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Prepublished online as a Blood First Edition Paper on July 18, 2002; DOI 10.1182/blood-2002-04-1284.
IMMUNOBIOLOGY
From the Laboratory of Immunology, National Institute
for Cancer Research (IST), Genoa; DIMI, University of Genoa; and
Laboratory of Tumor Immunology, Istituto Scientifico San Raffaele,
Milan, Italy.
The engagement of the activating isoforms of C-type lectin
inhibitory receptor (CLIR) or killer Ig-like receptor (KIR) by their
natural ligands, represented by soluble HLA-I (sHLA-I) molecules, induced programmed cell death of natural killer (NK) cells. Indeed, NK
cell apoptosis elicited by either putative HLA-E and HLA-F (sHLA-I
non-A, -B, -C, and -G) or sHLA-I-Cw4 or -Cw3 from untransfected or
-Cw4 or -Cw3 alleles transfected HLA-A It is generally accepted that the major
histocompatibility complex class I-specific inhibitory receptors on
natural killer (NK) cells prevent the lysis of healthy autologous
cells.1-3 This inhibitory receptor superfamily (IRS) can
be subdivided into 2 structural types of molecules. The first one
consists of immunoglobulin (Ig) superfamily receptors (ISIRs), the
other one of C-type lectin inhibitory receptors (CLIRs). IRS engagement
with the appropriate HLA-class I (HLA-I) allele delivers an inhibitory
signal that down-regulates several NK and T-cell functions such
as proinflammatory cytokine production, proliferation, and cytolytic
activity.1-3 Recently, we have demonstrated that IRS
members can function as survival receptors in NK cells. Indeed, the
apoptosis of NK cells induced by soluble HLA-I (sHLA-I), via the
engagement of CD8, is strongly down-regulated by ligation of sHLA-I
with IRS.4
Some members of the IRS include receptors with activating rather than
inhibitory function.1-3 These activating receptors are
represented by the killer cell Ig-like receptor (KIR) family with 2 Ig
domains (KIR2D) with a short cytoplasmic tail (KIR2DS or p50) or by
CLIR isoforms, such as CD94/NKG2C complex, which lack the
immunoreceptor tyrosine-based inhibition motif (ITIM).1-3 KIR2DS and NKG2C associate with a short disulfide-linked homodimer of
the protein called DAP12 that carries a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM).1-3 It has been
shown that these receptors Monoclonal antibodies and reagents
Indirect immunofluorescence
Isolation and culture of polyclonal and clonal NK cell populations Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were isolated by Ficoll-Hypaque gradient. CD3 CD4 cells were isolated after negative
immunodepletion as described.26 The resulting cell
population was 50% to 70% CD16+ (range of 8 different
experiments) but 99% CD3CD4. Highly
purified CD3CD4 cells were stimulated with
10 µg/mL phytohemagglutinin (PHA) and cultured in 96-well
U-bottomed microplates (Becton Dickinson) with complete medium in the
presence of 100 U/mL recombinant IL-2 (rIL-2) in a final volume of 200 µL per well in the presence of 105-per-well irradiated
allogeneic PBMCs and 5 × 103-per-well 721.221 lymphoblastoid cell line transfected with
HLA-G.26 CD3CD16+
clones were obtained by culturing highly purified
CD3CD4 NK cells under limiting dilution
conditions as previously reported.13,26 Cloning efficiency
was of 5% to 10% calculated as described.27 All
NK cell clones were analyzed for the expression of CD158a (p50.1),
CD158b (p50.2), p50.3 (CD158a and CD158b
but NKVFS1+), CD94/NKG2A (mAb Z199, Serotec), CD16, and
CD56. Each clone was analyzed in a killing assay using the
Fc R+ P815 murine mastocytoma cell line in the presence
of mAb recognizing either KIRs or C-type lectin inhibitory receptors at
the effector-target ratio (E/T) of 20:1 or 2:1 to identify clones with
inhibiting or activating forms of these HLA-I receptors,
respectively.1-3,10-13,22
Soluble HLA-I antigen preparations Soluble HLA-A2, -Cw3, and -Cw4 were prepared from culture supernatant of HLA-I-A, -B,
-C, and -G 721.221 cells transfected with
the corresponding HLA-I alleles 6,28,29 while soluble HLA
non-A, -B, -C, and -G was prepared from culture supernatant of
untransfected 721.221 cells by precipitation with ammonium sulfate,
low-medium pressure chromatography, strong anionic and strong cationic
ion exchange, and gel filtration as described 28,29 and
purified by affinity chromatography on anti-HLA class I mAb W6/32 (10 mg/mL) coupled to cyanogen bromide-activated Sepharose 4B
(Pharmacia). The purity of sHLA-I molecule preparations was
analyzed by 1-dimensional polyacrylamide gel electrophoresis (PAGE) under nonreducing/nondenaturing (Figure 2A) or
reducing/denaturing conditions (not shown) followed by silver
staining.28,29 721.221 untransfected cells appeared to be
faintly stained with W6/32 mAb followed by PE-conjugated GAM antiserum;
thus, in accordance with previous reports,30-36 it is
conceivable that the sHLA-I derived from these cells was represented,
at least in part, by HLA-E and HLA-F molecules.37
Determination of sFasL and IFN- present in supernatant from NK cell clones
incubated for 48 hours with medium alone, or under different
culture conditions as indicated in "Results," was evaluated by
ELISA (Bender MedSystems Diagnostics, Vienna).
Induction and detection of apoptosis Bulk NK cell populations or NK cell clones (105 per milliliter) were cultured in 24-well flat-bottomed plates with culture medium either alone or with different amounts of sHLA-I molecules (0.5 to 4 µg/mL) for different periods (6, 12, 24, 36, 48, 60, 72 hours) at 37°C in a 5% CO2 atmosphere. In some experiments cells were incubated with anti-CD158a or anti-CD158b or anti-CD94 or anti-CD54 mAb for 30 minutes at 4°C, washed, and either used in apoptotic assays (to analyze the effect of the covering of a given activating receptor) or further incubated for different times with 4-per-cell GAM-coated magnetic beads 6,28,29 (to obtain the optimal cross-linking of a given activating receptor). Early apoptotic events were evaluated by annexin V labeling method, and viable apoptotic cells were differentiated from necrotic cells by flow cytometry after PI staining of nonpermeabilized cells.6 Apoptotic cells were identified as annexin V+ PI cells6,28,29; 104 cells per
sample were analyzed and results plotted as the percentage of annexin
V+ cells and PI cells. Apoptosis was also
detected by PI staining after permeabilization (DNA content < 2n) and
by DNA laddering after DNA extraction and agarose gel
electrophoresis.6,28,29
Isolation of RNA, reverse transcription, and PCR amplification Total RNA was isolated from cell pellets by using the RNAzol B (Biotecx Laboratories, Houston, TX) method.28 Complementary DNA (corresponding to 2 µg RNA) was synthesized from oligo(dT)-primed RNA as described.28 The polymerase chain reaction (PCR) mixture was amplified using the following primer sequences: -actin 5'-ACTCCATCATGAAGTGTGACG, -actin
3'-CCTAGTCGTTCGTCCTCATAC (228-bp fragment); FasL
5'-CAAGTCCAACTCAAGGTCCATGCC, FasL 3'-CAGAGAGAGCTCAGATACGTTGAC (350-bp
fragment).6,28 PCR products were size-fractionated by
agarose gel electrophoresis and normalized according to the amount of
-actin detected in the same mRNA sample.
Cytolytic assays Cytolytic activity of CD3CD16+ NK cell
clones was tested in a 4-hour 51Cr-release assay as
previously described.13,22 Polyclonal or clonal
CD3CD16+ NK cell populations, selected for
the expression of activating forms of either KIR and/or CLIR, were used
as effector cells with the FcR+ murine mastocytoma cell
line P815 in the presence of mAb directed against activating receptor
for HLA, at an effector-target ratio (E/T) of 2:1, in a final volume of
200 µL RPMI 1640 medium in V-bottomed microwells.13,22
NK cell-mediated cytolysis was also analyzed against a panel of target
cells including K562 (erythroleukemia), Jurkat (T-cell lymphoma), and
721.221 lymphoblastoid cell line. The effect of cyclosporin A (5, 50, 500 ng/mL) or concanamycin A (0.3-3 µM) on NK cell-mediated
cytolytic activity was analyzed after pretreatment of NK cells for 15 minutes. The optimal concentration for each compound is indicated in
"Results." Cell viability was analyzed after incubation with the
different drugs or the appropriate dilution buffer and was always more
than 98%.
Soluble HLA-I induces NK cell apoptosis upon engagement of the activating isoforms of KIR or C-type lectin inhibitory receptor Besides inhibiting receptors for HLA-I, NK cells express at the cell surface the activating isoforms of these receptors.1-3 It appears that these activating receptors recognize the same HLA-I allele, which can interact with their inhibiting counterparts.1-3,7-13,30 NK cells bearing activating receptor can kill cells expressing the appropriate HLA-I allele,1-3,7-13,30 because their engagement delivers an activating signal that ultimately leads to NK cell degranulation of perforins and granzymes.1-3 Thus, these findings would suggest that the activating receptor for HLA-I is potentially harmful and, thus, control mechanisms that down-regulate activating receptor-mediated function should exist. It is conceivable that apoptosis can represent a useful tool to switch off effector cell-mediated activities.4 Thus, to define these mechanism(s), we first analyzed whether the engagement of activating receptor by its natural ligand, HLA-I, can induce NK cell apoptosis. To this aim, to demonstrate that HLA-I interacting with activating receptor can directly deliver an apoptotic signal, we incubated NK cell clones with the soluble form of HLA-I derived from the lymphoblastoid 721.221 HLA-I cell line either untransfected or stably
transfected with appropriate HLA-I allele35,36 (Figure
1A).
To select NK cells bearing activating receptor, NK cell clones were
first chosen on the basis of the homogeneous expression of receptors
for HLA-I. Secondly, each clone was tested in a killing assay against
the Fc Among the clones analyzed, about 10% expressed activating receptor
isoforms of either KIR or CLIR (35 of 350 tested) (Table 1). In addition, as we have demonstrated
that CD8 ligation with sHLA-I can induce NK cell apoptosis by
interacting with the
To determine whether the activating receptor may deliver an apoptotic
signal in NK cells, only NK cell clones with a dull expression of CD8
were further analyzed. As shown in Figure
2B, sHLA-I-Cw4 allele, the natural
ligand of CD158a, induced apoptosis of the clone 262, which expressed
the activating receptor form of CD158a. Preincubation of NK cells with
anti-CD158a mAb strongly reduced this apoptosis, indicating that the
interaction of sHLA-I-Cw4 with CD158a was necessary to induce cell
death. The finding that the optimal cross-linking of CD158a, achieved
by using anti-CD158a mAb followed by GAM-coated beads, led to NK cell
apoptosis further indicates that the engagement of activating receptor
may induce NK cell death. Noteworthy, the incubation of the NK cell
clone 262 with the sHLA-I-A2 allele, an HLA-I allele that does not
bind CD158a, did not induce apoptosis (Figure 2B), suggesting that the
apoptotic signal can be delivered only when the activating receptor is
engaged by its specific sHLA-I allele. Similar results were obtained
using the NK cell clone A1.25 bearing the activating receptor form of
CD158b and sHLA-I-Cw3 as specific ligand (Figure 2C). Because we have
previously demonstrated that the engagement of CD8 with sHLA-I at the
NK cell surface led to NK cell apoptosis, we further analyzed whether
apoptosis induced by ligation of activating receptor was independent on
CD8-mediated signaling. As shown in Figure 2D, we found that the
covering of CD8 with specific mAb did not affect activating
receptor-induced apoptosis.
Analogous results were obtained using NK cells bearing the activating
receptor forms of CLIR represented by CD94/NKG2 complex (Figure 2E).
However, in this case, we have used as specific sHLA-I the sHLA-I
isolated from HLA-A As depicted in Figure 3, that activating
receptor actually induced apoptosis in NK cells after interaction with
sHLA-I-specific allele was further supported by DNA analysis with PI
(Figure 3B) and DNA laddering (Figure 3C), besides staining of NK cells
with FITC-annexin V (Figure 3A). Indeed, the number of NK cells with a
DNA content less than 2n (apoptotic cells) after covering of activating
receptor form of CLIR (Figure 3Biii) with anti-CLIR mAb was strongly
reduced compared with that found upon incubation of NK cells with sHLA
non-A, -B, -C, and -G (Figure 3Bii; 85% vs 12%). Comparable results
were obtained by DNA laddering (Figure 3C, compare lanes 2 and 3). The
percentage of apoptotic cells was maximal at 4 µg/mL although it was
detectable at 1 µg/mL (Figure 3D). Finally, kinetics of apoptosis
revealed that the optimal incubation time of NK cells with the specific
sHLA-I allele was 48 hours (Figure 3E).
NK cell apoptosis induced by activating receptor of KIR or CLIR is mediated by FasL-Fas interaction It has been shown that FasL-Fas interaction is responsible for inducing apoptosis through the ligation of CD8 by sHLA-I both in T and NK cells.6,15,16,29 Thus, to define which surface receptor is involved in delivering an apoptotic signal upon engagement of activating receptor isoforms of KIR or CLIR, we incubated NK cells with sHLA-I alleles in the presence of either anti-Fas or anti-FasL mAb alone or in combination. As shown in Figure 4A, covering of Fas antigen at the NK cell surface and/or the incubation with anti-FasL mAb strongly reduced the activating receptor-mediated NK cell apoptosis. That FasL was implicated in this phenomenon was further supported by the finding that FasL was present in culture supernatants of NK cells incubated with the appropriate sHLA-I allele (Figure 4B-D). In addition, covering of CD8 with specific mAb did not reduce the amount of sFasL recovered from culture supernatant induced by incubation of the CD8dull NK cell clone A1.25 bearing activating receptor isoform of CD158b with its specific ligand sHLA-Cw3 (Figure 4C). This finding suggests that CD8 is not directly involved in activating receptor-mediated NK cell apoptosis. Furthermore, as shown in Figure 4E, the engagement of the activating receptor CD158a with sHLA-Cw4 (or upon cross-linking with anti-CD158a mAb plus GAM-coated beads) induced a strong increase of mRNA coding for FasL that was abolished by the covering of CD158a with specific mAb. Similar results were obtained by incubating NK cells bearing activating receptor of CD94 with sHLA non-A, -B, -C, and -G ligand (not shown). Altogether, these findings suggest that the engagement of activating receptor by sHLA-I-specific alleles delivers an apoptotic signal through the production of FasL that in turn is secreted in the extracellular milieu and eventually interacts with Fas at the NK cell surface, leading to NK cell apoptosis.
NK cell-mediated apoptosis via activating receptor isoforms of KIR or CLIR is CsA dependent Apoptosis induced by the activating isoforms of KIR and/or CLIR can represent a mechanism that limits cell tissue damage due to activation of NK cells against autologous cells. On the other hand, activating isoforms of KIR and/or CLIR can recognize at the cell surface of autologous tumor cells the corresponding HLA-I allele and, thus, this recognition can trigger target cell lysis. Thus, we analyzed whether the activating receptor-mediated NK cell apoptosis was sensitive to blocking drugs, different from activating receptor-mediated target cell lysis. In this context, it has been shown that NK cells lyse tumor target cells by means of secretion of perforins and granzymes present in intracellular discrete granules and that this process is sensitive to the specific inhibitor of vacuolar type H+-ATPase concanamycin (CMA).24,25 On the other hand, the FasL induction and its consequent secretion, in human NK cells, is strictly dependent on the activation of calcineurin and nuclear factors of activated T cell (NFAT), which in turn are highly cyclosporin A sensitive.23 Thus, we analyzed whether apoptosis and/or cytolysis induced through activating receptor were alternatively sensitive to CsA or CMA.NK cell treatment with CsA, in a dose-dependent fashion, led to a
strong reduction of sHLA-I-mediated apoptosis through ligation of
activating receptor (Figure 5A). Indeed,
the apoptosis induced by ligation of p50.3 on the NK cell clone 12, with either sHLA-Cw3 or with anti-p50 mAb followed by optimal
cross-linking with GAM-coated beads, was reduced by 99% and 92%,
respectively, in the presence of 500 ng/mL CsA (Figure 5A).
Importantly, CsA treatment of NK cells was accompanied by a strong
reduction of the amount of sFasL present in culture supernatant after
ligation of p50.3 with sHLACw3 or with anti-p50 mAb followed by optimal
cross-linking with GAM-coated beads (Figure 5B). Although not shown,
similar results were obtained with NK cell clones bearing the
activating receptor CD158a or CD158b or CD94. By contrast, CMA only
marginally influenced NK cell apoptosis induced by p50.3 (Figure 5C) or
via activating receptor isoforms of CD158a or CD158b or CD94 (not
shown).
As shown in Figure 6A-B, CMA strongly
reduced (by 50%), in a killing assay, lysis of P815 target cells
induced by the engagement of activating isoforms of KIR or CLIR,
whereas CsA had no effect. Furthermore, CsA did not inhibit NK
cell-mediated lysis of different tumor target cells including K562
(Figure 6C), 721.221 (Figure 6D) and Jurkat (Figure 6E) cell lines,
whereas CMA reduced lysis of these target cells by 50% to 80% (Figure
5C-E). Altogether, these findings indicate that apoptosis induced via
the engagement of activating receptor is selectively CsA sensitive but
this drug does not affect the activating receptor-mediated activation
of NK cell cytolysis.
Soluble HLA-I induces IFN- by ELISA. As shown in Figure
7A, a detectable amount of IFN- was
found in culture supernatant of the representative CD158a+
NK cell clone 262 incubated with sHLA-Cw4. Covering of CD158a with a
specific mAb almost completely inhibited sHLA-Cw4-induced IFN-
production (Figure 7A), indicating that ligation of CD158a with
corresponding sHLA-I allele was necessary to deliver the activating
signal leading to IFN- production. As clone 262 expressed CD8 at low
levels, we further analyzed whether IFN- found in culture
supernatant was due to the engagement of CD8 by sHLA-Cw4. As shown in
Figure 7A, anti-CD8 mAb had no effect on sHLA-Cw4-induced IFN-
production, indicating that CD8 was not involved in activating receptor-mediated IFN- production.
The amount of IFN-
Activating forms of KIR or CLIR can deliver an apoptotic signal in
human NK cells after their engagement with the appropriate ligand
represented by sHLA. This interaction leads to production and secretion
of FasL, which in turn induces apoptosis upon ligation with Fas at the
NK cell surface. This phenomenon is accompanied by the production of
the proinflammatory cytokine IFN- The physiological significance of the presence of activating receptor at the NK cell surface specific for discrete HLA-I allele is still debated.1-3 Conceivably, these activating receptors react with the same HLA-I allele reacting with their inhibiting counterpart, but their engagement can induce the activation of NK cell-mediated functional activities such as cytolysis and cytokine production.1-3 Several authors have claimed that, to avoid autologous tissue cell damage induced by activating receptor-triggered cytolysis, the inhibiting receptors for the HLA-I can down-regulate this effect because the inhibitory signal via KIR and/or CLIR should overcome the triggering signal via their activating counterpart.1-3 If this is the case, because the HLA-I NK cell receptors seem to recognize specific HLA-I allele,1-3 NK cells should bear at the same time inhibiting and activating receptors specific for the same HLA-I allele; otherwise, the interaction of different HLA-I alleles with different counter-receptors not necessarily should take place in close proximity at the NK cell surface in order to shut down cytolysis initiated upon the engagement of activating receptor. In fact, it has been stated that inhibiting signal can switch off the activating one only if these occur closely in NK cells.1-3 Among the NK cell clones analyzed in this and previous studies8-13 we found that it is not exceptional to find NK cell clones that express an activating receptor for one HLA-I allele and inhibiting receptors for other HLA-I alleles. Indeed, we found NK cell clones with activating receptor form of KIR but CLIR+, and vice versa, without any detectable expression (both in immunofluorescence and functional assays) of known inhibiting form of KIR (not shown). In addition, although to a very low frequency (in our culture conditions) some NK cell clones with only activating forms of KIR and CLIR can be found. In these instances, it would be very hard to block NK cell-mediated cytolysis of HLA-I+ autologous cells bearing the appropriate counter-receptor. Our present results can explain, at the same time, because NK cell clones with activating receptor do not usually exist at higher frequencies and because they do not kill all autologous cells. Indeed, a certain amount ranging from 0.5 to 2 µg/mL sHLA-I (composed of a mixture of HLA-A, -B, and -C alleles) is present in serum of healthy donors,17-19,27,41 this amount would be enough to induce apoptosis of activated NK cells with activating receptor without any NK cell interaction with autologous cells. In addition, during the interaction with self-HLA-I+ tissue cells, activating receptor on NK cells may deliver the apoptotic signal, thus leading to autolimitation of any self-reaction. If this were true, what is the physiological significance of activating receptor on NK cells? One can hypothesize that activating receptor plays a role in the elimination of tumor cells. Indeed, this study and others show that when tumor cells express the appropriate self-HLA-I antigens, it is possible that the engagement of activating receptor for HLA-I can efficiently trigger cytolysis.8-13 Thus, NK cells kill self-tumor target cells by means such as perforins and granzymes, and the consequent up-regulation of production and secretion of FasL leading to NK cell apoptosis can switch off innate immune response. Recently, several receptors responsible for NK cell-mediated cytolysis
have been identified.4,5,42 These receptors are represented by natural cytotoxicity receptors (NCR), including NKp30,
NKp44, and NKp46 molecules,4 which are specifically expressed only by NK cells, and NKG2D5,42 that is
present on NK cells and on cytolytic lymphocytes bearing Notably, we found that FasL-induced apoptosis of NK cells is strongly reduced by the immunosuppressive drug CsA. Indeed, CsA can block FasL expression induced via T-cell antigen receptor in cytolytic CD8+ T-cell clones.45 Thus, CsA treatment of NK cell clones with activating receptor may be a tool to maintain killing of autologous tumor target cells without the elimination of these really potent cytolytic effector cells. In fact, CsA did not inhibit activating receptor-mediated cytolysis as well as triggering of NK cells via CD16 (this study) or other activating NK cell receptors as CD69 (not shown), suggesting that CsA did not affect NK cell-mediated killing. However, it has been reported that cytolytic effector cells can lyse tumor target cells by 2 independent mechanisms: the first is represented by degranulation of perforin/granzyme, whereas the second one is FasL mediated. In fact, several tumor target cells express at the cell surface Fas antigen and can die upon Fas engagement by FasL.25,46-4 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Top
Abstract
Introduction
besides other triggering molecules
expressed on NK cells including CD16, CD2, CD69, NKp30, NKp44, NKp46,
and NKG2D
(astra102, IgG1) mAb, the NKVFS1 mAb, recognizing a common epitope of CD158a and
CD158b and p50.3, and the anti-CD69 mAb (31C4, IgG2a) were produced as
described.
) or the NK cell clone 10 (activating
receptor+ CD94+) (
) was incubated with
either sHLA-Cw4 or sHLA non-A, -B, -C, and -G (that is, putative HLA-E)
for the indicated periods of time, and apoptosis was evaluated by
staining cells with FITC-annexin V. 
indicates apoptosis of the NK
cell clone 255 incubated with medium alone. Results are expressed as
the percentage of apoptotic cells that are annexin V+ but
PI
indicates percentage of maximal apoptosis of the NK cell clone 45 incubated for 48 hours in medium alone. (B) Amount of sFasL present in
culture supernatant recovered after 24 hours of incubation under the
experimental conditions described in panel A with the same NK cell
clones. Results are expressed as nanograms per milliliter as determined
by ELISA. (C) Concanamycin A does not affect activating
receptor-mediated apoptosis. The NK cell clone 12 (p50.3+
activating) was incubated with sHLA-Cw3 or GAM-coated beads (4 per cell) after staining with anti-p50 mAb (1 µg/mL) (p50.3-XL) for
48 hours in the presence or absence of 3 µg/mL concanamycin A or with
500 ng/mL CsA and stained with FITC-annexin V. "nil" indicates
apoptosis in medium alone. Results are expressed as the percentage of
apoptotic cells (FITC-annexin V+ but PI
) or in the
presence of either 3 µg/mL CMA (
) for 24 hours alone or in the presence of increasing
doses of CsA (5, 50, 500 ng/mL). 
T-cell receptor (TCR).