Recruitment of activation receptors at inhibitory NK cell immune synapses

Abstract

Natural killer (NK) cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR) family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

Figure 1. Detection of inhibitory synapses using the cyt42/43 antiserum.

IL-2 activated polyclonal human NK cells were mixed with target cells for 10 minutes at 37°C, fixed, permeabilized and stained with the cyt42/43 rabbit polyclonal antiserum. NK cells and NK:target cell conjugates stained with the cyt42/43 antiserum were scored for KIR clustering. The number of cyt42/43-positive cell conjugates analyzed is given in parentheses over each bar. (A) Mixing with .221 and .221-Cw15 target cells, as indicated. (B) NK cells expressing KIR2DL2 and not KIR2DL1 mixed with .221-Cw3 and .221-Cw15 cells, as indicated. (C) Mixing with S2–Cw4 cells loaded with a peptide that is permissive for KIR2DL1 binding (peptide #1) or a peptide that is nonpermissive for KIR2DL1 binding (K8E), as indicated.

Figure 2. CD2 accumulates at both activating and inhibitory synapses.

IL-2 activated polyclonal human NK cells were mixed with target cells at 37°C for 10 minutes, fixed, permeabilized, and stained with the cyt42/43 antiserum and a mAb to CD2 followed by the relevant secondary antibodies. Confocal microscope z-series were obtained. (A) Mixed with .221-Cw15 as indicated. A single confocal section is shown. The cell labeled #1, which displays KIR expression and clustering, represents an inhibitory synapse while cell #2, which lacks KIR2DL1 expression, displays an activating synapse. (B) Mixed with S2–LFA-3/Cw4 target cells, as indicated. A single confocal section is shown. (C) The fluorescence intensity was scanned around the perimeter of conjugated NK cells. Profiles labeled 1, 2, and 3 are from the corresponding cells in Figures 2A and 2B. The green and red lines represent the cyt42/43 and anti–CD2 fluorescence, respectively. Vertical red and blue lines mark the boundaries of cell contact as determined in DIC images. (D) Confocal z-stacks were used to create an en face view of the zone of cell contact in 2 inhibitory synapses.

Figure 3. CD2 accumulates more frequently at inhibitory synapses than at activating synapses.

(A) Conjugates between IL-2 activated polyclonal human NK cells and 721.221-Cw15 cells or S2–LFA-3/Cw4 cells were formed and stained as in Figure 2. Activating (Act.) and inhibitory (Inhib.) synapses were scored for clustering of CD2 at the zone of contact. The number of conjugates scored in each condition is indicated in parentheses. (B) Conjugates between activated NK cells and 721.221-Cw15 cells were allowed to form for 1 minute or 10 minutes as indicated, stained with the cyt42/43 antiserum and an anti-CD2 antibody as in Figure 2, and scored for CD2 clustering.

Figure 4. 2B4 accumulates at both activating and inhibitory synapses.

Activated NK cells were mixed with target cells at 37°C for 10 minutes, fixed, permeabilized, and stained with the cyt42/43 antiserum and a mAb to 2B4 followed by the appropriate secondary antibodies. (A) Mixed with .221-Cw15 target cells. Confocal microscope z-series were obtained, and single sections are shown. The cells labeled #1 and #3, which display KIR expression and clustering, represent inhibitory synapses while cell #2, which lacks KIR2DL1 expression, is an activating synapse. The NK cells without a number in the top image were not analyzed, because they did not appear to form tight conjugates with target cells. (B) The fluorescence intensity was scanned around the perimeter of conjugated NK cells. Profiles labeled 1, 2, and 3 are from the corresponding cells in Figure 4A. The green and blue lines represent the cyt42/43 and anti–2B4 fluorescence, respectively. Vertical red and blue lines mark the boundaries of cell contact as determined in DIC images. (C) Confocal z-stacks were used to create an en face view of the zone of cell contact in 2 inhibitory synapses.

Figure 5. Surface level of CD11a is reduced at inhibitory synapses.

IL-2 activated polyclonal human NK cells and S2 cells expressing ICAM-1 and peptide-loaded HLA-Cw4 were mixed at 37°C for 10 minutes, fixed, permeabilized, and stained with cyt42/43 and a mAb to CD11a followed by the appropriate secondary antibodies. (A) Confocal microscope z-series were obtained, and single sections are shown. The cell labeled #1, which shows KIR expression and clustering, represents an inhibitory synapse while cell #2, which lacks KIR2DL1 expression, displays an activating synapse. (B) The fluorescence intensity was scanned around the perimeter of conjugated NK cells. Profiles labeled 1 and 2 are from the corresponding cells in Figure 5A. The third profile is another representative inhibitory synapse. The green and red lines represent the cyt42/43 and anti–CD11a fluorescence, respectively. Vertical red and blue lines mark the boundaries of cell contact as determined in DIC images. (C) Confocal z-stacks were used to create an en face view of the zone of cell contact in 2 inhibitory synapses. (D) The frequency of synapses displaying increased CD11a, reduced CD11a, or no change in CD11a intensity was determined for both activating and inhibitory synapses.