Intercellular protein transfer and regulation of inhibitory NK receptor accessibility

Sammanfattning: NK cells are important players of innate immunity and capable of promoting specific responses of the adaptive immune system. NK cells possess the ability to recognise and eliminate virus-infected cells, tumour cells and allogeneic bone marrow grafts. The effector functions of NK cells are regulated by a fine-tuned balance of signals from activating and MHC class I binding inhibitory receptors. In this thesis I investigated the interactions between inhibitory Ly49 receptors and their MHC ligands. In particular, effects of these interactions, like intercellular protein transfer and reduced cell surface expression of receptors, as well as the functional consequences thereof were studied. In addition a tumour therapy approach based on blockade of these interactions was explored. I: Bidirectional intercellular transfer of proteins across the inhibitory NK cell immunological synapse (IS). Here we show that for both murine and human cells, target cells expressing MHC class I ligands could acquire cognate inhibitory NK receptors. Along with these, other cell surface proteins could co-transfer. The extent of KIR acquired from NK cells correlated with the level of expression of cognate MHC class I protein on the target cells. Transfer of MHC molecules to the NK cell also occurred and the target cell cytoskeleton influenced intercellular transfer of proteins in both directions. Constitutively expressed KIR could not be removed via mild acid wash treatment while a fraction of acquired KIR could. However, an accumulation of phosphotyrosines at the location of the transferred KIR suggests a signalling capacity for NK cell proteins transferred to target cells. Recent data from our and other groups, regarding intercellular protein transfer, suggest that this kind of cellular communication might play an important role in immune surveillance. II: NK cells, expressing inhibitory Ly49A receptors, specifically acquire their cognate MHC class I ligands, H-2Dd, from surrounding cells in vivo. Here we introduce three different in vitro systems, supporting Ly49A+-dependent acquisition of H-2Dd by splenic NK cells. Kinetics experiments revealed that transfer of H-2Dd was observed already after 1 minute, while downmodulation of the Ly49A receptor occurred later, suggesting that MHC class I transfer precedes receptor downmodulation. Furthermore, the acquired H-2Dd molecules interfered with the capacity of Ly49A to receive inhibitory signals delivered by ligands on target cells. Interestingly, when Ly49C was co-expressed with Ly49A on NK cells, the ability to acquire H-2Dd increased, but only in the presence of the Ly49C ligand H-2Kb on the target cell. The transferred H-2Dd molecules may fine-tune, through cis interactions with Ly49A expressed on the same cell, the accessibility of inhibitory Ly49A receptors and thereby regulate the NK cell immune functions. III: The issue of accessibility of inhibitory receptors at the NK cell surface is an important question as the sensitivity of individual NK cells to inhibitory interactions is a critical determinant for NK cell function, not only at the effector stage, but also during NK cell development. The cis-interaction is formed between Ly49A and H-2Dd both expressed on the same NK cell surface. We quantified accessibility of the Ly49A receptors by using an established protein transfer assay, measuring the amount of H-2Dd-GFP molecules transferred to Ly49A expressing NK cells. Constitutive expression of H-2Dd molecules on B6.Dd NK cells reduced the ability to acquire H-2Dd-GFP molecules and decreased the clustering of H-2Dd-GFP molecules at the NK-target-cell contact site. This correlated to a reduced sensitivity to H-2Dd-mediated inhibition in cytotoxicity assays. Ly49A+ NK cells from B6.Dd mice showed a 90 % reduction in Ly49A accessibility that was caused both by absolute lower expression of Ly49A and interactions in cis between Ly49A and H-2Dd at the NK cell surface. Thus, endogenously expressed H-2Dd ligands regulate Ly49A receptor accessibility through interactions both in cis and in trans, in this manner regulate central developmental processes or peripheral tolerance mechanisms. IV: Therapeutic strategies for the treatment of cancer are being developed based on preventing NK cell inhibition or triggering NK cell receptors to activate NK cells. In this study we investigated, using a mouse model, whether it would be possible to identify a therapeutic interval for inhibitory receptor blockade, where NK cells would be induced to kill syngeneic tumours, but still leave normal cells untouched. Our approach was to block inhibitory Ly49C/I receptors that bind to MHC class I molecules (H-2Kb), with Ly49C/I specific F(ab )2 fragments both in vitro and in vivo. In vitro, this resulted in blockade of up to 80% of the Ly49C/I receptors and induced killing of syngeneic tumour cells and lymphoblasts by activated NK cells in vitro. In vivo, a 80-85% blockade of Ly49C/I caused NK cell-mediated selective rejection of i.v. inoculated fluorescence labelled syngeneic tumour cells but not of syngeneic spleen cells, bone marrow cells or lymphoblasts administered in a similar manner. The anti-tumor effect was maintained after 2 weeks of continuous receptor blockade without induction of autoreactivity or NK cell anergy. Our data demonstrate that inhibitory receptor blockade results in increased rejection of syngeneic tumour cells, but no killing of normal syngeneic cells in vivo.