Intercellular protein transfer and regulation of inhibitory NK receptor accessibility
Year: 2007
Type: dissertation
Abstract: NK cells are important players of innate immunity and capable of\npromoting specific responses of the adaptive immune system. NK cells\npossess the ability to recognise and eliminate virus-infected cells,\ntumour cells and allogeneic bone marrow grafts. The effector functions of\nNK cells are regulated by a fine-tuned balance of signals from activating\nand MHC class I binding inhibitory receptors. In this thesis I\ninvestigated the interactions between inhibitory Ly49 receptors and their\nMHC ligands. In particular, effects of these interactions, like\nintercellular protein transfer and reduced cell surface expression of\nreceptors, as well as the functional consequences thereof were studied.\nIn addition a tumour therapy approach based on blockade of these\ninteractions was explored.\n\nI: Bidirectional intercellular transfer of proteins across the inhibitory\nNK cell immunological synapse (IS). Here we show that for both murine and\nhuman cells, target cells expressing MHC class I ligands could acquire\ncognate inhibitory NK receptors. Along with these, other cell surface\nproteins could co-transfer. The extent of KIR acquired from NK cells\ncorrelated with the level of expression of cognate MHC class I protein on\nthe target cells. Transfer of MHC molecules to the NK cell also occurred\nand the target cell cytoskeleton influenced intercellular transfer of\nproteins in both directions. Constitutively expressed KIR could not be\nremoved via mild acid wash treatment while a fraction of acquired KIR\ncould. However, an accumulation of phosphotyrosines at the location of\nthe transferred KIR suggests a signalling capacity for NK cell proteins\ntransferred to target cells. Recent data from our and other groups,\nregarding intercellular protein transfer, suggest that this kind of\ncellular communication might play an important role in immune\nsurveillance.\n\nII: NK cells, expressing inhibitory Ly49A receptors, specifically acquire\ntheir cognate MHC class I ligands, H-2Dd, from surrounding cells in vivo.\nHere we introduce three different in vitro systems, supporting\nLy49A+-dependent acquisition of H-2Dd by splenic NK cells. Kinetics\nexperiments revealed that transfer of H-2Dd was observed already after 1\nminute, while downmodulation of the Ly49A receptor occurred later,\nsuggesting that MHC class I transfer precedes receptor downmodulation.\nFurthermore, the acquired H-2Dd molecules interfered with the capacity of\nLy49A to receive inhibitory signals delivered by ligands on target cells.\nInterestingly, when Ly49C was co-expressed with Ly49A on NK cells, the\nability to acquire H-2Dd increased, but only in the presence of the Ly49C\nligand H-2Kb on the target cell. The transferred H-2Dd molecules may\nfine-tune, through cis interactions with Ly49A expressed on the same\ncell, the accessibility of inhibitory Ly49A receptors and thereby\nregulate the NK cell immune functions.\n\nIII: The issue of accessibility of inhibitory receptors at the NK cell\nsurface is an important question as the sensitivity of individual NK\ncells to inhibitory interactions is a critical determinant for NK cell\nfunction, not only at the effector stage, but also during NK cell\ndevelopment. The cis-interaction is formed between Ly49A and H-2Dd both\nexpressed on the same NK cell surface. We quantified accessibility of the\nLy49A receptors by using an established protein transfer assay, measuring\nthe amount of H-2Dd-GFP molecules transferred to Ly49A expressing NK\ncells. Constitutive expression of H-2Dd molecules on B6.Dd NK cells\nreduced the ability to acquire H-2Dd-GFP molecules and decreased the\nclustering of H-2Dd-GFP molecules at the NK-target-cell contact site.\nThis correlated to a reduced sensitivity to H-2Dd-mediated inhibition in\ncytotoxicity assays. Ly49A+ NK cells from B6.Dd mice showed a 90 %\nreduction in Ly49A accessibility that was caused both by absolute lower\nexpression of Ly49A and interactions in cis between Ly49A and H-2Dd at\nthe NK cell surface. Thus, endogenously expressed H-2Dd ligands regulate\nLy49A receptor accessibility through interactions both in cis and in\ntrans, in this manner regulate central developmental processes or\nperipheral tolerance mechanisms.\n\nIV: Therapeutic strategies for the treatment of cancer are being\ndeveloped based on preventing NK cell inhibition or triggering NK cell\nreceptors to activate NK cells. In this study we investigated, using a\nmouse model, whether it would be possible to identify a therapeutic\ninterval for inhibitory receptor blockade, where NK cells would be\ninduced to kill syngeneic tumours, but still leave normal cells\nuntouched. Our approach was to block inhibitory Ly49C/I receptors that\nbind to MHC class I molecules (H-2Kb), with Ly49C/I specific F(ab )2\nfragments both in vitro and in vivo. In vitro, this resulted in blockade\nof up to 80% of the Ly49C/I receptors and induced killing of syngeneic\ntumour cells and lymphoblasts by activated NK cells in vitro. In vivo, a\n80-85% blockade of Ly49C/I caused NK cell-mediated selective rejection of\ni.v. inoculated fluorescence labelled syngeneic tumour cells but not of\nsyngeneic spleen cells, bone marrow cells or lymphoblasts administered in\na similar manner. The anti-tumor effect was maintained after 2 weeks of\ncontinuous receptor blockade without induction of autoreactivity or NK\ncell anergy. Our data demonstrate that inhibitory receptor blockade\nresults in increased rejection of syngeneic tumour cells, but no killing\nof normal syngeneic cells in vivo.
Source: Figshare
Author: Katja Andersson
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1550299076