Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D

Abstract

Cell surface proteins major histocompatibility complex (MHC) class I-related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein-tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.

Figure 1.

NKG2D ligands colocalize with cross-linked patches of CT-B in Daudi transfectants. (a) After cross-linking GM1, Daudi/Class I⁺ cells transfected with ULBP1, 2, 3, or MICA were fixed and stained for CT-B (second column) and the appropriate NKG2D ligand (third column). As a control, the distribution of CD45 was compared (last row). Images show single confocal planes. Bars, 5 μm. (b) Correlation coefficients calculated for colocalization of CT-B and NKG2D ligands in individual GM1-patched cells are shown (open circles). Filled circles mark the specific cell shown in panel a. Between 14 and 27 cells of each type that had clearly visible patching of GM1 were selected for quantitative analysis. The mean for each cell type is shown with standard errors. The positive control (+Ctrl) corresponds to labeling of CT-B with two different secondary antibodies.

Figure 2.

Probing the cell surface organization of NKG2D ligands in DRMs and by electron microscopy. DRMs were prepared from (a) Daudi/Class I⁺/ULBP3 and Namalwa or (b) Daudi/Class I⁺/MICA cells. 10 fractions were collected from the top of the gradient (fraction 1 denotes the top of the gradient and fraction 10 denotes the bottom), and equal volumes of each were resolved by reducing electrophoresis and probed by Western blotting for each molecule indicated. DRM and detergent-soluble (Det. Sol.) fractions are marked. Molecular masses are indicated in kilodaltons. For TfR, a fainter second band corresponding to the size of TfR dimers was also evident (not depicted). ULPB3, but not MICA, associates with DRMs. Ultrathin cryosections of Daudi/Class I⁺/ULBP1 were immunolabeled for (c) CD45 or (d) ULBP1. Primary antibodies were detected with 10 nm of gold particles conjugated to protein A. ULBP1 is expressed in clusters at the cell surface (arrowheads), as highlighted by the enlarged view of a small region of cell surface (inset, bottom right). Bar, 0.2 μm.

Figure 3.

Recruitment of ULBP3 and MICA to the NK cell immune synapse. (a) CD56⁺ CD3⁻ human peripheral blood NK cells were coincubated with Daudi/Class I⁺/MICA cells for 15 min, fixed, and stained with anti-MICA mAb, and the distribution at the immunological synapse was determined. The first row shows a single NK cell forming two activating synapses with two different Daudi/Class I⁺/MICA cells. Data shown are representative of three experiments in which 301 NK-Daudi/Class I⁺/MICA conjugates were assessed. (b) A CD56⁺ CD3⁻ human peripheral blood NK clone was coincubated with GPI-GFP–expressing Namalwa transfectants for 15 min, fixed, and stained for ULBP3. Images are representative of 13 out of 14 conjugates where ULBP3 was clearly seen to accumulate at the synapse. Bars, 5 μm.

Figure 4.

MICA is S-acylated, and a truncated form of MICA is unable to activate NK cells. (a) Daudi/Class I⁺/MICA cells were labeled for 5 h with either [³⁵S]methionine (35S MET) or with [³H]palmitate (3H PAL), lysed, immunoprecipitated with anti-MICA or anti-TfR mAb, and analyzed by SDS-PAGE and fluorography. Molecular weight markers (MW) are also shown. The two bands seen for TfR correspond to the size of TfR monomers and dimers. The mobility of MICA was higher in this gel than in that used in Fig. 2, probably due to the nonreducing conditions. Fluorograms were exposed for 81 d (3H PAL) or 7 d (35S MET). (b and c) Cell surface staining by anti-MICA mAb of (b) Daudi/Class I⁺/MICA and (c) Daudi/Class I⁺/MICA/C331*. Plots show staining with an isotype-matched control mAb (shaded) and staining with anti-MICA (line). (d) Susceptibility of Daudi (□), Daudi/Class I⁺ (♦), Daudi/Class I⁺/MICA (•), and Daudi/Class I⁺/MICA/C331* (▴) by a peripheral blood NK cell line, assessed in a 5-h [³⁵S]Met release assay. Data are representative of three independent experiments, each performed in triplicate.