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Blood, 1 March 2002, Vol. 99, No. 5, pp. 1723-1729
IMMUNOBIOLOGY
Analysis of natural killer cells in TAP2-deficient patients:
expression of functional triggering receptors and evidence for the
existence of inhibitory receptor(s) that prevent lysis of normal
autologous cells
Massimo Vitale,
Jacques Zimmer,
Roberta Castriconi,
Daniel Hanau,
Lionel Donato,
Cristina Bottino,
Lorenzo Moretta,
Henri de la
Salle, and
Alessandro Moretta
From the Istituto Nazionale per la Ricerca sul Cancro,
Genova, the Dipartimento di Medicina Sperimentale, Università di
Genova, and the Istituto Giannina Gaslini, Genova, Italy; INSERM EP
99-08 Etablissement Francais du Sang-Alsace, Strasbourg, and Service de
Pediatrie, Hopital de Hautepierre, Strasbourg, France.
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Abstract |
Natural killer (NK) cells are characterized by the ability to kill
cells that lack HLA class I molecules while sparing autologous normal
(HLA class I+) cells. However, patients with
transporter-associated antigen processing (TAP) deficiency, though
displaying strong reductions of HLA class I surface expression, in most
instances do not experience NK-mediated autoimmune phenomena. A
possible mechanism by which TAP / NK cells avoid
autoreactivity against autologous HLA class I-deficient cells could be
based on either quantitative or qualitative defects of surface
receptors involved in NK cell triggering. In this study we show that NK
cells derived from 2 patients with TAP2/ express normal
levels of all known triggering receptors. As revealed by the analysis
of polyclonal and clonal NK cells, these receptors display normal
functional capabilities and allow the killing of a panel of
NK-susceptible targets, including autologous B-LCLs. On the other hand,
TAP2/ NK cells were unable to kill either allogeneic
(HLA class I+) or autologous (HLA class I )
phytohemagglutinin (PHA) blasts even in the presence of anti-HLA class
I monoclonal antibody. These data suggest that TAP2/ NK
cells express still unknown inhibitory receptor(s) capable of
down-regulating the NK cell cytotoxicity on binding to surface ligand(s) expressed by T cell blasts. Functional analyses, both at the
polyclonal and at the clonal level, are consistent with the concept
that the putative inhibitory receptor is expressed by virtually all
TAP2/ NK cells, whereas it is present only in rare NK
cells from healthy persons. Another possibility would be that
TAP2/ NK cells are missing a still unidentified
triggering receptor involved in NK cell-mediated killing of PHA blasts.
(Blood. 2002;99:1723-1729)
© 2002 by The American Society of Hematology.
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Introduction |
The peptide transporter-associated antigen
processing (TAP)1,2 is a heterodimer (formed by TAP-1 and
TAP-2 subunits) that imports into the lumen of the endoplasmic
reticulum the peptides required for a correct assembly of HLA class I
molecules. Thus, cells derived from patients displaying defective
expression of either of the TAP subunits are characterized by a strong
reduction of mature HLA class I molecules at the cell
surface.3 It is well known that impaired HLA class I
expression renders target cells susceptible to NK-mediated
cytotoxicity.4-9 Therefore, in patients with TAP
deficiency, NK-mediated autoimmune reactions could occur unless unknown
fail-safe mechanisms prevent an attack against autologous normal cells
expressing insufficient amounts of HLA class I molecules. In agreement
with this concept, freshly isolated TAP2/ NK cells were
unable to kill autologous, HLA class I-negative, B-lymphoblastoid cell
lines (B-LCLs).10 However, this tolerance may be broken in
cases of inflammation. Indeed TAP/ patients have been
reported to have type 1 bare lymphocyte syndrome that is accompanied in
childhood by sinusitis and recurrent bronchitis and in adulthood by
chronic lung inflammation and bronchiectasia.3,10 In a
recent report, some TAP-deficient adult patients have been described
with necrotizing granulomatous lesions in the upper respiratory tract
and in the skin, with infiltrating, activated NK or T cell receptor
  + cells.11 Thus, at least in these
patients, a sustained activation of NK cells, which is likely to occur
in the context of recurrent infections and chronic inflammation, may
lead to the disruption of self-tolerance by NK cells and, consequently,
to autoimmune manifestations. In this context, after culture in the
presence of interleukin (IL)-2, TAP2/ NK cells have
been reported to acquire the ability to kill autologous Epstein-Barr
virus (EBV)-transformed B-LCLs10 or autologous fibroblasts.12 On the other hand, though some evidence
exists for the occurrence of autoimmune phenomena, it is conceivable that TAP/ NK cells adapt to the surrounding HLA class
I-negative microenvironment to avoid inappropriate attacks on
otherwise normal cells. A possible mechanism that could allow
TAP/ NK cells to spare autologous normal cells would be
based on the defective expression of one or another NK
cell-triggering receptor.
In this context, although NK cells from TAP2/ patients
have been shown to express a normally diversified repertoire of HLA class I-specific KIRs,10 no data are available on the
expression and function of the recently identified Natural Cytotoxicity
Receptors (NCRs).13 In healthy persons, these receptors
(including NKp46, NKp30, and NKp44)14-16 are responsible
for the induction of NK cell-mediated cytotoxicity against tumor cells
and normal allogeneic cells such as phytohemagglutinin (PHA)
blasts.17 In the current study, we show that NCRs are
expressed at normal levels in NK cells from 2 TAP2/
patients. More important, in activated TAP2/ NK cells,
NCRs allow killing of a wide range of target cells including allogeneic
tumor cell lines and autologous B-LCLs. However, TAP2/
NK cells did not lyse autologous PHA-induced T cell blasts in spite of
the lack of HLA class I expression. Our data also provide suggestive
evidence for the existence of receptor-ligand interactions that
prevent NK cell-mediated cytotoxicity against autologous normal cells.
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Materials and methods |
TAP2/ patients
The 2 patients analyzed (E.M.O. and E.F.A.) were previously
described.3,10 E.M.O. and E.F.A. are siblings (12 and 21 years old, respectively) homozygous for a stop mutation in the
TAP-2 gene. As a consequence, their cells express less than
3% of HLA class I molecules compared with normal cells. They
experience recurrent bacterial sinobronchial infections but have no
history of viral infections.
Approval was obtained from the Institutional Review Board for
these studies. Informed consent was provided according to the Declaration of Helsinki. The use of initials was approved by informed consent by both the patients and the healthy donor.
Purification of peripheral blood lymphocytes and generation of
polyclonal or clonal NK populations from patients and from healthy
donors
Peripheral blood lymphocytes (PBLs) were derived from healthy
donors or from patients by Ficoll-Hypaque gradients and depletion of
plastic-adherent cells. To obtain enriched NK cells, PBLs were incubated with anti-CD3 (JT3A), anti-CD4 (HP2.6), and anti-HLA-DR (D1.12) monoclonal antibodies (mAbs) (30 minutes at 4°C) followed by
goat antimouse-coated Dynabeads (Dynal, Oslo, Norway) (30 minutes at
4°C) and immunomagnetic depletion.14,16
CD34DR cells were cultured on
irradiated feeder cells in the presence of 100 U/mL recombinant IL-2
(Proleukin; Chiron, Emeryville, CA) and 1.5 ng/mL PHA (Gibco, Paisley,
Scotland) to obtain polyclonal NK cell populations or, after limiting
dilution, NK cell clones. To obtain polyclonal T cell populations, PBL
were cultured on irradiated feeder cells in the presence of 100 U/mL
rIL-2 and 1.5 ng/mL PHA.18
Monoclonal antibodies
The following mAbs were produced in our laboratory: JT3A
(immunoglobulin [Ig]G2a, anti-CD3), BAB281,14 and
KL24719 (IgG1 and IgM, respectively, anti-NKp46),
Z23116 and KS3820 (IgG1 and IgM, respectively,
anti-NKp44), AZ2015 (IgG1, anti-NKp30), MA15221
(IgG1, anti-NKp80), PP3519 (IgG1, anti-h2B4),
BAT22122 and ECM217 (IgG1 and IgG2b respectively,
anti-NKG2-D), QA7923 (IgG1, anti-p75-AIRM-1),
E59/12624 (IgG1, anti-IRP60), 1F1 (IgG1, anti-LAIR-1), c127
(IgG1, anti-CD16), c218 and A6/220 (IgG1 and IgM, respectively,
anti-CD56), A6-1366 (IgM, anti-HLA class I).
D1.12 (IgG2a, anti-HLA-DR) mAb was provided by Dr R. S. Accolla
(Pavia, Italy). MCA531 (IgM, anti-CD20) mAb was purchased from Serotec.
HP2.6 (IgG2a, anti-CD4) mAb was provided by Dr P. Sanchez-Madrid
(Madrid, Spain).
Flow cytofluorometric analysis
Cells were stained with the appropriate mAb followed by
phycoerythrin- or fluorescein isothiocyanate-conjugated,
isotype-specific, goat antimouse second reagent (Southern
Biotechnology, Birmingham, AL). Samples were analyzed by 1- or 2-color
cytofluorometric analysis (FACScan Becton Dickinson, Mountain View, CA)
as previously described.14,16
Cell lines, cytolytic assays, and interferon-
production
Targets used in the cytolytic assays were the following: MEL15
(MEL15392, human melanoma)9; M14 (human
melanoma)25; SMMC (human hepatocarcinoma)26;
BW1502 (murine thymoma); 721.221 (HLA class I-negative
B-lymphoblastoid cell line); LM EBV (B-lymphoblastoid cell line derived
from a healthy donor); ST-EMO EBV and ST-EFA EBV (B-lymphoblastoid cell
lines derived from the patients). The FcR-positive P815 (murine
mastocytoma) target cell line was used for redirected killing
experiments.14-16 Cells were tested for cytolytic activity
in a 4-hour chromium Cr 51 release assay in the absence or in the
presence of various mAbs. Concentrations of the various mAbs were 10 µg/mL for the masking experiments and 0.5 µg/mL for the redirected
killing experiments. E/T ratios are indicated in the text.
For the interferon (IFN)- assay, 8 × 105 cultured NK
cells derived from each patient or from 2 healthy donors were
stimulated overnight in plastic wells precoated or not with each of the
following mAbs: c218 (IgG1, anti-CD56, 3 µg/mL), BAB281
(IgG1, anti-NKp46, 3 µg/mL), or c127 (IgG1, anti-CD16 0.5 µg/mL).
Cell-free supernatants were harvested and analyzed for IFN- by an
enzyme immunoassay purchased from Biosource Europe s.a. Nivelles (Belgium).
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Results |
Cell surface expression of NK cell triggering receptors in
TAP2/ patients
Previous studies indicated that the cytolytic activity of normal
NK cells correlated with the surface density of NKp46.26 In view of these data, we analyzed whether a defective expression of
NKp46 could be responsible for the inability of TAP2/
NK cells to kill autologous cells. Thus, fresh or cultured NK cells
isolated from patients E.M.O. and E.F.A.,3,10 both
TAP2/, or from healthy donors were assessed by
cytofluorometric analysis for the surface expression of various NK cell
markers including NKp46.
Most (approximately 70%) of fresh TAP2/ NK cells
expressed high surface density of NKp46 (NKp46bright).
Thus, their surface phenotype did not significantly differ from those
of most healthy persons14 (Figure
1). Accordingly, the low levels of
cytolytic activity previously detected in fresh NK cells from these 2 patients10 did not appear to correlate with a defective
expression of NKp46. In line with the results on the surface expression
of NKp46, that of NKp3015 also did not reveal substantial
differences in comparison with healthy donors (not shown). However,
analysis of the surface expression of CD56 and CD16 revealed the
existence of high percentages of CD56++CD16
in TAP2/ NK cells (30%-40%). Moreover, most cells
expressing this phenotype were NKp46bright (Figure 1).
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| Figure 1.
Expression of NKp46 in freshly derived NK cells from
TAP2/ patients.
PBMC from the patients and from a healthy control donor were depleted
of CD3+, HLA-DR+, and adherent cells and were
analyzed by double fluorescence for the expression of NKp46, CD56, and
CD16. This analysis led to the identification of different NK cell
subsets. Histograms on the left show the percentages of the
CD56+CD16+NKp46bright cells (black
bars), CD56brightCD16NKp46bright
cells (gray bars), and
CD56+CD16+NKp46dull cells (white
bars) in the 2 patients and in the healthy donor. Cytofluorometric
profiles on the right show the overall surface expression of NKp46 in
the same donors.
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Next, polyclonal and clonal TAP2/ NK cell populations,
expanded in vitro in the presence of IL-2 for 15 days, were analyzed for the expression of various NK cell markers. Our data indicate that
more than 80% of polyclonal NK cells were represented by classical
CD56+ CD16+ NKp30 bright
NKp46bright cells (Figure 2).
This suggests that the CD56++ CD16 subset
detected in fresh NK cells may be characterized by a limited capability
to undergo proliferation in vitro. As in healthy controls, most NK
cells undergoing proliferation also expressed high levels of NKp44
(Figure 2), (expressed only on in vitro culture of NK cells).16 Polyclonal NK cell populations also expressed
normal levels of 2B4 and NKG2-D, whereas the expression of NKp80 was decreased (Figure 2) compared with that in most healthy
donors.21
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| Figure 2.
Expression of various triggering receptors in cultured
TAP2/ NK cells.
NK cells from patient E.M.O. were cultured in the presence of IL-2 for
15 days and were analyzed by cytofluorometry. (upper panels) Expression
of CD16 and NCRs (NKp46, NKp30, and NKp44). (lower panels) Expression
of 2B4, NKG2-D, and NKp80. Cells were analyzed by double fluorescence
for the expression of NKp46 in combination with CD16 or by single
fluorescence for the expression of the other indicated molecules.
Comparable results were obtained with NK cells from E.F.A.
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NK cell clones derived from the same TAP2/ patients
were further analyzed. In line with previous data indicating that
TAP2/ NK cells display poor proliferative
capacity,10 NK cells in E.M.O. and E.F.A. were
characterized by low clonal efficiency. However, sufficient numbers of
clones were obtained to allow further phenotypic and functional
analyses. In agreement with data on polyclonal cell populations, most
(approximately 80%) NK cell clones expressed the CD16+
NKp46bright surface phenotype. The remaining clones were
either CD16+ NKp46dull (approximately 17%) or
CD16 NKp46bright (3%).
TAP2/ NK cell clones were further studied for the
surface expression of various receptors and coreceptors known to be
involved in the regulation of the NK-mediated cytolytic
activity.17 Thus, in addition to NKp30 and NKp44, we also
analyzed 2B4, NKp80, and NKG2-D. All NK cell clones displayed a normal
2B4+ NKG2-D+ phenotype, whereas the levels of
surface expression of NKp30 and NKp44 correlated with that of NKp46, as
previously established in healthy donors.26 Similarly,
expression levels of NKp80 appeared to correlate, at least in part,
with those of NCR (not shown) though we could observe some clones
expressing an NCRbright phenotype that were
NKp80dull.
Analysis of the triggering capability of different receptors in
TAP2/ NK cells
We next assessed whether the various triggering receptors
expressed on TAP2/ NK cells were functionally competent
and could induce NK cell activation. To this end, polyclonal NK cell
populations or clones derived from patients or from healthy controls
were analyzed in a redirected killing assay using P815 target
cells14-16 (Figure 3A). These
experiments were performed in the absence or in the presence of mAbs
specific for various triggering receptors, including NKp46, NKp44,
NKp30, CD16, NKp80, 2B4, and NKG2-D. In the presence of mAb directed to
NCR, NKG2-D, or CD16, the cytolytic responses of TAP2/
polyclonal NK cells were similar to those elicited by normal NK cells,
whereas lower responses were elicited by anti-2B4 or anti-NKp80 mAbs.
As revealed by clonal analysis, TAP2/ NK cell clones
characterized by the NKp46bright phenotype were strongly
cytolytic in response to anti-CD16, anti-NKp44, anti-NKp30, or
anti-NKG2-D mAbs. At variance with healthy donors, some clones
expressing the NCRbright phenotype gave poor responses to
anti-2B4 or anti-NKp80 mAbs (see clone TAP3). As expected,
NCRdull clones were poorly responsive to most stimuli with
the exception of anti-CD16 and anti-NKG2-D
mAbs.19,22,26
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| Figure 3.
Cytolytic activity and IFN- production by
TAP2/ NK cells after stimulation with mAbs against
triggering receptors.
(A) Effector cells were analyzed in a redirected killing assay against
the FcR+ P815 target cells in the absence (CTR) or in
the presence of the following mAbs: BAB281 (IgG1, anti-NKp46); AZ20
(IgG1, anti-NKp30); Z231 (IgG1, anti-NKp44); PP35 (IgG1, anti-2B4);
MA152 (IgG1, anti-NKp80); c127 (IgG1, anti-CD16); BAT221 (IgG1,
anti-NKG2-D), and c218 (IgG1, anti-CD56). In this representative
experiment, the polyclonal NK cell populations derived from the 2 patients (E.M.O. and E.F.A.) and 2 healthy donors (A.M. and C.B.) and 4 clones from E.M.O. (TAP1, TAP3, TAP4, TAP5) are shown (similar results
could be obtained with E.F.A. clones). NK clones displayed the
following phenotypes: TAP1 (NCRbright 2B4+
NKp80+), TAP3 (NCRbright 2B4+
NKp80+), TAP4 (NCRdull 2B4+
NKp80+), TAP5 (NCRdull 2B4+
NKp80dull). The E:T ratio used in this experiment was 4:1.
(B) The same polyclonal NK cell populations were assessed for IFN-
production either in the absence of stimulation (CTR) or after
stimulation with each of the following mAb: c218 (IgG1, anti-CD56,
3µg/mL), BAB281 (IgG1, anti-NKp46, 3 µg/mL), c127 (IgG1, anti-CD16
0.5 µg/mL).
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We next assessed whether the cytolytic activity of
TAP2/ NK cells paralleled the ability to produce
cytokines (ie, IFN-). As shown in Figure 3B, polyclonal NK cells
from E.M.O. and E.F.A., after stimulation with anti-NKp46 or anti-CD16
mAbs, were able to produce levels of IFN- comparable to those of
healthy donors.
TAP2/ NK cells efficiently kill tumor or
EBV-transformed target cells
TAP2/ NK cells were analyzed for their spontaneous
cytotoxicity against a panel of NK-susceptible target cell
lines.9,25,26 These included the MEL14 and MEL15 melanomas
(Figure 4), the SMMC hepatocarcinoma (not
shown), the H9 T cell lymphoma, the BW1502 murine thymoma, and the HLA
class I-negative EBV-LCL 221 (Figure 4). Polyclonal NK cell
populations derived from TAP2/ patients did not display
significant differences in their ability to lyse these targets compared
with healthy controls. Similar results were obtained with
TAP2/ NK cell clones. In particular, the
NCRbright NK cell clones (see the representative TAP1 and
TAP3 clones) were highly cytolytic, whereas the NCRdull
clones (eg, TAP 4 and TAP 5 clones) displayed poor cytolytic activity
against the same targets (Figure 4). Remarkably, similar results were
also obtained when TAP2/ NK clones were analyzed
against autologous B-LCLs (Figure 4). These data clearly imply that
allogeneic tumors and autologous B-LCLs express ligands recognized by
the triggering receptors expressed by TAP2/ NK cells.
This was further confirmed by the finding that mAb-mediated masking of
the various NCR (using KL247, KS38, and AZ20 mAbs) could strongly
inhibit cytotoxicity against the above targets (not shown). Consistent
with previous results, killing of autologous B-LCLs was not modified by
the addition of anti-HLA class I mAb.10 This would mean
that the low levels of HLA class I molecules3 expressed by
TAP2/ B-LCLs, possibly associated with TAP-independent
viral peptides,27 do not affect the lysis of these targets
mediated by autologous NK cells. On the other hand, lysis of allogeneic
HLA class I+ B-LCLs (LM EBV) by TAP2/ NK
cells was increased in the presence of anti-HLA class I mAb (Figure 4).
This result is consistent with our previous observation that
TAP2/ NK cells express functional
KIR.10
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| Figure 4.
Cytolytic activity of TAP2/ NK cells
against tumor or EBV-transformed targets.
TAP2/ polyclonal NK cell populations and clones (TAP1,
TAP3, TAP4, TAP5) were assessed for cytotoxicity in comparison with 2 representative clones derived from a healthy donor: CB1
(NCRbright) and CB2 (NCRdull). (upper panels)
Effectors cells were analyzed in a cytolytic assay against the
indicated tumor target cells (E:T ratio, 4:1). (lower panels) The same
effector cells were analyzed against different EBV-transformed B-LCLs,
including the HLA class I-negative 721.221 LCL, ST-EMO LCL, ST-EFA LCL,
and LM-EBV LCL (derived from a healthy donor) (E:T ratio, 4:1). The
TAP2/ polyclonal NK cells were assessed for
cytotoxicity in the absence or in the presence of anti-HLA class I mAb
(A6/136 IgM).
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TAP2/ NK cells are unable to kill autologous
PHA blasts
Taken together, the data above suggest that fully competent and
potentially harmful NK cells do exist in TAP2/
patients. In view of these findings, we further analyzed whether such
potentially autoreactive TAP2/ NK cells were also
capable of killing normal, nonvirally infected (HLA class I negative)
autologous target cells. To this end, cultured polyclonal
TAP2/ NK cells were assessed for their ability to lyse
autologous PHA-induced T cell blasts. As shown in Figure
5A, though these target cells were highly
susceptible to cytotoxicity mediated by selected normal allogeneic NK
cells from different donors (note 2 representative clones, CB 1 and CB
2, derived from donor CB in Figure 5A), they were not killed by
TAP2/ polyclonal or clonal autologous NK cells. Thus,
contrary to EBV-infected cells (B-LCL), normal PHA blasts resulted
fully protected from autologous TAP2/ NK cells. As
shown in Figure 5B, TAP2/ PHA blasts expressed an HLA
class I surface density lower than that of TAP2/
B-EBV-transformed cells, thus suggesting that HLA class I molecules are unlikely to be important in the resistance of TAP2/
PHA blasts to autologous NK cells. Indeed, the addition of anti-HLA class I mAbs had no effect on the lysis of TAP2/ PHA
blasts mediated by either normal or TAP2/ NK cells. On
the other hand (as expected), in healthy donors (eg, donor C.B.) lysis
of PHA blasts by autologous NK cells occurred in the presence of
anti-HLA class I mAb.
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| Figure 5.
TAP2/ NK cells are unable to kill
autologous PHA blasts.
(A) Polyclonal NK cell populations derived from E.M.O. or from
E.F.A. and 2 clones from E.M.O. displaying the NCRbright
phenotype were assessed for cytolytic activity against E.M.O. PHA
blasts either in the absence (white bars) or in the presence (black
bars) of anti-HLA class I mAb. Controls were represented by 2 clones
(CB1 and CB2) derived from a healthy donor that were analyzed against
E.M.O. PHA blasts and against autologous PHA blasts (CB PHA blasts).
The E:T ratio used in this representative experiment was 10:1. (B) HLA
class I surface density on the indicated cell populations derived from
E.M.O. and from a healthy control donor. The different cell types were
stained with anti-HLA class I mAb (A6/136 IgM) and then were analyzed
by cytofluorometry. HLA class I expression levels in cells derived from
E.F.A. were comparable to those detected in E.M.O.
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As shown above, most TAP2/ NK cell clones were
characterized by reduced responses to anti-2B4 or anti-NKp80 mAbs in
redirected killing assays. The inability to kill autologous PHA blasts
could be related to the low triggering capability of these surface
molecules. This possibility, however, was ruled out by the finding that
some selected TAP2/ NK cell clones displayed normal
responses to anti-2B4 or anti-NKp80 mAbs, whereas they still failed to
lyse autologous PHA blasts (see clone TAP 1 in Figure 5). These data
suggest that PHA-induced T cell blasts (and possibly other normal
tissues) from TAP2/ patients are protected from NK
cells. This protection could reflect the expression of a still unknown
cell surface ligand that may be lost (or modified) in EBV-transformed
cells. We investigated whether such putative protective ligand was
selectively expressed by TAP2/ PHA blasts. Cultured
polyclonal NK cells derived from patient E.M.O. were analyzed for their
ability to lyse a panel of normal allogeneic PHA blasts (including CB
PHA blasts). These experiments were performed in the absence or in the
presence of anti-HLA class I mAbs (to avoid interferences by KIRs
expressed by TAP2/ NK cells) (Figure
6). Remarkably, all the allogeneic PHA
blasts analyzed were resistant to lysis by TAP2/ NK
cells, thus suggesting that the expression of the putative protective
ligand is not restricted to TAP2/ PHA blasts. The
presence of a ligand that protects PHA blasts from lysis by
TAP2/ NK cells implies that these cells express an
inhibitory receptor specific for this ligand. Is this inhibitory
receptor expressed also by NK cells from healthy controls? To answer
this question, NK cell clones derived from healthy donors were assessed
for their ability to lyse TAP2/ PHA blasts (Figure
7, left panel). Most such clones were
highly cytolytic; however, a minor subset (less than 10%) failed to
kill TAP2/ PHA blasts. Similar results were obtained
against autologous or allogeneic PHA blasts (in this case, in the
presence of anti-HLA class I mAb). It is of note that these rare NK
cell clones from healthy donors were strongly cytolytic against
TAP2/ EBV-infected target cells (Figure 7, right panel)
or against the various tumor cells analyzed above (not shown). In most
instances, these clones expressed the NCRbright phenotype.
Taken together, these data suggest that the putative inhibitory
receptor that allows TAP2/ NK cells to spare normal
autologous cells (PHA blasts) is expressed also by a minor
subpopulation of normal NK cells.
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| Figure 6.
Cytolytic activity mediated by TAP2/ NK
cells against a panel of allogeneic PHA blasts.
Polyclonal NK cells derived from E.M.O. or from a healthy donor (C.B.)
were assessed for cytolytic activity against PHA blasts derived from
different donors in the absence (white bars) or in the presence (black
bars) of anti-HLA class I mAb. The E:T ratio was 10:1.
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| Figure 7.
Rare NK clones derived from healthy donors are unable to
kill TAP2/ PHA blasts.
(left panel) Representative experiment in which a large number of NK
clones derived from a healthy donor (C.B.) were assessed for cytolytic
activity against E.M.O. PHA blasts in the presence of anti-HLA class I
mAb (each square represents the cytotoxicity of an individual clone).
(right panel) NK clones displaying no cytolytic activity against E.M.O.
PHA blasts were also unable to kill autologous or heterologous PHA
blasts but killed B-EBV target cells. In this experiment, a
representative clone (CB 520) was assessed for cytotoxicity against the
indicated target in the absence (white bars) or in the presence (black
bars) of anti-HLA class I mAb. The E:T ratio was 10:1 (PHA blasts) or
4:1 (B-EBV targets).
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Discussion |
In this study we provide evidence that the NK cells isolated from
TAP2/ patients do not present abnormalities in the
level of expression and function of various triggering receptors
involved in natural cytotoxicity. The finding that
TAP2/ NK cells can efficiently lyse tumor cells or
B-LCL while sparing normal PHA blasts is likely to reflect the
expression of a protective ligand on these normal cells. Moreover,
functional data indicate that this ligand interacts with a putative
inhibitory receptor expressed by all TAP2/ NK cells but
only by a minor fraction (less than 10%) of NK cells from healthy donors.
A mechanism by which NK cells from TAP2/ patients could
spare autologous cells is the lack or the down-regulation of triggering receptors responsible for NK cell activation in the lysis of HLA class
I-negative target cells.17 Although in healthy persons the loss of HLA class I occurs in cells undergoing tumor transformation or viral infection,28 in TAP2/ patients
the lack of expression of HLA class I is a feature common to all
tissues. Thus, in TAP2/ patients, to avoid the
NK-mediated attack to self-cells, the NK cell receptor repertoire could
be shaped toward the selection of NK cells with impaired cytolytic
activity. To analyze this possibility we first performed phenotypic
analysis of NK cells freshly isolated from 2 TAP2/
patients.3,10 These studies revealed a significantly
higher proportion of CD56++CD16 NK cells in
TAP2/ patients (30%-40%) than in healthy controls
(less than 10%). NK cells characterized by this phenotype have been
reported to be poorly cytolytic and are thought to represent relatively
immature NK cells.29-31 In this context, the tolerance of
resting TAP2/ NK cells toward autologous cells could
be, at least in part, the result of a block of NK cell maturation.
However, this cannot fully explain why fresh NK cells are poorly
cytolytic against HLA class I-negative targets (such as K562) or
autologous (TAP2/) B-LCL.10 An additional
explanation could be the absence or low level of expression of
triggering NK receptors. As established by previous
studies,26 in freshly isolated NK cells a strict correlation exists between the level of surface expression of NKp46
(bright vs dull) and the magnitude of NK-mediated cytotoxicity against
HLA class I-negative target cells such as K562. Thus, we analyzed the
NKp46 phenotype of resting TAP2/ peripheral NK cells.
NKp46 was expressed at high density in most NK cells. Thus, it appears
that, in TAP2/ patients, no direct correlation exists
between low levels of cytotoxicity of fresh NK cells and their NKp46
phenotype. Because approximately 40% of the NKp46bright NK
cells from TAP2/ donors expressed the
CD56++CD16 phenotype, it is conceivable that
the low cytolytic activity of resting TAP2/ NK cells
may be explained on the basis of their stage of maturation.
It has been suggested that autoreactive phenomena described in some
TAP/ patients with chronic inflammation are mediated by
activated NK cells.11 Thus, we analyzed in more detail the
expression and the function of various triggering NK receptors in
activated NK cells derived from TAP2/ patients.
Polyclonal NK cell populations and NK cell clones cultured in the
presence of IL-2 have been analyzed. These studies revealed normal
levels of expression and function of the natural cytotoxicity receptors
(including NKp46, NKp30, and NKp44)13 and of
NKG2-D.22,32 On the other hand, in some instances a
defective functional response to stimuli acting through
2B419,33-36 and NKp8021 could be observed. Remarkably, most of the cultured TAP2/ NK cells or
clones expressed CD16. This implies that the "immature" CD56++CD16 subset in fresh NK cells underwent
poor proliferation in vitro (or underwent maturation?). The cytolytic
activity of TAP2/ NK cell clones against NK-susceptible
tumor target cells was comparable to that of normal NK clones. This
would mean that triggering receptors expressed by cultured
TAP2/ NCRbright NK cells can induce NK cell
activation on interaction with tumors or virally infected cells (eg,
autologous B-LCLs). Although not shown, TAP2/ NK cells
could kill autologous fibroblasts, thus confirming our previous
report.12 This capability was restricted to
NKp46bright NK cells, and lysis was inhibited by
mAb-mediated masking of NKp46. These data raise the question of whether
the NKp46-dependent killing of autologous fibroblasts may be involved
in the pathogenesis of the autoimmune manifestations in some HLA class
I-deficient patients. In this context, it will be interesting to test
whether the development of vasculitis or granuloma in TAP-deficient
patients correlates with higher proportions of NKp46bright cells.
The potential autoreactivity of TAP2/ NK cells was
further evaluated against a different source of normal autologous cells represented by PHA-induced T cell blasts. Significantly,
TAP2/ PHA blasts were resistant to autologous NK cells,
yet they were highly susceptible to lysis by NK cells from healthy
donors. This strongly suggests that TAP2/ PHA blasts
express ligands recognized by triggering NK receptors. In agreement
with this concept, mAb-mediated masking of NCRs prevented the killing
of TAP2/ PHA blasts by normal NK cells (as shown by
previous studies, NCRs are required for the NK-mediated lysis of PHA
blasts).15 Thus, our current data on TAP2/
NK cells are clearly reminiscent of previous data obtained in  2-microglobulin-deficient or TAP-1 knockout mice.37,38
These studies showed that 2-m NK cells, while sparing
autologous concanavalin A blasts and bone marrow cells, could lyse
allogeneic major histocompatibility complex class I
tumors. In view of our current finding that TAP2/ NK
cells express functional NCR, it is unlikely that they fail to kill PHA
blasts because of the lack of still undefined triggering receptor(s).
We favor the hypothesis that the observed phenomenon may reflect the
occurrence of an interaction between a still undefined inhibitory
receptor expressed on TAP2/ NK cells and its ligand on
PHA blasts. This explanation is also supported by a series of
additional data: (1) lysis of PHA blasts could not be restored by the
addition of anti-HLA class I mAbs (Figures 5, 6) or by the mAb-mediated
masking of one or another HLA class I-specific inhibitory receptor
(data not shown); (2) TAP2/ NK cells failed to lyse
autologous and allogeneic PHA blasts, thus suggesting that the putative
protective ligand(s) is not confined to TAP2/ cells;
(3) NK cells that fail to kill PHA blasts are present also in healthy
donors, but they are scarcely represented within the total NK cell
pool; (4) in healthy persons, these rare NK cells display an
NCRbright phenotype, indicating that the lack of cytolytic
activity against PHA blasts is not the result of a general inability to
lyse but rather of the occurrence of an inhibitory interaction (this is also supported by the finding that NK cells displayed a strong cytolytic activity against various NK-susceptible tumor target cells).
Taken together, our data indicate that to avoid attacks on normal
cells, shaping of the NK cell receptor repertoire in
TAP2/ patients is not based on the down-regulation of
triggering receptors. Rather, it appears to be skewed toward the
selection of NK cells expressing a still undefined inhibitory receptor.
Remarkably, in healthy persons, this putative receptor is expressed on
a minor fraction of NK cells. The restricted expression of this
inhibitory receptor rules out the possible involvement of previously
identified, non-HLA-specific inhibitory receptors including
LAIR-1,39,40 p75/AIRM1,23 and
IRp60,24 because they are expressed by virtually all
normal NK cells. That they are not involved in this phenomenon was also
directly demonstrated by experiments in which mAb-mediated masking of
these receptors did not allow TAP2/ NK cells to lyse
autologous PHA blasts (not shown).
Regarding the nature of the putative ligand(s) for the inhibitory
receptor, it does not seem to be related to classical HLA class I
molecules. Indeed, it is expressed on TAP2/ cells, and
lysis of target cells could not be reversed by the addition of
conventional anti-class I mAbs. Notably, its expression seems to be
altered in B-LCLs (because these cells are lysed by TAP2/ NK cells). This may suggest down-regulation of
the ligand as a consequence of viral infection, or it may suggest that
its expression is constitutively different in T cells and B cells.
Future studies should focus on the identification and molecular
characterization of receptor(s) and ligand(s) involved in this novel
inhibitory interaction.
| |
Acknowledgments |
We thank Tiziana Baffi for secretarial assistance.
| |
Footnotes |
Submitted June 14, 2001; accepted October 23, 2001.
Supported by grants from Fondazione Italiana per la Ricerca sul Cancro,
Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di
Sanità, Ministero della Sanità, and Ministero
dell'Università e della Ricerca Scientifica e Tecnologica,
Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie,
MURST CNR 5% CNR Biotechnology program 95/95, Telethon-Italy (grant
E.0892), Etablissement Francaise du Sang-Alsace, and Fondation Touraine.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: Alessandro Moretta, Dipartimento di Medicina
Sperimentale, Sezione di Istologia, Via G.B. Marsano 10, 16132 Genova,
Italy; e-mail: alemoret{at}unige.it.
| |
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Abstract
Introduction