|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2396-2402
Modulation of T-Cell Functions in KIR2DL3 (CD158b) Transgenic Mice
By
Anna Cambiaggi,
Sylvie Darche,
Sophie Guia,
Philippe Kourilsky,
Jean-Pierre Abastado, and
Eric Vivier
From the Unité de Biologie Moléculaire du Gène,
Institut Pasteur, Paris, France; the Centre d'Immunologie INSERM/CNRS
de Marseille-Luminy, Marseille, France; and the Institut Universitaire
de France, France.
 |
ABSTRACT |
In humans, a minor subset of T cells express killer cell Ig-like
receptors (KIRs) at their surface. In vitro data obtained with
KIR+   and   T-cell clones showed that
engagement of KIR molecules can extinguish T-cell activation signals
induced via the CD3/T-cell receptor (TCR) complex. We analyzed the
T-cell compartment in mice transgenic for KIR2DL3
(Tg-KIR2DL3), an inhibitory receptor for HLA-Cw3. As
expected, mixed lymphocyte reaction and anti-CD3 monoclonal antibody
(MoAb)-redirected cytotoxicity exerted by freshly isolated splenocytes
can be inhibited by engagement of transgenic KIR2DL3 molecules. In
contrast, antigen and anti-CD3 MoAb-induced cytotoxicity exerted by
alloreactive cytotoxic T lymphocytes cannot be inhibited
by KIR2DL3 engagement. In double transgenic mice, Tg-KIR2DL3 × Tg-HLA-Cw3, no alteration of thymic differentiation could be
documented. Immunization of double transgenic mice with Hen egg white
lysozime (HEL) or Pigeon Cytochrome-C (PCC) was indistinguishable from
immunization of control mice, as judged by recall antigen-induced in
vitro proliferation and TCR repertoire analysis. These results indicate
that KIR effect on T cells varies upon cell activation stage and show
unexpected complexity in the biological function of KIRs in vivo.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
MOLECULES ENCODED by the class I major
histocompatibility complex (MHC) are recognized by 3 distinct groups of
cell surface immunoreceptors: the CD3/T-cell receptor (TCR) complex, CD8 dimers, and natural killer cell receptors (NKRs).1,2 NKRs belong to 2 families of molecules: human killer cell Ig-like receptors (KIRs) and leukocyte Ig-like receptors (LIRs; also called Ig-like transcripts [ILTs]) are Ig-like receptors, whereas CD94-NKG2 heterodimers and mouse Ly-49 homodimers are C-type
lectins.3-7 In contrast to the exquisite recognition of
antigen-MHC complex by TCR polypeptides, the recognition of MHC class I
molecules by KIRs, LIRs, and Ly-49 receptors is degenerated, because
these NKRs interact with the products of multiple MHC class I alleles. Inhibitory and activating NKRs isoforms have been
described.8,9 Inhibitory NKRs harbor intracytoplasmic
immunoreceptor tyrosine-based inhibition motifs (ITIMs) that recruit
and activate the protein tyrosine phosphatases SHP-1 and/or
SHP-2.10,11 Activating NKRs lack ITIMs and associate with
polypeptides containing immunoreceptor tyrosine-based activation motifs
(ITAMs), such as DAP-12/KARAP or FcR, which recruit and activate the
protein tyrosine kinases ZAP-70 and/or p72 Syk.9,12-16
Although the biological function of activating NKRs remains obscure, it
is proposed that inhibitory NKRs confer to NK cells a molecular sensor
device that allows the selective lysis of autologous cells with altered
MHC class I expression.1,2 Such quantitative and/or
qualitative defects in MHC class I surface expression are common
alterations that occur upon tumor transformation and viral
infection.17,18
A minor subset of T cells, in most instances CD8+ T cells,
express KIRs.19 KIR+ T lymphocytes display a
cell surface phenotype typical of memory T cells, and analysis of the
TCR V repertoire showed the oligoclonal or monoclonal nature of
these populations, suggesting that KIRs are expressed de novo by T
cells as a consequence of chronic antigen-driven activation in
vivo.20-31 Consistent with these observations is the fact
that KIR+ T cells are present in adult peripheral lymphoid
tissue and blood, but absent in thymus or cord blood.21
Analysis of KIR+ T-cell clones has shown that KIR
engagement inhibits CD3/TCR-mediated T-cell activation in different
experimental systems.19-31 In particular, it has been
reported that KIR+ cytotoxic T lymphocytes
(CTLs) fail to lyse antigen-presenting cells that express
the KIR-cognate HLA class I molecules.26,30
We have generated transgenic mice for KIR2DL3 using an MHC class I
promoter and an Ig enhancer.32 KIR2DL3 transgenic mice express the transgene on all NK cells, T cells, and a proportion of B
cells. Because both CD4+ and CD8+ express
KIR2DL3 at their surface, the KIR2DL3 transgenic mice represent a
unique model to dissect the function of HLA class I-specific inhibitory
receptors on T lymphocytes.
| |
MATERIALS AND METHODS |
Mice.
The KIR2DL3 transgenic mice (Tg-KIR2DL3) were described
elsewhere.32 The transgenic founder mice
([B6×CBA]F2 mice) were backcrossed to both B6 and
CBA backgrounds, and offspring were typed for H-2 alleles with
monoclonal antibody (MoAb) specific for H-2Kb (20.8.4) and
H-2Kk (11.4.1). The HLA-Cw3 transgenic mice of B6
background (Tg-HLA-Cw3) have been described.32 The
Tg-HLA-Cw3 were backcrossed with CBA and offspring typed for H-2
alleles. All the mice were maintained at the Animal Facility of the
Pasteur Institute (Paris, France). Nontransgenic wild-type mice (WT)
were also used as controls.
Immunofluorescence analysis.
Spleen cells and thymocytes were stained as previously described and
analyzed on a FACScan apparatus (Becton Dickinson, Mountain View,
CA).32 Cells were stained with the following MoAbs:
anti-HLA class I fluorescein isothiocyanate (FITC)-conjugated;
anti-KIR2DL3 phycoerythrin (PE)-conjugated (Immunotech, Marseille,
France); anti-CD3 FITC- or PE-conjugated; anti-CD4 FITC- or
PE-conjugated; anti-CD8 biotine-, FITC- or PE-conjugated (Pharmingen,
San Diego, CA); and tricolor (TC)-conjugated streptavidin (Caltag, San
Francisco, CA).
Mixed lymphocyte reaction (MLR) and interleukin-2 (IL-2) production.
Unfractionated spleen cells (2 × 105) from WT and
Tg-KIR2DL3 mice were stimulated with 4 × 105
irradiated (3,000 rads) allogeneic spleen cells. No cytokines were
added in the assay. After 3 days of culture, the cells were pulsed
overnight with 1 µCi/well [3H]Thymidine (Amersham Life
Science, Amersham, UK). The results of the assay were expressed as the
mean of cpm of triplicate culture ± standard deviation. For the
determination of IL-2 production, 100 µL of day-3 MLR supernatant was
added to 104 CTLL-2, an IL-2-dependent cell line. After 24 hours of culture, the cells were pulsed overnight with 1 µCi/well
[3H]Thymidine. Data represent the mean cpm from
triplicate cultures ± standard deviation.
Generation of alloreactive CTLs and cytotoxicity assay.
Recipient mice were injected with 107 irradiated (3,000 rads) allogeneic splenocytes into the hind foot pads. After 3 days, the
draining lymph nodes were collected and the cells were cultured for 4 more days in vitro in the absence of any stimulating cell in culture
medium containing 40 U/mL recombinant IL-2
(rIL-2).33
Con-A-activated T-cell blasts were used as target cells in a standard
4-hour 51Cr release in vitro assay with primary in
vivo-induced CTLs. Briefly, 107 spleen cells were
cultivated for 48 hours in culture medium containing 2 µg/mL Con-A
(Sigma Chemicals Co, St Louis, MO). Con-A blasts were labeled with 500 µCi 51Cr (Amersham) for 1 hour and added to the CTLs at
various effector:target (E:T) ratios in 96-well U-bottom plates. After
4 hours at 37°C, 100 µL of supernatant was collected from each
well and counted in a -counter for the determination of
51Cr release and the percentage of specific lysis.
For the antibody-redirected killing assay, the murine mastocytoma cell
line P815 was used as a source of target cells. Briefly, effector cells
(alloreactive CTLs or freshly isolated splenocytes) were incubated with
anti-CD3 and anti-KIR2DL3 MoAbs, alone or in combination, before the
addition of 51Cr-labeled P815 target cells at an E:T ratio
of 5:1 for the CTLs and of 200:1 for the splenocytes. When freshly
isolated splenocytes were used as effector cells, mice (WT and
Tg-KIR2DL3) were injected with poly I:C 24 hours before the assay as
described.32
Skin grafts.
Donor tail skin (1 cm2) was grafted onto the flank of
recipient mice, which was then covered with pansement dressing. A
plaster was applied and secured with an autoclip. Bandaging was removed 7 days later, and grafts were observed every day for rejection. Grafts
were scored as being rejected when no viable tissue was visible.
Hen egg white lysozime (HEL) immunization.
HEL protein was purchased from Appligene (Illkrich, France). Mice were
immunized in the hind foot pads with 3.5 nmol/L HEL in
phosphate-buffered saline (PBS) emulsified 1:1 with
complete Freund adjuvant (CFA). Nine days later, popliteal
lymph nodes were collected.34 Lymph nodes cells were
cultured (5 × 105/well) in fetal calf serum
(FCS)-free medium HL-1 (Ventrex Laboratories, Portland,
ME) supplemented with 2 mmol/L glutamine, 100 U/mL
penicillin, and 100 µg/mL streptomycin in the presence of different
concentrations of HEL (from 0.1 to 50 µmol/L). The cells were
incubated with 1 µCi/well [3H]Thymidine for the last 18 hours of a 4-day culture, and [3H]Thymidine incorporation
was assayed by liquid scintillation counting. The results were
expressed as the mean cpm of triplicate cultures ± standard deviation.
T-cell repertoire analysis.
Mice were immunized twice at 1-week intervals at the base of the tail
either with 100 µg Pigeon Cytochrome-C (PCC) emulsified 1:1 in CFA or
with CFA alone. After 1 week from the last immunization, the draining
lymph nodes were collected and mRNA was extracted using Trizol reagent
(GIBCO BRL, Gaithersburg, MD), as recommended by the manufacturer. cDNA
was synthesized using (dT)17 primer and Moloney's murine
leukemia virus (M-MLV) reverse transcriptase (GIBCO BRL)
in the presence of RNasin (Promega, Madison, MI). Immunoscope analysis
and C-, V-, and J-specific primers have been previously
described.35 Briefly, 1/10 of the cDNA was amplified in a
50 µL polymerase chain reaction (PCR) with C and V3.1
oligonucleotides. Each amplified product was then used as a template
for elongation with J-specific oligonucleotides with a fluorescent
tag (run-off reaction), as described. The fluorescent run-off products
were loaded onto a polyacrylamide gel and subjected to electrophoresis in an automated DNA sequencer. The CDR3 size distribution and signal
intensities were then analyzed with the Immunoscope
software.36
| |
RESULTS |
Inhibition of in vitro T-cell activation by engagement of KIR2DL3.
In Tg-KIR2DL3 mice, both CD4+ and CD8+ T cells
express high levels of KIR2DL3 at their surface, and no
KIR CD3+ T cells could be detected
(Table 1). We have previously shown that
KIR2DL3 is able to confer specific inhibition of anti-CD3 MoAb-redirected cytotoxicity to T lymphocytes freshly isolated from
these transgenic mice.32 To investigate whether other
T-cell functions could be controlled by engagement of the KIR2DL3
molecule, we analyzed in vitro T-cell proliferation and IL-2 production in response to allogeneic stimulation. MLR were established using unfractionated spleen cells from WT and Tg-KIR2DL3 of H-2b
haplotype as responders. Allogeneic stimulator splenocytes from WT
H-2k haplotype mice were able to induce proliferation of
H-2b splenic T cells as well as H-2b splenic
KIR2DL3+ T cells (Fig 1A). In
contrast, when allogeneic H-2k cells expressing HLA-Cw3
were used as stimulators, a significant inhibition of T-cell
proliferation was observed with responder T cells isolated from KIR2DL3
transgenic mice, but not with responder T cells isolated from WT mice
(Fig 1A). The inhibition of T-cell proliferation correlates with a
lower IL-2 production by Tg-KIR2DL3 responder T cells in the presence
of HLA-Cw3+ stimulator cells (Fig 1B).
View larger version (26K):
[in this window]
[in a new window]
| Fig 1.
KIR2DL3 expression on T cells inhibits MLR. (A) The
proliferation of splenic T cells from Tg-KIR2DL3 and WT mice of
H-2b haplotype in response to allogeneic (H-2k)
stimulator spleen cells was assessed by (3H)thymidine
incorporation after 3 days of MLR. (B) IL-2 secretion in the
supernatant of MLR at day 3 was assessed by measuring the proliferation
of the IL-2-dependent CTLL-2 cell line. Data represent the mean values ± standard deviation of triplicate determinations of 1 out of 3 representative experiments. ( ) None; ( ) WT H2b; ( )
WT H2k; ( ) Tg-HLA-Cw3 H2k.
|
|
Regulation of in vivo T-cell activation by engagement of KIR2DL3.
We generated mouse alloreactive CTLs by injecting irradiated spleen
cells from allogeneic mice into the hind foot pads.33 In
vivo-generated CTLs from WT and Tg-KIR2DL3 mice of H-2k
haplotype were then tested for their cytolytic activity against syngeneic and allogeneic target cells. Both WT-derived and
Tg-KIR2DL3-derived H-2k CTLs were able to efficiently lyse
target cells from WT H-2b mice
(Fig 2A), but not syngeneic target cells
from WT H-2k mice (Fig 2B). Surprisingly, the expression of
HLA-Cw3 on allogeneic H-2b target cells does not protect
them from lysis by Tg-KIR2DL3-derived H-2k CTLs (Fig 2C).
View larger version (17K):
[in this window]
[in a new window]
| Fig 2.
Cytolytic activity of in vivo-generated CTL against
allogeneic and syngeneic target cells. WT ( ) and Tg-KIR2DL3 ( )
mice of H-2k haplotype were injected into the hind foot
pads with irradiated splenocytes from WT H-2b mice.
Draining lymph node cells were tested against WT (A) and Tg-HLA-Cw3 (C)
of H-2b haplotype and WT H-2k (B) ConA blasts.
Data shown are representative from 3 independent experiments.
|
|
We further tested the in vitro cytotoxicity of in vivo-derived CTLs
against P815 target cells in the presence of anti-CD3 and anti-KIR2DL3
MoAbs. As shown in Fig 3A, anti-CD3
MoAb-mediated cytotoxicity of CTLs derived from Tg-KIR2DL3 mice is not
inhibited in the presence of anti-KIR2DL3 MoAb. In contrast, splenic T
cells freshly isolated from Tg-KIR2DL3 mice showed a decreased lytic activity against P815 target cells in the presence of anti-KIR2DL3 MoAb
(Fig 3B), as we previously reported.32
View larger version (30K):
[in this window]
[in a new window]
| Fig 3.
Antibody-redirected cytolytic activity of freshly
isolated splenocytes and in vivo-generated CTL. In vivo-generated CTL
(A) and freshly isolated nonadherent splenocytes injected (B) from WT
(left panels) and Tg-KIR2DL3 mice (right panels) were tested in a
redirected killing assay against P815 target cells at an E:T ratio of
5:1 for the CTL and 200:1 for the splenocytes. () Anti-CD3; ( )
anti-CD3 + anti-KIR2DL3.
|
|
To investigate the ability of KIR2DL3 to modulate T-cell functions in
vivo, we studied allogeneic skin graft rejection. Tail skin grafts from
Tg-HLA-Cw3 H-2k mice were grafted onto syngeneic Tg-HLA-Cw3
H-2k mice or allogeneic Tg-HLA-Cw3 or Tg-KIR2DL3 of
H-2b haplotype. As shown in
Table 2, the syngeneic grafts were
accepted. In contrast, all H-2b allogeneic recipients (ie,
Tg-HLA-Cw3 or Tg-KIR2DL3) rejected the skin grafts between 14 and 15 days. These results indicate that KIR2DL3 expressed on T cells from
allogeneic recipient mice is unable to control T-cell-mediated skin
graft rejection, despite cell surface expression of HLA-Cw3 on donor
skins.
Analysis of KIR2DL3 × HLA-Cw3 double transgenic mice.
Double transgenic mice for KIR2DL3 and its ligand HLA-Cw3 were
generated and analyzed for their T-cell compartment. All thymocytes and
T lymphocytes express the KIR2DL3 receptor, and all bone marrow-derived as well as stroma-derived cells express the HLA-Cw3
molecule.32,37 Despite the early expression of both
transgenic molecules during T-cell ontogeny, no alteration in the
numbers and percentages of thymocytes and splenic T cells could be
detected when comparing WT, simple transgenic littermates (Tg-HLA-Cw3
and Tg-KIR2DL3), and double transgenic animals
(Table 3 and data not shown). Moreover, surface expression analysis of CD4 and CD8 molecules did not show any
modification of T cells in double transgenic animals as compared with
WT mice or single transgenic animals (Fig 4
and data not shown).
View larger version (54K):
[in this window]
[in a new window]
| Fig 4.
Flow cytometric analysis of CD4 and CD8 expression on
thymocytes and splenocytes of WT and Tg-KIR2DL3 × HLA-Cw3 mice.
Freshly isolated thymocytes and splenocytes from WT and Tg-KIR2DL3 × HLA-Cw3 mice were stained with anti-CD4 and anti-CD8 MoAbs and analyzed
by flow cytometry.
|
|
Tg-HLA-Cw3, Tg-KIR2DL3, and Tg-KIR2DL3 × HLA-Cw3 mice of
H-2k haplotype were immunized with HEL. Nine days after HEL
injection, the draining lymph nodes were collected and the lymph node
cells were purified.34 The number of recovered lymph node
cells after HEL immunization was similar in the 3 groups of mice
(number of lymph node cells recovered × 106, mean ± SD, n = 6: 31 ± 7 for Tg-HLA-Cw3, 33.5 ± 5.4 for
Tg-KIR2DL3, and 27.5 ± 7 for Tg-KIR2DL3 × HLA-Cw3). When
lymph node cells were restimulated in vitro with various concentrations
of HEL, the proliferative responses were similar in the 3 groups of
mice (Fig 5). These results indicate that
the double transgenic mice are able to respond to antigen immunization
despite the presence of KIR2DL3 on T cells and HLA-Cw3 on
antigen-presenting cells.
View larger version (19K):
[in this window]
[in a new window]
| Fig 5.
Proliferative response of HEL-primed Tg-HLA-Cw3,
Tg-KIR2DL3, and Tg-KIR2DL3 × HLA-Cw3 mice lymph node cells.
Tg-HLA-Cw3 (), Tg-KIR2DL3 (), and Tg-KIR2DL3 × HLA-Cw3 ( )
mice were immunized with HEL, and the draining lymph node cells were
isolated 9 days after the immunization. Lymph node cells were then
restimulated in vitro in the presence of the indicated concentrations
of HEL and cell proliferation was measured after 4 days of culture.
Data shown are representative of 3 independent experiments.
|
|
We then asked whether the simultaneous presence of KIR2DL3 and HLA-Cw3
within the thymus could induce changes in T-cell repertoire by
modifying the threshold of positive and negative selection. It has been
previously shown that the immune response against PCC in an
H-2k background is characterized by the emergence of a
dominant V3-J1.2 rearrangement with a 9 amino acid long
CDR3.35 To characterize the TCR repertoire of T
lymphocytes specific for PCC, we immunized Tg-HLA-Cw3, Tg-KIR2DL3, and
Tg-KIR2DL3 × HLA-Cw3 of H-2k haplotype with PCC. As
shown in Fig 6, lymph node cells from all
groups of mice immunized with PCC displayed an expansion corresponding to the described PCC-specific response. These results suggest that the
TCR repertoire in Tg-KIR2DL3 × HLA-Cw3 mice does not display
major modifications.
View larger version (30K):
[in this window]
[in a new window]
| Fig 6.
CDR3 size distribution in PCC immunized mice.
Tg-HLA-Cw3, Tg-KIR2DL3, and Tg-KIR2DL3 × HLA-Cw3 mice were immunized
with PCC emulsified in CFA (PCC) as indicated. As a control, a group of
Tg-KIR2DL3 × HLA-Cw3 mice were injected with CFA alone. The mRNA was
extracted from the draining lymph node cells and reversely transcribed
into cDNA. The cDNA from the indicated mice was subjected to PCR using
V3.1- and C-specific primers followed by a run-off with
fluorescent J1.2- and J2.3-specific oligonucleotides. The size
distribution was analyzed with the Immuoscope software. Data shown are
representative from 3 mice per group. Analysis of V3-J2.3 was
performed as a negative control. The arrows indicate the V3-J1.2
rearrangement with a 9 amino acid long CDR3 that is dominant upon
PCC immunization.
|
|
| |
DISCUSSION |
Inhibitory KIRs have been initially characterized on NK cells as
MHC-specific receptors whose engagement leads to inhibition of NK
cytolytic activity.38 Consistent with these seminal
observations, the transgenic expression of KIR2DL3 confers a negative
control to NK cells upon interaction with cognate HLA-Cw3 molecules
expressed on target cells.32 On NK cells, KIRs are thus
thought to act as sensor molecules to detect the quality as well as the
quantity of self MHC class I molecules expressed on autologous cells
and to restrict the NK effector function towards cells expressing no or
altered self MHC class I molecules. Discrete subsets of T cells also
express KIRs, but the biological significance of KIR expression on T
lymphocytes remains elusive. KIR+ T cells have been shown
to present a cell surface phenotype typical of memory T cells as
indicated by expression of CD45R0 and CD29 as well as by the lack of
CD28 and CD45RA.24 KIRs can act as inhibitory molecules on
T-cell clones, as shown in several experimental in vitro models, such
as CD3/TCR-mediated cytolysis.19-22,27-30 It has also been
shown that treatment of KIR+ T-cell clones with anti-KIR
and anti-HLA class I MoAb leads to an increased tumor necrosis  (TNF) and interferon- (IFN-) production by CD4+
and CD8+ T-cell clones.23 These results
suggested that recognition of autologous MHC class I molecules by
KIR+ T cells might provide a mechanism for limiting T-cell
activation, which could be involved in the maintenance of tolerance or
prevention of autoimmunity.23 Melanoma-specific
KIR+ CTL cell clones have also been
described.26 Such KIR+ CTLs are active only
against tumor cells showing a partial MHC class I loss, eg, the cognate
MHC class I molecule that interacts with the given KIR.26
In these circumstances, expression of KIRs on T cells appears to be
deleterious for antitumor immunity.
We have analyzed the T-cell compartment in Tg-KIR2DL3 and Tg-KIR2DL3 × HLA-Cw3 mice, which represent a unique model to dissect the
consequence of a single KIR expression on T cells in the presence or
absence of the cognate HLA-Cw3 ligand. Unexpectedly, the transgenic expression of KIR2DL3 on mouse T cells reveals that the inhibition of
T-cell activation induced by KIR cannot be detected in vitro using in
vivo-primed T cells (Figs 2 and 3A). Furthermore, no attenuation of in
vivo T-cell response can be detected in KIR2DL3 transgenic mice (Table
2) or in double transgenic mice coexpressing KIR2DL3 and its ligand
HLA-Cw3 (Figs 5 and 6).
We previously demonstrated that the transgenic KIR2DL3 molecule is
capable of coupling to mouse NK cell inhibitory pathways, because NK
cells isolated from KIR2DL3 transgenic mice are tolerant in vitro and
in vivo to HLA-Cw3+ target cells.32 Similarly,
engagement of KIR2DL3 can inhibit anti-CD3 MoAb-redirected cytotoxicity
exerted by mouse splenocytes freshly isolated from KIR2DL3 transgenic
mice (Cambiaggi et al32 and Fig 3B), as well as MLR towards
HLA-Cw3+ targets (Fig 1). Therefore, we can rule out the
possibility that the heterologous expression of human KIRs in mouse
lymphocytes and the level of expression of KIR2DL3 and HLA-Cw3 in
transgenic mice are responsible for the lack of inhibitory function
exerted by the transgenic KIR2DL3 molecules on mouse T cells.
T-cell activation is controlled by a dynamic balance between activating
and inhibitory signals.39 A feature of all ITIM-bearing molecules, including KIRs, is that their engagement sets a threshold of
cell activation, which can be bypassed using increasing amounts of
activating reagents, such as antigen or agonist
MoAbs.10,32,40 It is therefore possible that the absence of
inhibition mediated by the transgenic KIR2DL3 molecule on mouse T cells
is merely the consequence of a quantitative insufficiency of KIR
inhibitory mechanisms to antagonize full-blown T-cell activation. In
this regard, the potent in vitro CTL activation induced by alloantigen is refractory to KIR inhibition (Fig 2), and KIR2DL3 transgenic mice
are not tolerant to HLA-Cw3+ allogeneic skins (Table 2).
Consistent with these data, anti-CD3 MoAb-redirected lysis exerted by
primed CTLs is not inhibited by KIR engagement (Fig 3A), in contrast to
anti-CD3 MoAb-redirected lysis of freshly isolated splenocytes (Fig
3B). This discrepancy may be the direct consequence of the higher
efficiency of primed CTLs to exert cytotoxicity as compared to freshly
isolated splenocytes.
Alternatively, it is also possible that the lack of KIR inhibitory
function on T cells is the consequence of the coupling of KIRs to
downstream effector molecules distinct from SHP-1 and SHP-2 protein
tyrosine phosphatases. In particular, it has been shown that the p85
regulatory subunit of phosphatidylinositol (PI) 3-kinase can be
recruited by KIR phospho-ITIMs.41 It is therefore possible
that, depending on the activation status of lymphocytes, KIRs may
either couple to SHP-1/SHP-2 or to PI 3-kinase. Such a differential
recruitment may occur through a variable KIR accessibility to these
downstream effector molecules. Similarly, engagement of FcRIIB,
another ITIM-bearing molecule has been shown either to extinguish
B-cell activation through the recruitment of SHIP or to result in
enhanced apoptosis through a still-undefined mechanism.42
Such developmental regulation of KIR accessibility to protein tyrosine
phosphatases or other downstream effector molecules remains to be
further dissected.
It has been suggested that KIR expression on T cells allows the
cells to downmodulate or terminate an immune response and might therefore be involved in the control of peripheral tolerance. This hypothesis needs to be re-evaluated, because our results show that
the signals induced upon KIR engagement are not sufficient to modulate
in vivo T-cell response to antigen. Moreover, once T cells have been
activated in vivo, they become refractory to KIR-mediated inhibition.
Similarly, KIR2DL3 engagement is unable to control T-cell cytotoxicity
on a fraction of Epstein-Barr virus (EBV)-specific
KIR+ T-cell clones derived from EBV-infected patients
(Couedel et al, unpublished results). Therefore,
strategies aimed at controlling T-cell activation through the
manipulation of KIR expression on T cells should be reconsidered. In
contrast to KIRs, the cell surface expression of lectin-like NKRs, such
as CD94-NKG2 dimers, is heavily controlled by
cytokines.31,43,44 It will be of interest to investigate
whether the in vivo function of lectin-like NKRs, such as CD94-NKG2
dimers, correlates with their in vitro inhibitory function on
CD3/TCR-induced T-cell activation.45,46
| |
ACKNOWLEDGMENT |
The authors thank Drs Margarita Salcedo, Philippe Bousso (Institut
Pasteur, Paris, France), Marc Bonneville (INSERM U463, Nantes, France),
and Sophie Ugolini (CIML, Marseille, France) for helpful
discussions and thank Corinne Béziers La Fosse (CIML) for graphics.
| |
FOOTNOTES |
Submitted April 15, 1999; accepted June 3, 1999.
Supported by institutional grants from INSERM, CNRS, and
Ministère de l'Enseignement Supérieur et de la Recherche
and by specific grants from Association pour la Recherche contre le
Cancer (E.V. and P.K.), Ligue Nationale contre le Cancer (E.V. and P.K. subvention: axe immunologie des tumeurs), and the Training and Mobility
of Researcher Programme (A.C.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Eric Vivier, DVM, PhD, Centre
d'Immunologie INSERM/CNRS de Marseille-Luminy, Case 906, 13288 Marseille cedex 09, France; e-mail: vivier{at}ciml.univ-mrs.fr.
| |
REFERENCES |
1.
Moretta A, Bottino C, Vitale M, Pende D, Biassoni R, Mingari MC, Moretta L:
Receptors for HLA-class-I molecules in human natural killer cells.
Annu Rev Immunol
14:619, 1996[Medline]
[Order article via Infotrieve]
2.
Lanier LL:
NK cell receptors.
Annu Rev Immunol
16:359, 1998[Medline]
[Order article via Infotrieve]
3.
Long EO, Burshtyn DN, Clark WP, Peruzzi M, Rajagopalan S, Rojo S, Wagtmann N, Winter CC:
Killer cell inhibitory receptors: Diversity, specificity, and function.
Immunol Rev
155:135, 1997[Medline]
[Order article via Infotrieve]
4.
Lee N, Llano M, Carretero M, Ishitani A, Navarro F, Lopez-Botet M, Geraghty DE:
HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A.
Proc Natl Acad Sci USA
95:5199, 1998[Abstract/Free Full Text]
5.
Yokoyama WM:
Natural killer cell receptors.
Curr Opin Immunol
10:298, 1998[Medline]
[Order article via Infotrieve]
6.
Borges L, Hsu M-L, Fanger N, Kubin M, Cosman D:
A family of human lymphoid and myeloid Ig-like receptors, some of which bind to MHC class I molecules.
J Immunol
159:5192, 1997[Abstract]
7.
Vitale M, Castriconi R, Parolini S, Pende D, Hsu ML, Moretta L, Cosman D, Moretta A:
The leukocyte Ig-like receptor (LIR)-1 for the cytomegalovirus UL18 protein displays a broad specificity for different HLA class I alleles: Analysis of LIR-1 + NK cell clones.
Int Immunol
11:29, 1999[Abstract/Free Full Text]
8.
Biassoni R, Cantoni C, Falco M, Verdiani S, Bottino C, Vitale M, Conte R, Poggi A, Moretta A, Moretta L:
The human leukocyte antigen (HLA)-C-specific "activatory" or "inhibitory" natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions.
J Exp Med
183:645, 1996[Abstract/Free Full Text]
9.
Olcese L, Cambiaggi A, Semenzato G, Bottino C, Moretta A, Vivier E:
Killer-cell activatory receptors for MHC class I molecules are included in a multimeric complex expressed by human killer cells.
J Immunol
158:5083, 1997[Abstract]
10.
Vivier E, Daëron M:
Immunoreceptor tyrosine-based inhibition motifs (ITIMs).
Immunol Today
18:286, 1997[Medline]
[Order article via Infotrieve]
11.
Burshtyn DN, Long EO:
Regulation through inhibitory receptors: Lessons from natural killer cells.
Trends Cell Biol
7:473, 1997[Medline]
[Order article via Infotrieve]
12.
Tomasello E, Olcese L, Vely F, Geourgeon C, Blery M, Moqrich A, Gautheret D, Djabali M, Mattei MG, Vivier E:
Gene structure, expression pattern, and biological activity of mouse killer cell activating receptor-associated protein (KARAP)/DAP-12.
J Biol Chem
273:34115, 1998[Abstract/Free Full Text]
13.
Lanier LL, Corliss BC, Wu J, Leong C, Phillips JH:
Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells.
Nature
391:703, 1998[Medline]
[Order article via Infotrieve]
14.
Lanier LL, Corliss B, Wu J, Phillips JH:
Association of DAP-12 with activating CD94/NKG2C NK cell receptors.
Immunity
8:693, 1998[Medline]
[Order article via Infotrieve]
15.
Campbell KS, Cella M, Carretero M, Lopez-Botet M, Colonna M:
Signaling through human killer cell activating receptors triggers tyrosine phosphorylation of an associated protein complex.
Eur J Immunol
28:599, 1998[Medline]
[Order article via Infotrieve]
16.
Nakajima H, Samaridis J, Angman L, Colonna M:
Human myeloid cells express an activating ILT receptor (ILT1) that associates with Fc receptor gamma-chain.
J Immunol
162:5, 1999[Abstract/Free Full Text]
17.
Garrido F, Ruiz-Cabello F, Cabrera T, Pérez-Villar JJ, Lòpez-Botet M, Duggan-Keen M, Stern PL:
Implications for immunosurveillance of altered HLA class I phenotypes in human tumors.
Immunol Today
18:89, 1997[Medline]
[Order article via Infotrieve]
18.
Tay CH, Szomolanyi-Tsuda E, Welsh RM:
Control of infections by NK cells.
Curr Top Microbiol Immunol
230:193, 1998[Medline]
[Order article via Infotrieve]
19.
Mingari MC, Vitale C, Cambiaggi A, Schiavetti F, Melioli G, Ferrini S, Poggi A:
Cytolytic T lymphocytes displaying natural killer (NK)-like activity: Expression of NK-related functional receptors for HLA class I molecules (p58 and CD94) and inhibitory effect on the TCR-mediated target cell lysis or lymphokine production.
Int Immunol
7:697, 1995[Abstract/Free Full Text]
20.
Ferrini S, Cambiaggi A, Meazza R, Sforzini S, Marciano S, Mingari MC, Moretta L:
T cell clones expressing the natural killer cell-related p58 receptor molecule display heterogeneity in phenotypic properties and p58 function.
Eur J Immunol
24:2294, 1994[Medline]
[Order article via Infotrieve]
21.
Mingari MC, Ponte M, Cantoni C, Vitale C, Schiavetti F, Bertone S, Bellomo R, Tradori Cappai A, Biassoni R:
HLA-class I-specific inhibitory receptors in human cytolytic T lymphocytes: Molecular characterization, distribution in lymphoid tissues and co-expression by individual T cells.
Int Immunol
9:485, 1997[Abstract/Free Full Text]
22.
Phillips JH, Gumperz JE, Parham P, Lanier LL:
Superantigen-dependant, cell-mediated cytotoxicity inhibited by MHC class I receptors on T lymphocytes.
Science
268:403, 1995[Abstract/Free Full Text]
23.
D'Andrea A, Chang C, Phillips JH, Lanier LL:
Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles.
J Exp Med
184:789, 1996[Abstract/Free Full Text]
24.
Mingari MC, Schiavetti F, Ponte M, Vitale C, Maggi E, Romagnani S, Demarest J, Pantaleo G, Fauci AS, Moretta L:
Human CD8+ T lymphocyte subsets that express HLA class I-specific inhibitory receptors represent oligoclonally or monoclonally expanded cell populations.
Proc Natl Acad Sci USA
93:12433, 1996[Abstract/Free Full Text]
25.
De Maria A, Ferraris A, Guastella M, Pilia S, Cantoni C, Polero L, Mingari MC, Basseti D, Fauci AS, Moretta L:
Expression of HLA class I-specific inhibitory natural killer cell receptors in HIV-specific cytolytic T lymphocytes: Impairment of specific cytolytic functions.
Proc Natl Acad Sci USA
94:10285, 1997[Abstract/Free Full Text]
26.
Ikeda H, Lethé B, Lehmann F, Van Baren N, Baurain J-F, De Smet C, Chambost H, Vitale M, Moretta A, Boon T, Coulie PG:
Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor.
Immunity
6:199, 1997[Medline]
[Order article via Infotrieve]
27.
Mingari MC, Ponte M, Bertone S, Schiavetti F, Vitale C, Bellomo R, Moretta A, Moretta L:
HLA-class I specific inhibitory receptors in T lymphocytes. IL-15 induced expression of CD94/NKG2A in superantigen- or alloantigen-activated human T cells.
Proc Natl Acad Sci USA
95:1172, 1998[Abstract/Free Full Text]
28.
Halary F, Peyrat M-A, Champagne E, Lopez-Botet M, Moretta A, Moretta L, Vié H, Fournié J-J, Bonneville M:
Control of self-reactive cytotoxic T lymphocytes expressing T cell receptors by natural killer inhibitory receptors.
Eur J Immunol
27:2812, 1997[Medline]
[Order article via Infotrieve]
29.
Battistini L, Borsellino G, Sawicki G, Poccia F, Salvetti M, Ristori G, Brosnan CF:
Phenotypic and cytokine analysis of human peripheral blood gamma delta T cells expressing NK cell receptors.
J Immunol
159:3723, 1997[Abstract]
30.
Bakker AB, Phillips JH, Figdor CG, Lanier LL:
Killer cell inhibitory receptors for MHC class I molecules regulate lysis of melanoma cells mediated by NK cells, gammadelta T cells, and antigen-specific CTL.
J Immunol
160:5239, 1998[Abstract/Free Full Text]
31.
Mingari MC, Moretta A, Moretta L:
Regulation of KIR expression in human T cells: A safety mechanism that may impair protective T-cell responses.
Immunol Today
19:153, 1998[Medline]
[Order article via Infotrieve]
32.
Cambiaggi A, Verthuy C, Naquet P, Romagne F, Ferrier P, Biassoni R, Moretta A, Moretta L, Vivier E:
Natural killer cell acceptance of H-2 mismatch bone marrow grafts in transgenic mice expressing HLA-Cw3 specific killer cell inhibitory receptor.
Proc Natl Acad Sci USA
94:8088, 1997[Abstract/Free Full Text]
33.
Noun G, Reboul M, Abastado JP, Kourilsky P, Sigaux F, Pla M:
Strong alloantigenicity of the alpha-helices residues of the MHC class I molecule.
J Immunol
161:148, 1998[Abstract/Free Full Text]
34.
Gapin L, Bravo de Alba Y, Casrouge A, Cabaniols JP, Kourilsky P, Kanellopoulos J:
Antigen presentation by dendritic cells focuses T cell responses against immunodominant peptides: Studies in the hen egg-white lysozyme (HEL) model.
J Immunol
160:1555, 1998[Abstract/Free Full Text]
35.
Cochet M, Pannetier C, Regnault A, Darche S, Leclerc C, Kourilsky P:
Molecular detection and in vivo analysis of the specific T cell response to a protein antigen.
Eur J Immunol
22:2639, 1992[Medline]
[Order article via Infotrieve]
36.
Pannetier C, Delassus S, Darche S, Saucier C, Kourilsky P:
Quantitative titration of nucleic acids by enzymatic amplification reactions run to saturation.
Nucleic Acids Res
21:577, 1993[Abstract/Free Full Text]
37.
Dill O, Kievits F, Koch S, Ivanyi P, Hämmerling GJ:
Immunological function of HLA-C antigens in HLA-Cw3 transgenic mice.
Proc Natl Acad Sci USA
85:5664, 1988[Abstract/Free Full Text]
38.
Moretta A, Moretta L:
HLA class I specific inhibitory receptors.
Curr Opin Immunol
9:694, 1997[Medline]
[Order article via Infotrieve]
39.
Renard V, Cambiaggi A, Vély F, Blery M, Olcese L, Olivero S, Bouchet M, Vivier E:
Transduction of cytotoxic signals in natural killer cells: A general model of fine tuning between activatory and inhibitory pathways in lymphocytes.
Immunol Rev
155:205, 1997[Medline]
[Order article via Infotrieve]
40.
Bléry M, Delon J, Trautmann A, Cambiaggi A, Olcese L, Biassoni R, Moretta L, Chavrier P, Moretta A, Daëron M, Vivier E:
Reconstituted killer-cell inhibitory receptors for MHC class I molecules control mast cell activation induced via immunoreceptor tyrosine-based activation motifs.
J Biol Chem
272:8989, 1997[Abstract/Free Full Text]
41.
Marti F, Xu CW, Selvakumar A, Brent R, Dupont B, King PD:
LCK-phosphorylated human killer cell-inhibitory receptors recruit and activate phosphatidylinositol 3-kinase.
Proc Natl Acad Sci USA
95:11810, 1998[Abstract/Free Full Text]
42.
Ono M, Okada H, Bolland S, Yanagi S, Kurosaki T, Ravetch JV:
Deletion of SHIP or SHP-1 reveals two distincts pathways for inhibitory signaling.
Cell
90:293, 1997[Medline]
[Order article via Infotrieve]
43.
Ponte M, Bertone S, Vitale C, Tradori-Cappai A, Bellomo R, Castriconi R, Moretta L, Mingari MC:
Cytokine-induced expression of killer inhibitory receptors in human T lymphocytes.
Eur Cytokine Netw
9:69, 1998[Medline]
[Order article via Infotrieve]
44.
Bertone S, Schiavetti F, Bellomo R, Vitale C, Ponte M, Moretta L, Mingari MC:
Transforming growth factor-beta-induced expression of CD94/NKG2A inhibitory receptors in human T lymphocytes.
Eur J Immunol
29:23, 1999[Medline]
[Order article via Infotrieve]
45.
Le Dréan E, Vély F, Olcese L, Cambiaggi A, Guia S, Krystal G, Gervois N, Moretta A, Jotereau F, Vivier E:
Inhibition of antigen-induced T cell response and antibody-induced NK cell cytotoxicity by NKG2A: Association of NKG2A with SHP-1 and SHP-2 protein-tyrosine phosphatases.
Eur J Immunol
28:264, 1998[Medline]
[Order article via Infotrieve]
46.
Noppen C, Schaefer C, Zajac P, Schutz A, Kocher T, Kloth J, Heberer M, Colonna M, De Libero G, Spagnoli GC:
C-type lectin-like receptors in peptide-specific HLA class I-restricted cytotoxic T lymphocytes: Differential expression and modulation of effector functions in clones sharing identical TCR structure and epitope specificity.
Eur J Immunol
28:1134, 1998[Medline]
[Order article via Infotrieve]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
E. Merck, R. B. Voyle, and H. R. MacDonald
Ly49D Engagement on T Lymphocytes Induces TCR-Independent Activation and CD8 Effector Functions That Control Tumor Growth
J. Immunol.,
January 1, 2009;
182(1):
183 - 192.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Henel, K. Singh, D. Cui, S. Pryshchep, W.-W. Lee, C. M. Weyand, and J. J. Goronzy
Uncoupling of T-cell effector functions by inhibitory killer immunoglobulin-like receptors
Blood,
June 1, 2006;
107(11):
4449 - 4457.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Tanaka, T. Toubai, Y. Tsutsumi, Y. Miura, N. Kato, S. Umehara, K. Kahata, A. Mori, N. Toyoshima, S. Ota, et al.
Cytolytic activity and regulatory functions of inhibitory NK cell receptor-expressing T cells expanded from granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells
Blood,
August 1, 2004;
104(3):
768 - 774.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y.-J. Chwae, M. J. Chang, S. M. Park, H. Yoon, H.-J. Park, S. J. Kim, and J. Kim
Molecular Mechanism of the Activation-Induced Cell Death Inhibition Mediated by a p70 Inhibitory Killer Cell Ig-Like Receptor in Jurkat T Cells
J. Immunol.,
October 1, 2002;
169(7):
3726 - 3735.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Miller, M. Peters, A. E. Oran, G. W. Beresford, L. Harrington, J. M. Boss, and J. D. Altman
CD94/NKG2 Expression Does Not Inhibit Cytotoxic Function of Lymphocytic Choriomeningitis Virus-Specific CD8+ T Cells
J. Immunol.,
July 15, 2002;
169(2):
693 - 701.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Van Beneden, A. De Creus, F. Stevenaert, V. Debacker, J. Plum, and G. Leclercq
Expression of Inhibitory Receptors Ly49E and CD94/NKG2 on Fetal Thymic and Adult Epidermal TCR V{gamma}3 Lymphocytes
J. Immunol.,
April 1, 2002;
168(7):
3295 - 3302.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Gati, N. Guerra, J. Giron-Michel, B. Azzarone, E. Angevin, A. Moretta, S. Chouaib, and A. Caignard
Tumor Cells Regulate the Lytic Activity of Tumor-specific Cytotoxic T Lymphocytes by Modulating the Inhibitory Natural Killer Receptor Function
Cancer Res.,
April 1, 2001;
61(8):
3240 - 3244.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
F. Vely, M.-A. Peyrat, C. Couedel, J.-F. Morcet, F. Halary, F. Davodeau, F. Romagne, E. Scotet, X. Saulquin, E. Houssaint, et al.
Regulation of Inhibitory and Activating Killer-Cell Ig-Like Receptor Expression Occurs in T Cells After Termination of TCR Rearrangements
J. Immunol.,
February 15, 2001;
166(4):
2487 - 2494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION