A modified FCCS procedure applied to Ly49A-MHC class I cis-interaction studies in cell membranes.

The activity of natural killer (NK) cells is regulated by a fine-tuned balance between activating and inhibitory receptors. Dual-color fluorescence cross-correlation spectroscopy (FCCS) was used to directly demonstrate a so-called cis-interaction between a member of the inhibitory NK cell receptor family Ly49 (Ly49A), and its ligand, the major histocompatibility complex (MHC) class I, within the plasma membrane of the same cell. By a refined FCCS model, calibrated by positive and negative control experiments on cells from the same lymphoid cell line, concentrations and diffusion coefficients of free and interacting proteins could be determined on a collection of cells. Using the intrinsic intercellular variation of their expression levels for titration, it was found that the fraction of Ly49A receptors bound in cis increase with increasing amounts of MHC class I ligand. This increase shows a tendency to be more abrupt than for a diffusion limited - three dimensional bimolecular reaction, which most likely reflects the two-dimensional confinement of the reaction. For the Ly49A- MHC class I interaction it indicates that within a critical concentration range the local concentration level of MHC class I can provide a distinct regulation mechanism of the NK cell activity.

Quantifying the reduction in accessibility of the inhibitory NK cell receptor Ly49A caused by binding MHC class I proteins in cis.

Murine natural killer (NK) cells are inhibited by target cell MHC class I molecules via Ly49 receptors. However, Ly49 receptors can be made inaccessible to target cell MHC class I by a cis interaction with its MHC class I ligand within the NK cell membrane. It has recently been demonstrated that MHC class I proteins transfer from the target cells to the NK cell. Here, we establish that the number of transferred MHC class I proteins is proportional to the number of Ly49A receptors at the NK cell surface. Ly49A+ NK cells from mice expressing the Ly49A ligand H-2D(d) showed a 90% reduction in Ly49A accessibility compared to Ly49A+ NK cells from H-2D(d)-negative mice. The reduction was caused both by lower expression of Ly49A and interactions in cis between Ly49A and H-2D(d) at the NK cell surface. Approximately 75% of the Ly49A receptors on H-2D(d)-expressing NK cells were occupied in cis with endogenous H-2D(d) and only 25% were free to interact with H-2D(d) molecules in trans. Thus, H-2D(d) ligands control Ly49A receptor accessibility through interactions both in cis and in trans.

Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells.

Intercellular transfer of proteins across the immunological synapse is emerging as a common outcome of immune surveillance. We previously reported that target-cell MHC class I protein transfers onto natural killer (NK) cells expressing cognate killer Ig-like receptors (KIRs). We now show that, for both murine and human cells, target cells expressing inhibitory MHC class I ligands acquire cognate inhibitory NK receptors. Other cell-surface proteins, but not a cytoplasmic dye, also transferred from human NK cells to target cells across an inhibitory immunological synapse. The number of KIRs acquired from NK cells correlated with the level of expression of cognate MHC class I protein on target cells. Treatment with cytoskeletal inhibitors demonstrated that the target-cell cytoskeleton influences intercellular transfer of proteins in both directions. In contrast to constitutively expressed KIRs, a fraction of acquired KIRs could be removed by mild acid wash, demonstrating a difference between some of the acquired KIRs and constitutively expressed KIRs. An accumulation of phosphotyrosine at the location of the transferred KIRs implies a signaling capacity for NK cell proteins transferred to target cells. Thus, intercellular protein transfer between immune cells is bidirectional and could facilitate new aspects of immune cell communication.

The protean immune cell synapse: a supramolecular structure with many functions.

Heterogeneity in the supramolecular organization of immunological synapses arises from the involvement of different cells, distinct environmental stimuli, and varying levels of protein expression. There may also be heterogeneity in the types and amounts of cell surface proteins and lipids that transfer between lymphocytes during immune surveillance. In addition, immune cells can be involved in the assembly of a 'viral synapse', such that micrometer-scale organization of proteins at intercellular contacts occurs during transmission of a virus between T cells. Thus, while there may be unity in molecular mechanisms underlying the organization of cell surface receptors at immune cell synapses, there is diversity in their function.