Ly49A inhibitory receptors redistribute on natural killer cells during target cell interaction

Abstract

When T effector cells meet antigen-bearing target cells, there is a specific accumulation of T-cell receptors, co-receptors and structural proteins at the point of cell-cell contact. Ly49 inhibitory receptors bind to murine major histocompatibility complex (MHC) class I molecules and prevent natural killer-(NK) cell cytotoxicity. In this study we have tested whether inhibitory receptors accumulate at the point of cell-cell contact when NK cells encounter target cells bearing MHC class I ligands for those inhibitory receptors. We have used RNK-16 effector cells that express Ly49A receptors and have found that there was a specific accumulation of Ly49A receptors at the point of NK cell-target cell contact when the target cells expressed H-2Dd. We also observed that engagement of Ly49A on NK cells resulted in an altered redistribution of potential triggering receptors CD2 and NKR-P1. These data indicate that inhibitory receptors, like activating receptors, may specifically aggregate at the point of cell-cell contact which may be necessary for them to mediate their full inhibitory effect.

Figure 1

Ly49A accumulates at cell–cell contact point on EL4KK cells. EL4KK cells were incubated for 15 min with EL4-D^d (a–c) or EL4 cells (d–f). The EL4KK-target conjugates were then acetone fixed and stained for Ly49A (c,f). (a,d) and (b,e) show phase contrast images and Cascade Blue^© staining on target cells, respectively.

Figure 2

Prelabelling of target cells with Cascade Blue^© does not alter NK cell recognition. RNK-16 49A effector cells were assayed for their ability to lyse YB or YB-D^d tumour target cells. The target cells were surface biotinylated and labelled with Cascade Blue^© prior to use in 4-hr ⁵¹Cr-release assays. The results are representative of three independent experiments.

Figure 3

Analysis of cell surface receptors on RNK-16 49A cells. RNK-16 49A cells were incubated for 2 min with YB-D^d (a–c), (g–i) and (m–o) or with YB (d–f), (j–l) and (p–r). The effector–target conjugates were then fixed in acetone (a–f) or in paraformaldehyde (g–r), and stained for Ly49A (c,f), CD2 (i,l) or NKR-P1 (o,r). (a, d, g, j, m and p) show phase contrast images of effector–target conjugates, and (b, e, h, k, n and q) show Cascade Blue^© staining on target cells.

Figure 4

NKR-P1 accumulates inside RNK-16 49A effector cells. Surface- and intracellular staining of NKR-P1 in non-fixed (b), acetone-fixed (d), and paraformaldehyde fixed (f) RNK-16 49A cells. (a, c, and e) show phase contrast images of the antibody-labelled effector cells.