1 proteins E and G (6, 7, 8). Although expression of some NKG2D ligands can be found on healthy tissue, these molecules play an important role in the regulation of NK cell and T cell activity, as their expression is often induced on transformed or infected cells (9, 10, 11, 12). MICA, for example, is a highly glycosylated, transmembrane protein normally restricted to some intestinal epithelium, but is up-regulated on many tumors and also upon infection by human CMV or Escherichia coli (10, 11, 12, 13). Most recently, NKG2D ligands were shown to be induced by signal cascades involved in the DNA damage pathway (14).

Expression of NKG2D ligands is therefore generally regarded as a "danger signal," marking cells for immune cell attack (4, 15). Indeed, ex vivo studies with human cells and in vivo tumor models in mice demonstrated that expression of NKG2D ligands on tumor cells results in an increased susceptibility to NK cell and cytotoxic T cell attack (16, 17, 18, 19). NKG2D ligand expression even resulted in killing of MHC class I-positive cells, demonstrating that the increased expression of activating ligands can override inhibitory signals (8, 19). Additionally it was reported that the expression of MICA and MHC class I-related chain B can be induced on dendritic cells (20). In this case, ligation of NKG2D on NK cells serves to promote NK cell activation and influence the adaptive immune response.

The engagement of immune cells with potential target cells or APC commonly results in specific micrometer scale organization of proteins at the intercellular contact, i.e., assembly of an immunological synapse (21, 22). NK cells form activating or inhibitory synapses that involve distinct supramolecular arrangements of proteins correlating with distinct outcomes of the interaction (23, 24, 25, 26). A common, but less-studied, aspect of synapse formation is the exchange of molecules between the interacting cells (27, 28, 29, 30, 31, 32). T and B cells acquire peptide/MHC class I proteins and costimulatory molecules or Ag, respectively (33, 34). Human, as well as mouse, NK cells acquire cognate MHC class I molecules in vitro and in vivo (35, 36, 37). This transfer is bidirectional, as target cells will take up inhibitory NK cell receptors in the same interactions (38). While this study was under peer review, it was reported that there was an intercellular exchange of NKG2D and MHC class I-related chain B in NK cell-target cell interactions, which correlated with reduced NKG2D-dependent cytotoxicity in subsequent NK cell-target cell interactions (39).

We report here that the NKG2D ligand MICA transfers from target cells to NK cells, further demonstrating that activating ligands are exchanged. Importantly, we provide evidence that the acquisition of MICA enables NK cells to engage NKG2D on neighboring NK cells, suggesting a novel immunoregulatory mechanism between NK cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells and reagents

Human NK cells were isolated from peripheral blood and cultured as previously described (26). 721.221 cells (40), in this study referred to as 221 cells, were transfected to stably express full length MICA lacking the stop codon, tagged with yellow fluorescent protein (YFP) at the C terminus (221/MICA-YFP). For this, MICA (allele as previously described in Ref. 8) was amplified with the stop codon removed by RT-PCR and cloned as a KpnI-BamHI fragment into pcDNA3.1/neo (Invitrogen Life Technologies) directly upstream and in frame with YFP, inserted between BamHI and XhoI. HLA-Cw6 already lacking the stop codon (23) was amplified by RT-PCR and ligated into pcDNA3.1/hygro, between KpnI and BamHI, directly upstream and in frame with the cyan fluorescent protein (CFP) sequence, inserted between BamHI and XhoI. The 221 cells already stably transfected to express HLA-Cw6-CFP were subsequently transfected by electroporation to express MICA-YFP (221/MICA-YFP/Cw6-CFP). Cells were selected for expression of CFP, YFP, or both by flow cytometry. Untransfected 221 cells were found not to express NKG2D ligands ULBP-1, ULBP-2, ULBP-3, or MICA (data not shown).

mAbs used were as follows: mouse anti-MICA (clone M673), a gift from D. Cosman and J. Chalupny (Amgen, Seattle, WA), anti-NKG2D (clone 149810; R&D Systems), anti-GFP (clone GFP20; Sigma-Aldrich), anti-phosphotyrosine (4G10; Upstate Cell Signaling Solutions), FITC-conjugated anti-CD69 (clone FN50), anti-CD86 (clone 2331), anti-CD19 (clone HIB19), PE-conjugated anti-CD107a, PE-conjugated mouse IgG1{kappa}, FITC-conjugated goat anti-mouse (all from BD Biosciences), Cy5-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories), and HRP-conjugated goat anti-mouse IgG (Pierce). PP2 (Biomol) was used as an Src kinase inhibitor and 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo(2,3-d)pyrimidin-4-ylamine (Sigma-Aldrich) as specific Lck inhibitor.

Conjugate formation and cell staining

A total of 2 x 105 NK cells and target cells were coincubated in 50 µl of prewarmed culture medium in a 96-well V-bottom plate for 1 h at 37°C, 5% CO2. For immunofluorescence labeling and confocal microscopy, conjugates were directly fixed with buffer containing paraformaldehyde and saponin (Cytofix/Cytoperm; BD Biosciences) for 15 min at 4°C before cells were stained as previously described (26). For flow cytometry, conjugates were separated in 0.5 mM EDTA/PBS on ice for 30 min, which allowed NK cells to be clearly distinguished from target cells due to their smaller size. For conjugation assays, NK cells were labeled with DiD (Invitrogen Life Technologies) before incubation with 221/MICA-YFP. After 60 min, cells were fixed with paraformaldehyde and analyzed by flow cytometry. Conjugates were identified as being YFP- and DiD-positive. The percentage of NK cells in conjugate was calculated as (NK cells in conjugate)/(total number of NK cells), where the total number is NK cells in conjugate plus NK cells not in conjugate.

Live cell imaging

Live cell imaging was performed by resonance scanning confocal microscopy (TCS SP2 RS; Leica Microsystems) using an inverted microscope, a x63 oil immersion objective (NA 1.2), and an environmental chamber (Solent Scientific) to keep the cells at 37°C and under 5% CO2. Conjugates were scanned in the xy-direction every 0.3 µm throughout the z-plane. Where appropriate, images of conjugates were then reconstructed using a maximum intensity projection (Volocity 3D; Improvision).

Cell mixing and Western blotting

A total of 107 NK cells were mixed with target cells at an E:T ratio of 1:1 and incubated for 1 h at 37°C, 5% CO2. Cells in conjugate were separated in 0.5 mM EDTA/PBS on ice for 30 min and NK cells were isolated from target cells by FACS for CD19-negative cells. NK cells were then lysed in RIPA buffer for 30 min on ice. Lysates were cleared, separated by SDS-PAGE, and analyzed by Western blotting (SuperSignal West Femto; Pierce).

Transwell assay for intercellular contact dependence of MICA transfer

A total of 2 x 105 NK cells were placed in the upper compartment of a Transwell chamber with membrane of pore size of 0.4 µm (Millicell-PC; Millipore). A total of 2 x 105 target cells in 1 ml of prewarmed culture medium were either added to the NK cells in the upper chamber or separated from NK cells in the lower compartment. Cells were incubated for 1 h at 37°C, 5% CO2, before cells from the upper chamber were collected in 0.5 mM EDTA/PBS and placed on ice for 30 min to split cells in conjugate. NK cells were then assessed for YFP fluorescence by flow cytometry.

Assay for NKG2D down-regulation and NK cell degranulation

NK cells were incubated with target cells at an E:T ratio of 1:1, for 1 h at 37°C, 5% CO2, before cells in conjugate were separated in 0.5 or 5 mM EDTA/PBS on ice for 30 min. Cells were then incubated with CD56 microbeads (Miltenyi Biotec) or CD56 Ab followed by magnetic colloid (StemCell Technologies) according to the manufacturer’s instructions. NK cells were purified by magnetic separation and were then labeled with either CFSE or SNARF and incubated with unlabeled autologous NK cells for 4 h at 37°C, 5% CO2. Thereafter, cells in conjugate were separated by the addition of EDTA. For NKG2D down-regulation assays, CFSE-negative NK cells were assessed for NKG2D expression by flow cytometry. NKG2D expression was calculated for mean fluorescence intensity (MFI) as (MFI of fresh NK cells coincubated with either CFSE+ NK/221 or NK/221 MICA)/(MFI of fresh NK cells coincubated with CFSE+ NK cells) x 100, wherein MFI is the geo-MFI corrected by the geo-MFI obtained with an isotype control Ab. To assay for degranulation, expression of CD107a was compared on YFP-positive and YFP-negative fresh NK cells (i.e., NK cells that were CFSE- or SNARF-negative).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MICA transfers to NK cells during target cell contact

It has recently been established that killer Ig-related receptor (KIR) and their corresponding ligands, class I MHC proteins, exchange between NK cells and target cells in contact (35, 36, 37, 38). To determine whether activating ligands transfer from target cells to NK cells during activating interactions, we stably expressed YFP-tagged MICA, a ligand for the activating NK cell receptor NKG2D, in the EBV transformed B cell line 721.221 (221/MICA-YFP). The 221 cell line is well characterized as a good target for NK cells because it lacks endogenous expression of inhibitory class I MHC proteins. Over-expression of MICA in 221 augmented their susceptibility to lysis by several, although not all, NK clones (data not shown). Live cell imaging clearly demonstrated that MICA is recruited to the contact between 221/MICA-YFP and human peripheral blood NK cells (Fig. 1A), which is consistent with previously published data from our group (41). It may be interesting to note that before contact with NK cells, MICA-YFP was predominantly located at the back of the cell, i.e., the cell uropod (Fig. 1, A and B). The leading edge, clearly apparent when 221 cells migrate, was largely free of MICA-YFP. Approximately 2–4 min after initial contact with an NK cell, blebbing of the 221 cell membrane was apparent (Fig. 1A), indicative of the cytolytic response.


Figure 1
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  		FIGURE 1. MICA transfers from target cells to NK cells. A and B, Representative images, reconstructed from a series of optical slices, show conjugate formation and intercellular transfer of MICA between peripheral blood NK cells and 221/MICA-YFP. Rapid live cell imaging by resonance scanning confocal microscopy was used to acquire nearly 40 optical slices throughout a cell-cell conjugate up to every 12–18 s. MICA-YFP has a distinctly polarized distribution (large cell surface "green" patches, top panels) before NK cell docking. On contact, MICA-YFP distribution becomes more uniform over the cell surface and then accumulates at the immunological synapse before transferring to the NK cell. Time is noted in minutes. C, Conjugates formed between peripheral blood NK cells and 221/MICA-YFP were fixed and stained with an anti-MICA mAb. Red line histogram quantifies MFI along the red line shown in bottom left image of YFP and the anti-MICA label. For reference, arrows in both the image and the MFI plot indicate the same region within the cell membrane. Image is representative of 33 conjugates, of which 26 showed transfer of MICA-YFP. Twenty three of these 26 showed colocalization of anti-MICA staining. D, Peripheral blood NK cells were incubated with 221 or 221/MICA-YFP, cells in conjugate were separated, and NK cells isolated. A total of 107 NK cells were lysed and analyzed by Western blotting for MICA (left). Lysates of 221 or 221/MICA-YFP served as references (right). Purity of the NK cell isolation was confirmed by probing the membrane for CD86, expressed on 221 cells but not transferred to NK cells that are mostly CD28-negative. E, Conjugates formed between peripheral blood NK cells and HeLa were fixed and stained with anti-MICA mAb. Scale bars represent 5 µm for A–C or 10 µm in E.

 
By live cell imaging, transfer of YFP from 221/MICA-YFP onto an apposing NK cell in conjugate was readily apparent, suggesting that MICA transferred from the target to the NK cell (Fig. 1, A and B, and shown in supplemental videos 1–4).7 Accumulation of MICA at the target cell contact preceded the transfer of MICA, consistent with recruitment to the immunological synapse helping efficient intercellular transfer. To test whether the transferred YFP reflects transfer of MICA-YFP, conjugates were fixed and stained using mAb against an extracellular epitope of MICA (Fig. 1C). Analysis showed colocalization of YFP and anti-MICA mAb on the NK cell, suggesting that indeed the transfer of YFP does report transfer of MICA-YFP. MICA-YFP colocalized with both anti-CD56 mAb and cholera toxin B subunit, which binds the GM1 ganglioside, suggesting that MICA-YFP remained on the NK cell surface (data not shown).

To test whether the full size of MICA-YFP transferred to NK cells, we first incubated human NK cells with 221/MICA-YFP, separated cells in conjugate and purified NK cells by FACS for CD19-negative cells. Lysates were then probed for MICA by Western blotting. The purity of NK cells in this assay was confirmed by Western blotting for CD86, which is expressed in 221 and not NK cells, and also does not transfer to NK cells that mostly do not express the CD28 receptor for CD86 (Fig. 1D). The apparent molecular mass of MICA-YFP, between 100 and 120 kDa, as detected in the transfectants, was larger than that predicted by the amino acid sequence alone, likely due to extensive glycosylation as reported previously (41). Most importantly, MICA was detectable by Western blotting in the lysate of NK cells incubated with 221/MICA-YFP, but not those incubated with untransfected 221 (Fig. 1D, top panels). This size of MICA-YFP found in NK cells was identical with the size of the protein detected from the 221/MICA-YFP lysate, confirming that fully sized MICA-YFP transfers from target to NK cells.

To check whether the intercellular transfer of MICA-YFP could be an artifact of overexpression or the addition of C-terminal YFP, HeLa that expresses MICA endogenously was coincubated with human peripheral blood NK cell for 30 min, fixed, and stained for MICA. Staining with an anti-MICA mAb was clearly evident on NK cells conjugated to HeLa, confirming that MICA transfers to NK cells from a cell line endogenously expressing MICA (Fig. 1E).

Next, we set out to determine the kinetics of the intercellular transfer of MICA by flow cytometry (Fig. 2). Incubation of human peripheral blood NK cells with 221/MICA-YFP resulted in YFP fluorescence being detected on NK cells (Fig. 2A). With increasing coincubation times, the fluorescence of YFP on the NK cells increased such that the amount of YFP on the NK cell population reached ~3% of that on 221/MICA-YFP by 30 min of coincubation (Fig. 2B). Longer periods of coincubation did not result in a further increase of MICA on NK cells.


Figure 2
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  		FIGURE 2. Kinetics for the intercellular transfer of MICA. A, Peripheral blood NK cells were incubated with 221/MICA-YFP for either 2 or 60 min, and YFP fluorescence on NK cells was analyzed by flow cytometry. Fluorescence of 221/MICA-YFP is shown for reference. B, Peripheral blood NK cells were incubated with 221/MICA-YFP for different times and YFP fluorescence on NK cells was analyzed by flow cytometry. The YFP fluorescence transferred to NK cells is expressed as a percentage of YFP fluorescence on 221/MICA-YFP before contact with NK cells. The mean from four independent experiments is shown and error bars are SE.

 
Characterization of MICA transfer

To investigate whether the transfer of MICA from target to NK cells was dependent on intercellular contact, Transwell assays were used. NK cells were separated from 221/MICA-YFP by a porous membrane that would allow small fragments of the cell membrane or vesicles but not cells to pass or NK cells were coincubated with 221/MICA-YFP directly (Fig. 3A). Although significant amounts of MICA-YFP transferred to NK cells directly coincubated with target cells, no YFP transferred to NK cells separated from target cells by the Transwell membrane. Thus, transfer of MICA from target cells to NK cells is highly dependent on intercellular contact.


Figure 3
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  		FIGURE 3. Characterization of MICA transfer. A, Peripheral blood NK cells and 221/MICA-YFP were incubated in a Transwell chamber and separated either by a porous membrane (gray filled histogram) or left unseparated (open histogram). After 1 h, cells in conjugate were separated and YFP fluorescence on NK cells was analyzed by flow cytometry. Data are representative for three independent experiments. B and C, Peripheral blood NK cells were incubated with 221/MICA-YFP for 1 h, cells in conjugate were separated and stained with an anti-GFP mAb (black line histogram), an anti-MICA mAb (gray line histogram), or an isotype-matched control mAb (gray filled histogram), in the presence of nonpermeabilizing (B) or cell-permeabilizing (C) buffer. The level of YFP fluorescence on NK cells was analyzed by flow cytometry. D, Representative images from live cell imaging of KIR2DL1-positive human NK cell clones in conjugate with 221/MICA-YFP/Cw6-CFP. Panels show fluorescence of MICA-YFP (red), HLA-Cw6-CFP (green), and colocalization of both (yellow) for the corresponding bright-field images of the cells (top). Time is shown in minutes. Of 96 conjugates analyzed, 70 showed colocalization of MICA and HLA-Cw6. E, Conjugates, formed within 20 min, between peripheral blood NK cells and 221/MICA-YFP were fixed and stained with an anti-phosphotyrosine mAb, followed by a Cy5-conjugated secondary Ab. Of 130 conjugates, 94 showed transfer of MICA-YFP and 81 of these showed colocalization of phosphotyrosine staining. Two representative conjugates are shown. Scale bar, 10 µm.

 
To test whether MICA transferred to NK cells could be functional in its new host cell membrane, we set out to determine whether the N-terminal extracellular binding domain of MICA (42) remained available for ligation. NK cells were coincubated with 221/MICA-YFP, cells in conjugate were separated, fixed, and stained with one mAb against an extracellular epitope of MICA and a second mAb directed against YFP under either nonpermeabilizing (Fig. 3B) or permeabilizing (Fig. 3C) conditions. Although MICA was stained under both conditions, YFP was only detectable when cells were permeabilized before staining. Thus, nearly all transferred MICA-YFP is oriented in its new host cell membrane such that the extracellular binding domain of MICA remains accessible for recognition, while YFP remains unexposed.

We next used transfectants of 221 cells expressing YFP-tagged MICA and CFP-tagged HLA-Cw6 (221/MICA-YFP/Cw6-CFP) to determine whether both inhibitory and activating NK ligands transfer to NK cells concurrently. Strikingly, both proteins transferred to NK cells and colocalized in discrete domains of the NK cell surface (Fig. 3D). A similar distribution was observed when MICA transferred in the absence of HLA-C, as revealed by confocal microscopy of fixed cell conjugates (Fig. 3E). Interestingly, clusters of MICA-YFP on the NK cell colocalized with anti-phosphotyrosine staining (Fig. 3E). Thus, tyrosine-phosphorylated proteins cotransfer with MICA and/or signaling can persist in regions where transferred MICA accumulated.

NK clones that are KIR2DL1+ are generally not able to lyse 221/MICA-YFP/Cw6-CFP, consistent with an inhibitory signal being able to dominate MICA/NKG2D ligation in at least some circumstances (43) and (J. Endt, F. E. McCann, C. R. Almeida, D. Urlaub, R. Leung, D. M. Davis, and C. Watz, submitted for publication). However, MICA-YFP still efficiently transferred from 221/MICA-YFP/Cw6-CFP onto KIR2DL1+ NK clones (data not shown) and time-lapse microscopy of NK cell interactions (shown in supplemental videos 1–4) shows that MICA-YFP transfers onto NK cells rapidly upon intercellular contact, before an obvious signature of lysis.7 Thus, transfer of MICA-YFP is not dependent on cell lysis.

The transfer of MICA is dependent on NKG2D

Previously, we have determined that bidirectional exchange of HLA-C and KIR between NK cell and target cells is facilitated by cognate recognition between appropriate alleles of these proteins (35, 38). To investigate whether the transfer of MICA is dependent on NKG2D engagement, we incubated human NK cells with 221/MICA-YFP for different times in the presence of either a blocking anti-NKG2D mAb or an isotype-matched control mAb (Fig. 4, A and B). In the presence of anti-NKG2D mAb but not the isotype-matched control, acquisition of YFP fluorescence by the NK cell was blocked (Fig. 4, A and B). Moreover, anti-NKG2D mAb blocked transfer of MICA-YFP in a dose-dependent manner with strong inhibition of the transfer of MICA being obtained with only 0.1–0.5 µg/ml anti-NKG2D mAb (Fig. 4B). At the same time, mAb concentrations between 0.01 and 0.5 µg/ml had no effect on conjugate formation between NK cells and 221/MICA-YFP (data not shown). Thus, intercellular transfer of MICA-YFP is largely facilitated by specific recognition via NKG2D.


Figure 4
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  		FIGURE 4. NKG2D facilitates transfer of MICA onto NK cells, and itself transfers to target cells. A, Peripheral blood NK cells were coincubated with 221/MICA-YFP for 2 or 60 min in the presence of 20 µg/ml anti-NKG2D mAb (black line histogram) or isotype-matched control mAb (gray line histogram). Conjugates were separated, and YFP fluorescence on NK cells was analyzed by flow cytometry. B, Peripheral blood NK cells were coincubated with 221/MICA-YFP for 60 min in the presence of increasing concentrations of either anti-NKG2D mAb ({blacksquare}) or isotype-matched control mAb ({blacktriangleup}). YFP fluorescence on NK cells was analyzed by flow cytometry and is expressed as a percentage of YFP fluorescence on 221/MICA-YFP cells before conjugation with NK cells. Data are representative of five independent experiments. C, Peripheral blood NK cells were incubated for 1 h with either 221 (black line histogram) or 221/MICA-YFP (gray lined histogram). Cells in conjugate were separated and stained with anti-NKG2D mAb (open histograms) or isotype-matched control mAb (gray filled histograms). MFI was analyzed by flow cytometry.

 
NKG2D transfers to target cells expressing MICA

To investigate next whether NKG2D is also transferred to the target cell, we incubated human NK cells with untransfected 221 cells or 221/MICA-YFP for 1 h. Cells in conjugate were separated and 221 cells were analyzed for the presence of NKG2D by flow cytometry. NKG2D was clearly detectable on 221/MICA-YFP but not untransfected 221 (Fig. 4C). Thus, NKG2D can transfer from NK cells to target cells expressing MICA. Acquisition of NKG2D by target cells in this particular circumstance is perhaps unlikely to be important for immune cell function because the target cell is anyway lysed. Nevertheless, it is possible that the transfer of NKG2D during noncytolytic interactions with other cell types can be functionally important, or alternatively that the loss of NKG2D from NK cells could be functionally significant.

The transfer of MICA is partially dependent on kinase signaling

To investigate whether the acquisition of MICA is dependent on intracellular signaling, we tested whether the Src kinase inhibitor PP2 influenced the amount of MICA transferred onto NK cells (Fig. 5, A and B). As little as 5–10 µM PP2 considerably reduced the transfer of MICA from the target cell to the NK cell (Fig. 5A). The 30 µM PP2 did not influence the number of conjugates between NK cells and 221/MICA-YFP after coincubation for 1 h (Fig. 5C), ruling out that the reduction of MICA transfer is caused by reduced conjugation. Interestingly, however, transfer of MICA was not completely blocked even with higher concentrations of PP2. We next tested whether the Src kinase Lck was specifically important using a selective inhibitor of Lck, 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo(2,3-d)pyrimidin-4-ylamine (Fig. 5, D and E). These data suggest that the transfer of MICA to NK cells is augmented by Src kinase, and specifically Lck signaling, but a low level of transfer can occur in the absence of Src kinase signaling.


Figure 5
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  		FIGURE 5. Intercellular transfer of MICA is impaired by Src kinase inhibition. A, Peripheral blood NK cells were incubated with 221/MICA-YFP in the presence of increasing concentrations of a potent Src kinase inhibitor PP2. Conjugates were separated, and YFP fluorescence on NK cells was analyzed by flow cytometry. The level of fluorescence is expressed as a percentage found on NK cells in the absence of PP2, i.e., in the presence of the DMSO vehicle only. B, Peripheral blood NK cells were incubated with either 221 (control, dashed line histogram) or 221/MICA-YFP in the presence of 8.25 or 16.5 µM PP2 (black line histogram), or the DMSO vehicle only (gray filled histogram). C, Peripheral blood NK cells were labeled with DiD and incubated with 221/MICA-YFP in the presence of increasing concentrations of PP2. After 60 min, cells were fixed and the percentage of NK cells in conjugate was analyzed by flow cytometry. D, Peripheral blood NK cells were incubated with 221/MICA-YFP as in A and coincubated in the presence of a specific Lck inhibitor, 7-cyclopentyl-5-(4-phenoxyphenyl)-7H-pyrrolo(2,3-d)pyrimidin-4-ylamine. E, Peripheral blood NK cells were incubated with 221/MICA-YFP in the presence of 0.3 or 2.5 µM of the specific Lck inhibitor (black line histogram), or the DMSO vehicle only (gray filled histogram). After 1 h of coincubation, YFP fluorescence on NK cells was analyzed by flow cytometry. As a control, YFP fluorescence was analyzed on NK cells after 1 min coincubation in the presence of DMSO (dashed line histogram). Data are representative of three independent experiments.

 
Transferred MICA engages NKG2D on neighboring NK cells

Constitutive expression of MICA in mice leads to a dramatic decrease in NKG2D expression on NK and T cells (44, 45). Also in mice, recognition of macrophage MICA by NK cells results in down-modulation of NKG2D expression at the cell surface (46). In line with these data, soluble MICA protein found in the serum of cancer patients was reported to be responsible for NKG2D down-regulation on human T cells (47). In this study, we found that expression of NKG2D at the NK cell surface was substantially decreased after NK cells were coincubated for 1 h with 221/MICA-YFP but not 221, whereas the level of CD69 expression remained similar (Fig. 6A). Longer periods of incubation did not reduce NKG2D surface expression any further (data not shown).


Figure 6
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  		FIGURE 6. MICA transferred onto NK cells from target cells is subsequently recognized by NKG2D on other NK cells. A, Peripheral blood NK cells were incubated with 221 cells, 221/MICA-YFP, or left alone (none) for 1 h. Cells in conjugate were then separated and stained with an anti-NKG2D mAb (open histograms, top), an anti-CD69 mAb (open histograms, bottom), or an isotype-matched control mAb (gray filled histograms). MFI on NK cells were analyzed by flow cytometry. B, Analogous experiments were performed, except that cells were fixed and permeabilized before staining. C, Peripheral blood NK cells were incubated with either 221 cells (NK/221) or 221/MICA-YFP (NK/221-MICA) for 1 h. Conjugates were separated and NK cells were isolated by magnetic cell sorting. Isolated NK cells were then labeled with CFSE and incubated with autologous fresh NK cells for 4 h. Conjugates were again separated and cells were stained with an anti-NKG2D mAb. NKG2D fluorescence was then assessed on CFSE-negative cells. D, Peripheral blood NK cells were incubated with either 221 cells (NK/221) or 221/MICA-YFP (NK/221-MICA) for 1 h. Conjugates were separated and NK cells were isolated by magnetic cell sorting. Isolated NK cells were then labeled with either CFSE or SNARF and incubated with fresh NK cells for 4 h in the presence of either PE-conjugated CD107a or isotype control mAb. CD107a fluorescence was then assessed on YFP-positive and YFP-negative cells within the fresh (CFSE- or SNARF-negative) NK cell population. Symbols represent different experiments and horizontal bar represents the mean.

 
To determine the fate of NKG2D lost from the cell surface after contact with MICA-expressing target cells, we next stained for the presence of NKG2D in NK cells permeabilized after conjugation with target cells (Fig. 6B). There was an apparent reduction in staining for either MICA or CD69 in permeabilized cells compared with unpermeabilized cells, likely reflecting a loss of epitope recognition by each mAb under permeabilizing conditions. Nevertheless, under permeabilizing conditions, NKG2D staining was lower for NK cells coincubated with 221/MICA-YFP compared with NK cells incubated with 221 (Fig. 6B). Thus, the total amount of NKG2D in these cells has been lowered by contact with 221/MICA-YFP. Because the amount of NKG2D transferred from NK cells to target cells is very low (Fig. 4C), these data suggest that NKG2D is rapidly degraded after MICA-triggered internalization.

We next exploited NKG2D down-regulation as a marker for receptor engagement, as previously used (46), to determine whether MICA that had transferred from target cells to NK cells could be recognized in subsequent intercellular contacts. NK cells were incubated with untransfected 221 or 221/MICA-YFP, cells in conjugate were separated, and NK cells were isolated by magnetic cell sorting for CD56-positive cells. Sorted NK cells were then labeled with CFSE and incubated with fresh NK cells from the same donor for 4 h, after which NKG2D expression was analyzed on CFSE-negative cells by flow cytometry (Fig. 6C). Down-regulation of NKG2D was observed on those NK cells that were incubated with NK cells that had acquired MICA from target cells (NK/221-MICA plus NK). In comparison to NKG2D down-regulation after direct contact with 221/MICA-YFP (~80%) (Fig. 6A), there was generally less down-regulation of NKG2D caused by NK cells that have acquired MICA from target cells. This response likely reflects the different levels of MICA on 221/MICA-YFP and NK cells that have captured MICA from target cells (Fig. 2A). Nevertheless, NK cells that have been in contact with MICA-expressing target cells can clearly use MICA acquired from those targets to engage NKG2D on other NK cells and this could represent a novel mechanism for NK cell immunoregulation.

NK cells that acquired MICA from target cells cause degranulation in subsequent interactions with other NK cells

Next, we incubated NK cells with 221 or 221/MICA-YFP, separated conjugates, and isolated NK cells by magnetic cell sorting. Sorted NK cells were then labeled with a cytosolic dye (CFSE or SNARF) and incubated with a second population of unlabeled autologous NK cells in the presence of anti-CD107a mAb, a marker for degranulation (48). After 4 h, we detected YFP fluorescence on a small number (1–6%) of fresh, i.e., CFSE- or SNARF-negative, NK cells (data not shown). Thus, MICA-YFP transferred between the two populations of NK cells.

In most (8/10) experiments we also observed degranulation in fresh NK cells, which had acquired MICA-YFP from the NK cells initially incubated with target cells (Fig. 6D). Thus, NK cells that have captured proteins from target cells triggered degranulation in a second population of fresh NK cells. This is reminiscent of "fratricide" in which CTL captured agonist pMHC from APC and were lysed by neighboring CTL (33). Increased degranulation detected by anti-CD107a Ab, correlates strongly with NK cell-mediated lysis (48). Thus, our observations of NK cell degranulation indicate that NK cell fratricide could occur, by recognition of MICA or other activating proteins captured from target cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Intercellular transfer of cell surface proteins and membrane has been observed between several cell types, including T cells, B cells, and NK cells (27, 28, 29, 30, 31, 32). We and others previously demonstrated bidirectional transfer of inhibitory NK cell receptors and ligands at inhibitory immune synapses (35, 38). We now demonstrate transfer of an activating ligand, MICA (in agreement with another study (39) demonstrating transfer of MHC class I-related chain B, which was published while our report was under review). Moreover, we report in this study that NK cells that have acquired MICA from target cells can engage NKG2D in subsequent interactions.

Intercellular transfer of MICA onto NK cells was observed some minutes after intercellular contact, and after accumulation of MICA at the immunological synapse, consistent with the transfer of MICA occurring via the synapse. Indeed, transfer of MICA was contact dependent and the fact that the transfer of MICA was greatly reduced by mAb-mediated blocking of NKG2D, clearly demonstrates a dependence on receptor engagement. Inhibition of MICA transfer with the Src kinase inhibitor PP2 or a specific Lck inhibitor clearly indicates that receptor signaling augments the transfer of MICA. Thus, it is likely that receptor signaling directly influences the intercellular transfer of MICA, perhaps by increasing the extent of NKG2D accumulation, and hence MICA engagement at the immunological synapse.

It is intriguing, that once transferred, MICA is not evenly distributed throughout its new cell membrane but is retained in discrete domains. Hence the attachment of MICA to the NK cell membrane is somehow distinct from how endogenously expressed MICA is tethered to the target cell surface. A distinct mechanism for association with the cell surface for transferred proteins was also evident for KIR since transferred KIR, but not endogenously expressed KIR, was removed by a mild acid wash (38). Regions of the cell surface where transferred MICA accumulated had a high density of phosphotyrosine. Thus, phosphorylated proteins directly acquired from the target cell or ligation of NK receptors by ligands captured from target cells could sustain signals in NK cells after disassembly of the immunological synapse.

Despite being associated in its new host cell membrane in a manner distinct from endogenously expressed MICA, the extracellular, receptor binding domain of MICA is accessible to mAb. Moreover, NKG2D was down-regulated on NK cells incubated with NK cells that had acquired MICA in a prior target cell interaction. Thus the levels of acquired MICA found on the surface of NK cells are sufficient to be detected by NKG2D on neighboring NK cells. Furthermore these interactions are likely to induce signaling, as NKG2D down-regulation was previously reported to be at least in part dependent on DAP10 function (49). This homotypic NK cell interaction could have several functional consequences. One possibility is that MICA-positive NK cells stimulate neighboring NK cells, or subsets of T cells that express NKG2D, to proliferate or secrete IFN-{gamma}. In this scenario NK cells that already encountered target cells would augment the NK cell response by stimulating other cells. However, we were not able to detect increased IFN-{gamma} resulting from homotypic NK cell interactions (data not shown).

Alternatively, it is possible that the acquisition of MICA results in NK cell fratricide, similar to that observed by T cells after acquisition of MHC class I molecules from APCs (33). In support of this possibility, a large fraction of NK cells that had acquired MICA-YFP from other NK cells had degranulated. Thus in summary, we have demonstrated that activating ligands transfer from target cells onto NK cells during immune surveillance, which could facilitate a novel mechanism for immunoregulation of NK cell activity.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Medical Research Council, the Biotechnology and Biological Science Research Council, a Lister Institute Research Prize (to D.M.D.), and by a fellowship from The Wenner-Gren Foundations (to B.Ö.). Back

2 F.E.M. and P.E. contributed equally to this work. Back

3 Current address: Kennedy Institute of Rheumatology, Imperial College London, 1 Aspenlea Road, London W6 8LH, U.K. Back

4 Current address: Strategic Research Center for Studies of Integrative Recognition in the Immune System, Microbiology and Tumor Biology Center, Karolinska Institute, Box 280, S-171 77 Stockholm, Sweden. Back

5 Address correspondence and reprint requests Dr. Daniel M. Davis, Division of Cell and Molecular Biology, Sir Alexander Fleming Building, Imperial College London, South Kensington Campus, London SW7 2AZ, U.K. E-mail address: d.davis{at}imperial.ac.uk Back

6 Abbreviations used in this paper: MICA, MHC class I-related chain A; ULBP, UL-16 binding protein; YFP, yellow fluorescent protein; MFI, mean fluorescence intensity; KIR, killer Ig-related receptor; CFP, cyan fluorescent protein. Back

7 The online version of this article contains supplemental material. Back

Received for publication July 5, 2006. Accepted for publication December 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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