Intercellular protein transfer and regulation of inhibitory NK receptor accessibility
Author: Andersson, Katja
Date: 2007-04-27
Location: Hillarpsalen vid Institutionen för Neurovetenskap, Retzius väg 8, Karolinska Institutet
Time: 09.30
Department: Institutionen för mikrobiologi, tumör- och cellbiologi / Department of Microbiology, Tumor and Cell Biology
View/ Open:
thesis.pdf (2.138Mb)
Abstract
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.
List of papers:
I. Vanherberghen B, Andersson K, Carlin LM, Nolte-t Hoen EN, Williams GS, Hoglund P, Davis DM (2004). "Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells." Proc Natl Acad Sci U S A 101(48): 16873-8. Epub 2004 Nov 18
Pubmed
II. Andersson KE, Sjöström-Douagi A, Höglund P (2007). "Intercellular transfer of target cell MHC class I proteins to NK cells leads to occupation of Ly49 receptors in cis and impaired ligand recognition." (Manuscript)
III. Andersson KE, Williams GS, Davis DM, Hoglund P (2007). "Quantifying the reduction in accessibility of the inhibitory NK cell receptor Ly49A caused by binding MHC class I proteins in cis." Eur J Immunol 37(2): 516-27
Pubmed
IV. Vahlne G, Andersson K, Brennan F, Galsgaard E, Wickström S, Wagtmann N, Karre K, Johansson MH (2007). "In vivo blocking of inhibitory MHC class I receptors triggers selective NK cell-mediated rejection of syngeneic leukemia cells without breaking tolerance towards normal syngeneic cells." (Manuscript)
I. Vanherberghen B, Andersson K, Carlin LM, Nolte-t Hoen EN, Williams GS, Hoglund P, Davis DM (2004). "Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells." Proc Natl Acad Sci U S A 101(48): 16873-8. Epub 2004 Nov 18
Pubmed
II. Andersson KE, Sjöström-Douagi A, Höglund P (2007). "Intercellular transfer of target cell MHC class I proteins to NK cells leads to occupation of Ly49 receptors in cis and impaired ligand recognition." (Manuscript)
III. Andersson KE, Williams GS, Davis DM, Hoglund P (2007). "Quantifying the reduction in accessibility of the inhibitory NK cell receptor Ly49A caused by binding MHC class I proteins in cis." Eur J Immunol 37(2): 516-27
Pubmed
IV. Vahlne G, Andersson K, Brennan F, Galsgaard E, Wickström S, Wagtmann N, Karre K, Johansson MH (2007). "In vivo blocking of inhibitory MHC class I receptors triggers selective NK cell-mediated rejection of syngeneic leukemia cells without breaking tolerance towards normal syngeneic cells." (Manuscript)
Issue date: 2007-04-06
Rights:
Publication year: 2007
ISBN: 978-91-7357-183-8
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