Transfer of NKG2D and MICB at the cytotoxic NK cell immune synapse correlates with a reduction in NK cell cytotoxic function

Abstract

Although transfer of membrane proteins has been shown to occur during immune cell interactions, the functional significance of this process is not well understood. Here we describe the intercellular transfer of NKG2D and MHC class I chain-related molecule (MIC) B proteins at the cytotoxic natural killer cell immune synapse (cNK-IS). MICB expressed on the 721.221 cell line induced clustering of NKG2D at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing F-actin. Moreover, natural killer (NK) cell membrane-connective structures formed during cytotoxic interactions contained F-actin, perforin, and NKG2D. NKG2D transfer depended on binding to MICB and was specific because transfer of other molecules not involved in NK-IS formation was not observed. Transfer of MICB to NK cells also was noted, suggesting a bidirectional exchange of receptor/ligand pairs at cNK-IS. Experiments designed to test the functional significance of these observations revealed that brief interactions between NK cells and MICB expressing target cells led to a reduction in NKG2D-dependent NK cytotoxicity. These data demonstrate interchange of an activating receptor and its ligand at the cNK-IS and document a correlation between synapse organization, intercellular protein transfer, and compromised NK cell function after interaction with a susceptible target cell.

Fig. 1.

MICB-induced clustering and cSMAC segregation of NKG2D at cNK-IS. (A) NKL/221 and NKL/221B conjugates were stained for NKG2D. Fluorescence and bright-field images (a) and quantification (%) of cell conjugates in which NKG2D was localized at the contact site (b) are shown. One representative experiment of at least three independent ones is shown. (B–D) Conjugates formed with NKL (B) or primary-activated NK cells (C and D) and 221 or 221B cells were costained for NKG2D (red) and F-actin (green). (C) NKG2D distribution patterns shown in NK/221B or NK/221 interface are representative of 11 conjugates analyzed over three independent donors. (D) Four confocal sections of two representative NK/221B conjugates showing intracellular NKG2D-containing vesicles (arrows). In all experiments, target cells were identified by their bigger size or by labeling with CMAC (blue) in experiments done with the NKL cell line. (Scale bars: 5 μm.)

Fig. 2.

NKG2D transfer to 221B cells. (A–C) NKL/221B (A) or NK/221B (B and C) cell conjugates were costained for NKG2D (red) and F-actin (green). A confocal section (merged channels in A and B) shows NKG2D clusters on target cells (see arrowheads). (B) Three-dimensional max projection shows the synapse face of the NK cell pointed by an arrow. (C) Zoom of merged channels shows NKG2D clusters at the edge of F-actin rich lamelipodia. (D) NK/221B conjugates costained for NKG2D (red) and DAP10 (green). One confocal section and the 3D max projection are shown. An arrowhead points to an NKG2D/DAP10-containing cluster distant to the synapse face. (E) Confocal microscopy analysis of NK/221B samples shows NKG2D-containing clusters transferred to an isolated 221B target cell (Left, arrow). As a negative control, NKG2D staining obtained in 221B plated alone on coverslips is shown (Right). (Scale bars: Left, 5 μm; Right, 20 μm.) (F) Quantification of target cells containing NKG2D or 2B4 membrane clusters in NK/221 or NK/221B conjugates. Histogram bars represent the arithmetic mean ± SD (n = 100). (G) NK/221B conjugates were costained for 2B4 (green) and NKG2D (red). Arrowheads point to colocalization of NKG2D and 2B4 on interacting cells. One confocal section and the differential interference contrast image are shown. 221B cells were labeled with CMAC (blue) in experiments done with NKL cells. (Scale bars: 5 μm except E Right.)

Fig. 3.

MCS formed in NK/221B conjugates contain NKG2D. (A) The indicated cell conjugates were stained for NKG2D. Confocal microscopy analysis shows NKG2D containing MCS (arrowheads) and clusters transferred to 221B target cells but not to 221 ones (arrows). (B–D) NK/221B conjugates were costained for NKG2D (red)/F-actin (green) (B), perforin (red)/F-actin (green) (C), and perforin (green)/NKG2D (red) (D). (B) Arrowheads and yellow arrows inside the zoom of merged channels indicate NKG2D spots and overlapping NKG2D and F-actin, respectively. Two arrows point to the MCS in the differential interference contrast image. (C) A magnified view of the area of the MCS is shown. (D) A yellow arrow points to the area magnified that shows the partial overlapping between NKG2D and perforin. Confocal sections and differential interference contrast images are shown. (Scale bars: 5 μm.)

Fig. 4.

NKG2D transfer to 221B cells depends on MICB binding. (A) Shown are NKG2D (phycoerythrin) and MHC class I (FITC) double staining of cell mixtures containing NK and 221B cells at 4°C (Left) and NK cells previously incubated with 221 or 221B at 37°C (Center and Right). Numbers within plots indicate the FL2 geomean fluorescence of the 221 or 221B gated population. (B) Quantification of the NKG2D transfer in NK/221 or NK/221B conjugates. Histogram bars represent the arithmetic mean ± SD of NKG2D/negative staining geomean ratio obtained in five experiments done with five different donors. Statistical significance of data was analyzed by the Mann–Whitney U test. (C) Abrogation of NKG2D transfer by anti-NKG2D blocking mAb in NK/221B conjugates. As a negative control, an anti-CD56 mAb was used. Concentrations of the antibodies are indicated. One representative experiment of at least three independent ones is shown. (D) Down-modulation of NKG2D surface expression in NK cells exposed to 221B cells. Means ± SD of three independent experiments are compared by the Student t test. P > 0.05 was considered nonsignificant (ns). (E) Histograms of one representative experiment showing the surface expression of NKG2D in NK cells before (a) and after an encounter with either 221 (b) or 221B (c) cells. Numbers indicate the geomean fluorescence of NKG2D staining. Thin lines represent the negative staining. (Ed) NKG2D histograms of a (thin lane) and c (thick lane) are overlays.

Fig. 5.

MICB transfers to NK cells. (A–C) The indicated samples were costained for MICB in red and F-actin (A) or NKG2D (B and C) in green. Arrowheads point to MICB at the contact interface (A) and at MCS and interacting NK cells (B). Yellow arrows point to NKG2D/MICB colocalization on NK cells (B and C). (C) Cross-sections (zx and zy) show the colocalization between NKG2D and MICB on the cell surface of an isolated NK cell. (D) Negative MICB staining on activated NK cells plated alone. (A–D) One confocal section and differential interference contrast images are shown. (E Left) A representative example of MICB staining on NK cells previously incubated with CSFE-labeled 221 or 221B cells is shown (Materials and Methods). (E Right) The quantification of the MICB transfer to NK cells in NK/221 or NK/221B cell mixtures is shown. Histogram bars represent the arithmetic mean ± SD of MICB geomean fluorescence obtained from three independent experiments done with three different donors. NK/221 (MICB-GFP) samples were stained for NKG2D (red). A confocal plane and 3D max projection are shown. Arrowheads point to NKG2D/MICB-GFP colocalization on NK cell surface. (Scale bars: A–C, E, and F, 5 μm; D, 10 μm.)

Fig. 6.

MICB expressed on 721.221 cells reduce NKL cytotoxic function. (A) Cytotoxic activity, against ⁵¹Cr-labeled 221B cells, of NKL cells previously incubated with 221 or 221B cells at 37°C for 5 min (E:T ratio 1:1) was evaluated (N1/221B and NB/221B) and compared with the cytotoxic activity of NKL (not incubated with any target) against 221 and 221B cells (NKL/221 and NKL/221B). Cytotoxic assays were done at an E:T ratio of 5:1. Histogram bars represent the arithmetic mean ± SD of three independent experiments done in triplicate. Means were compared by the Student t test. (B) FACS analysis of perforin expression in effector cells. Numbers in histogram plots indicate the geomean fluorescence of the positive population under the line. Dotted lines represent the isotype control staining.