By
§
From the * Department of Cancer Biology, Hematology/Oncology, Beth Israel-Deaconess Medical
Center, Boston, Massachusetts 02215; the Division of Rheumatology, Immunology, and Allergy,
Brigham and Women's Hospital, Boston, Massachusetts 02215; and the § Harvard Medical School,
Boston, Massachusetts 02115
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Abstract |
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A population of human T cells expressing an invariant V24J
Q T cell antigen receptor
(TCR)
chain and high levels of CD161 (NKR-P1A) appears to play an immunoregulatory
role through production of both T helper (Th) type 1 and Th2 cytokines. Unlike other
CD161+ T cells, the major histocompatibility complex-like nonpolymorphic CD1d molecule
is the target for the TCR expressed by these T cells (V
24invt T cells) and by the homologous
murine NK1 (NKR-P1C)+ T cell population. In this report, CD161 was shown to act as a
specific costimulatory molecule for TCR-mediated proliferation and cytokine secretion by
V
24invt T cells. However, in contrast to results in the mouse, ligation of CD161 in the absence of TCR stimulation did not result in V
24invt T cell activation, and costimulation
through CD161 did not cause polarization of the cytokine secretion pattern. CD161 monoclonal antibodies specifically inhibited V
24invt T cell proliferation and cytokine secretion in
response to CD1d+ target cells, demonstrating a physiological accessory molecule function for
CD161. However, CD1d-restricted target cell lysis by activated V
24invt T cells, which involved a granule-mediated exocytotic mechanism, was CD161-independent. In further contrast to the mouse, the signaling pathway involved in V
24invt T cell costimulation through
CD161 did not appear to involve stable association with tyrosine kinase p56Lck. These results
demonstrate a role for CD161 as a novel costimulatory molecule for TCR-mediated recognition of CD1d by human V
24invt T cells.
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Introduction |
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cell subsets which express CD161 (NKR-P1) are
found in humans and mice. In rodents there are three
NKR-P1 molecules, NKR-P1A, -B, and -C, which are
"NK locus"-encoded C-type lectins (1). Murine NK1
(NKR-P1C)+ T cells are either CD4+ or double negative
(DN)1 and constitutively express a range of additional
markers, such as Ly-49C and B220, which are not found
on conventional T cells (5). They also express a highly
restricted TCR repertoire consisting of an invariant
V14J
281
chain in association with a restricted repertoire of V
genes (7, 10). Genetic and reconstitution studies demonstrated that NK1+ T cells are positively selected by and recognize the MHC-unlinked
2-microglobulin-associated protein, CD1d (5, 6, 11, 13). Of human T
cell populations expressing the single known human NKR-P1
molecule, NKR-P1A (CD161), the subset that is analogous to murine NK1+ T cells expresses an invariant V
24J
Q
TCR
chain paired predominantly with V
11 (14).
These human T cells, referred to here as V
24invt T cells,
have been shown to specifically recognize CD1d (19).
TCR-mediated stimulation of human V24invt T cells
results in simultaneous production of large amounts of both
IL-4 and IFN-
, and hence they have been described as
Th0 cells (18). Similarly, murine NK1+ T cells can
produce large amounts of cytokines, notably IL-4, early in
immune responses (9, 21), and a role for NK1+ T cells in
promoting Th2 responses has been proposed (22). However, the murine NK1+ T cell population is clearly not essential for all Th2 responses, since
2-microglobulin-deficient mice, which lack detectable NK1+ as well as most
CD8+ T cell populations, can still mount such responses
(23, 24). CD1d knockout mice, which similarly lack NK1+
T cells, are also able to generate model Th2 responses, such as nonspecific production of IgE (25). Murine NK1+ T
cells have also been shown to have NK-like cytotoxic activity (8, 9, 12, 28). This NK-like activity is induced by IL-12 (29) and appears to play a role in IL-12-mediated tumor rejection, a Th1-like cell-mediated response (30). Although the precise functions of human V
24invt T cells remain to be defined, quantitative and qualitative defects in
these T cells or the corresponding murine population are
predictive of progression in certain human and murine autoimmune conditions (28, 31).
It has been established that NK locus-encoded C-type
lectins can mediate NK cell activation, and that rodent
NK1, but not human CD161, acts as an autonomous NK
cell stimulatory structure (3, 4, 36). Direct stimulation
of murine NK1+ T cells through NK1 rather than the
TCR results in a cytokine switch to IFN- (13, 39, 40),
suggesting that precisely how these cells are activated may
contribute to determining the composition of the immune
response. In this study, the role of the human NK1 homologue CD161 and other candidate accessory molecules in
regulation of human DN V
24invt T cell responses to
CD1d was assessed. The results demonstrated that CD161
functions as a costimulatory receptor for CD1d recognition by V
24invt T cells. However, in contrast to murine NK1+
T cells, ligation of human CD161 on V
24invt T cells did
not directly activate cytokine secretion, and CD161 costimulation did not result in the selective production of
IFN-
. Our results identify CD161 expressed by V
24invt
T cells as a costimulatory molecule for this unique T cell
population.
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Materials and Methods |
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T Cell Clones and Cell Lines.
VAntibodies.
Antibodies used were anti-VFunctional Analysis of T Cells.
For activation of T cells (105/ well), anti-CD3 mAb OKT3 was bound overnight in PBS (50 µl/well) to 96-well flat-bottomed tissue culture plates, and unbound antibody was washed off. Coating mAb concentrations were 1 µg/ml OKT3 for subsequent incubations with no PMA and 0.1 µg/ml for incubations with PMA (Sigma Chemical Co., St. Louis, MO) at 1 ng/ml, unless otherwise indicated. Plate-bound (50 µl/well) or soluble costimulatory mAbs at 10 µg/ml or indicated concentrations were then added for at least 4 h. Subsequently, rested T cells at 2-4 wk after PHA stimulation were incubated with plate-bound mAb and IL-2 at 0.3 nM. In the case of soluble mAb, an equal amount of cross-linking anti-murine IgG antibody was added after the T cells had been allowed to settle on the limiting plate-bound anti-CD3 mAb. For CD1d responses, equal numbers of CD1d+ human C1R B cell transfectants or control mock-transfected C1R cells were incubated with the rested T cells, PMA (1 ng/ml unless otherwise stated), and IL-2 at 0.3 nM, as described previously (19). Released cytokine levels at 48 h were determined in triplicate by ELISA with matched antibody pairs in relation to cytokine standards (PharMingen; Endogen, Inc., Cambridge, MA) and converted to nanograms or picograms per milliliter using the Softmax program (Molecular Devices Corp., Sunnyvale, CA). Similarly, T cell proliferation between 48 and 72 h was determined by [3H]thymidine incorporation (1 µCi/well), using target cells pretreated with mitomycin C (0.09 mg/ml) for 1 h. Results are shown with SEM. Cytolytic activity of VAssessment of Protein Interactions of NK Locus Molecules.
Interaction of membrane and cytosolic proteins was assessed as described previously (44). In brief, V ![]() |
Results |
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Human V24invt T cells express high levels of CD161 and
variable levels of other members of the NK locus C-type
lectin family (14). FACS® profiles of two representative
V
24invt T cell clones, DN2.D5 and DN1.10B3, are
shown in Fig. 1. CD161 (NKR-P1A) was strongly expressed by these two clones derived from two different
donors (Fig. 1), as well as by all of six additional CD1d-
reactive V
24invt T cell clones (19). CD69, another C-type
lectin encoded in the NK locus, was also expressed by all of
the V
24invt T cell clones (Fig. 1; reference 19). Although
transiently expressed after activation of conventional T
cells, CD69 showed prolonged expression on V
24invt T
cell clones for at least several months after PHA stimulation (data not shown). CD94, a third NK locus-encoded
C-type lectin, was expressed by seven out of eight V
24invt
T cell clones (not DN1.10B3; Fig. 1). Analysis of other potential V
24invt T cell accessory molecules showed that p40
(45) and p38 C1.7 proteins (46), both previously found on
NK cells and some cytolytic T cells, were expressed by
some V
24invt T cell clones. However, these molecules
were also found on several control V
24+ noninvariant
cells and other T cell clones not belonging to this subset
(data not shown). V
24invt T cells had variable expression
of CD28, from barely detectable on some clones to levels
comparable to conventional T cells (Fig. 1, and data not
shown). Finally, as shown previously, the V
24invt T cells
did not express the NK cell-associated p58 or p70 killer inhibitory receptors or the other NK cell markers CD16,
CD56, and CD57 (19). Thus, established CD1d-reactive
V
24invt T cell clones were consistently CD161+CD69+,
with more variable expression of other candidate accessory
molecules.
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Ligation of murine NK1 (NKR-P1C) alone activates
murine NK1+ T cells and, in contrast to TCR stimulation,
results in an exclusively IFN--secreting Th1-biased phenotype (13, 39, 40). Therefore, we examined the effect of
direct ligation of CD161 on stimulation and costimulation
of human V
24invt T cells. Proliferative responses were
measured in the presence of appropriate suboptimal concentrations of immobilized CD3 mAb. Proliferation of all
V
24invt T cell clones tested (DN2.D5, DN2.D6, DN2.D7,
and DN1.10B3) and the V
24invt T cell line DN2.V
11+
was substantially augmented by CD161 mAbs 191.B8 (47)
and DX-1 (36) in the absence of PMA (Fig. 2 A, and data
not shown). With the addition of phorbol ester, which is
required for activation of these cells by CD1d+ targets in
vitro (19), similar costimulation by CD161 ligation was also
observed (Fig. 2 B). PMA lowered the concentrations of CD3 mAb required ~10-fold (Fig. 2, A and B). Under
both conditions, costimulation of V
24invt T cells by
CD161 was readily seen over a 25-fold range of anti-CD3 mAb concentrations (Fig. 2, A and B). Optimal costimulation via plate-bound CD161 required >1 µg/ml 191.B8
coating mAb and was not seen with soluble 191.B8, even
at up to 10 µg/ml in the presence of a soluble cross-linking
secondary antibody (Fig. 2 C). In no experiment was proliferation by human V
24invt T cell clones observed in response to plate-bound CD161 mAb in the absence of CD3
mAb (Fig. 2, A, B, and D; Table 1; and data not shown).
Similar lack of direct stimulation was observed using two
different CD161 mAbs (DX-1 and 191.B8) at concentrations up to 20 µg/ml (Fig. 2, Table 1, and data not shown).
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As with proliferative responses, and unlike in the mouse,
there was no IL-4 or IFN- secretion by human V
24invt
T cell clones in response to plate-bound CD161 mAb in
the absence of CD3 mAb, either in the presence or absence
of PMA (Fig. 2 D, Table 1, and data not shown). However, both IL-4 and IFN-
production by V
24invt T cell
clones induced by limiting anti-CD3 mAb were substantially augmented by CD161 mAbs 191.B8 and DX-1 in
both the presence and absence of PMA (Table 1, and data
not shown). Antibody-mediated CD161 ligation did not
alter the pattern of cytokines produced by suboptimal
TCR stimulation. IL-4 to IFN-
secretion ratios between
the CD161-costimulated and CD1d-specific responses varied by only approximately threefold, and indicated that
there was no polarization of cytokine secretion toward
IFN-
production induced by CD161 costimulation of the
human V
24invt T cells (Table 1).
Similarly to CD161 mAb, CD94 mAb HP-3D9 also
produced significant costimulation of V24invt T cell proliferation (Fig. 2, A and B) and IFN-
and IL-4 secretion (Table 1) in both the absence and presence of PMA.
V
24invt T cell clone DN1.10B3, which expressed CD161
but no detectable cell surface CD94 (Fig. 1 A), was
strongly costimulated by CD161 mAb, but not by CD94
mAb (not shown). However, as with CD161, there was no
direct activation of V
24invt T cell proliferation or cytokine
secretion through CD94 alone or in combination with
CD161, using several different antibodies (Fig. 2, A, B, and
D; Table 1; and data not shown). In no case was synergistic
or even additive costimulation of proliferation by CD161
and CD94 mAbs seen (Fig. 2 D, and Table 1), and in no case was significant alteration of cytokine secretion observed with simultaneous addition of both mAbs (Table 1).
This was true even at lower levels of CD3 mAb and suboptimal levels of costimulation of proliferation by more recently activated V
24invt T cells (Fig. 2 E). Anti-CD28
mAb (CD28.2), which potently costimulated control conventional T cell clones (not shown), showed only weak costimulation of the proliferation and cytokine secretion of
the CD28+ V
24invt T cell clones, and only in the absence
of PMA (Fig. 2, A and B). Anti-p40 mAb NKTA255 was
also costimulatory for V
24invt T cells, but only in the absence of PMA (not shown). CD69 mAb (Fig. 2, A and B),
p38 C1.7 mAb (not shown), MHC class I mAb, and isotype-matched nonbinding control mAb had no costimulatory or direct stimulatory activity (Fig. 2, A and B, and data
not shown). Therefore, human V
24invt CD1d-reactive T
cells differed from their murine counterparts in lack of
direct activation in response to CD161, or indeed, other
C-type lectin ligation, whereas both CD161 and CD94
mAbs were specifically costimulatory.
Since CD1d is a natural ligand of V24invt T
cells, it was important to determine whether CD161 or
other molecules contributed to T cell activation in response
to CD1d+ target cells. V
24invt T cells were incubated with
CD1d transfectants, and proliferation and cytokine responses were measured. Recognition of CD1d+ human B
cell transfectants by V
24invt T cells, measured as proliferation, or IFN-
or IL-4 cytokine secretion, was inhibited by
CD1d mAbs 51.1 (Fig. 3, A-C) and 42.1 (not shown).
Proliferation in response to CD1d was comparably inhibited with CD161 mAb DX-1 (Fig. 3 A). Similarly, secretion of both IFN-
and IL-4 was inhibited by CD161 mAb
DX-1 (Fig. 3, B and C). Each of three different CD161
mAbs tested inhibited proliferative and cytokine secretion
responses to CD1d recognition, with HP-3G10 consistently the most potent, followed by 191.B8, and then
DX-1 (Fig. 3, D and E, and data not shown). Inhibition of
proliferation and cytokine secretion in response to CD1d
by CD161 mAb was seen over a wide range of PMA concentrations (0.05-5 ng/ml; Fig. 3, and data not shown) and
not just under suboptimal conditions (<1 ng/ml PMA).
In contrast to these results with CD161 mAb, the costimulatory CD94 mAb HP-3D9 did not inhibit T cell proliferation or cytokine secretion in response to CD1d (Fig. 3, A-C). Other mAbs specific for CD94 (DX-22 and HP-3B1), CD69 (FN50), p38 (C1.7), and the weakly costimulatory p40 mAb (NKTA255) had no consistent inhibitory effect on CD1d-dependent T cell proliferation or cytokine secretion (not shown). The HLA class I control mAb (W6/32) had a small inhibitory effect (Fig. 3, D and E), which was also seen using CD1d+ CHO cells as targets (not shown). Since the class I mAb does not bind to cells of hamster origin, this result appears to reflect mAb binding directly to the T cells in the assay.
Involvement of molecules other than CD1d on the target
cell and CD161 on the T cell was tested by incubation with
mAbs against various molecules preferentially expressed on
resting and activated B and/or T cells. mAbs against CD19,
CD20, CD22, CD23, CD24, CD25, or CD28 did not affect
activation of invariant TCR+ T cells by CD1d+ B cells,
whether measured as proliferation, or IFN- or IL-4 secretion (not shown). Consistent with lack of effect of CD28 mAb, CTL-associated antigen 4 (CTLA4)-Ig fusion protein,
which blocks both B7-1 and B7-2 costimulation, had no significant effect on CD1d-dependent T cell stimulation (S.B.
Wilson, personal communication). Therefore, of the molecules studied, only CD1d itself and CD161 were found to
contribute to V
24invt T cell responses to CD1d+ target cells.
Recently activated V24invt T cell clones
displayed potent and specific cytolytic activity against C1R
CD1d+ transfectants (Fig. 4). V
24invt T cell clones induced 20-70% of maximal 51Cr release from CD1d+ C1R
cells at E/T ratios of 10:1 (Fig. 4 A, and data not shown). Cytotoxicity of the same T cell clones against C1R mock
transfectants was <10% at these E/T ratios, demonstrating
CD1d specificity of cytolysis. As seen for proliferative and
cytokine secretory responses above, the CD1d-specific cytolytic effector response of the T cell clones was inhibited
in a dose-dependent manner by CD1d-specific mAbs 42.1 and 51.1. These CD1d mAbs had an IC50 of ~1 µg/ml
(Fig. 4 A) and could reduce cytolysis to nearly background
levels at higher concentrations of mAb (Fig. 4, A and B).
This confirmed that cytolytic activity, like proliferation and
cytokine secretion, was a response to the intact CD1d molecule. The cytolytic activity of V
24invt T cells was abolished by EGTA, indicating a Fas-independent mechanism requiring release of cytolytic granules (Fig. 4 B).
To determine the role of CD161 in cytolytic activity,
CD161 mAbs were also included. No effects of any of the
three CD161 mAbs on CD1d-specific cytolytic activity
were seen at up to 10 µg/ml. This was true even when a
limiting amount of CD1d mAb (0.08 µg/ml) was included
to amplify any inhibition (Fig. 4 B), after preliminary experiments showed no inhibition by CD161 alone. Cytolytic responses were also PMA-independent. These results
demonstrated that costimulatory pathways activated by
CD161 ligation and PMA were not required for CD1d-specific cytolytic activity of V24invt T cells. These observations parallel conventional CTLs, for which costimulatory molecules such as CD28 are not required to induce cytolysis by recently activated T cells.
Because DN V24invt T cells lack CD4 and
CD8
, which are essential for physiological activation of
conventional T cells through p56Lck, an association between
V
24invt T cell p56Lck and certain accessory molecules might
be expected. Association between murine NK1 and p56Lck
has been described (48), but human CD161 (36) does not
contain the cytoplasmic tail p56Lck binding motif found in
CD4 and CD8 (49) and in all of the murine NKR-P1 molecules (1) (see Fig. 5 A). Therefore, we directly tested for
interaction of CD161 with V
24invt T cell p56Lck by immunoprecipitation and subsequent Western blotting.
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In preliminary experiments, it was confirmed that murine NK1+ T cell hybridoma DN32.D3 (7) did show association of NK1.1 with p56Lck (Fig. 5 B). Human p56Lck was
also expressed by DN V24invt T cells, and Con A precipitation of Triton X-100 lysates followed by Western blot
showed that p56Lck was constitutively associated with glycoprotein(s) (Fig. 5 C). However, CD161 immunoprecipitates did not contain detectable p56Lck (Fig. 5 C). Furthermore, in the reciprocal experiment in which Triton X-100
lysates were immunoprecipitated with p56Lck antibody and
immunoblotted with CD161 mAb, there was also no detectable association of CD161 with p56Lck (Fig. 5 D). We
conclude that p56Lck was not stably associated with CD161
in V
24invt T cells. Taken together, the results presented
support the model that human CD161 functions as a novel
costimulatory molecule for human V
24invt T cells.
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Discussion |
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CD161+ V24invt T cells are likely to play an important
immunoregulatory role (28, 31), presumably through
interactions with CD1d+ target cells (19). However, it is
unclear how activation and effector functions of this T cell
population in response to CD1d recognition are regulated.
By analogy with conventional MHC-restricted T cells, it
appears likely that activation of V
24invt T cells is regulated
by the engagement of accessory molecules on the T cell
surface by specific ligands expressed by appropriate target
cells. In the absence of CD4 and CD8
and with highly variable levels of CD28, therefore, CD161 and other related molecules were investigated as potential costimulatory
or accessory molecules for V
24invt T cells. The results reported here indicate that CD161-ligand interactions positively regulate CD161+ V
24invt T cell activation.
CD161, the single known human NKR-P1 molecule,
which was first characterized on NK cells and some T cell
populations (36), is expressed at high levels by V24invt T
cells (18). In contrast to results in the mouse (39), anti-CD161 mAb did not directly activate human V
24invt T
cells. However, activation with limiting quantities of anti-CD3 mAb revealed costimulatory activity of CD161 ligation for CD1d-reactive V
24invt T cell proliferation and
cytokine secretion. Phorbol ester lowered the threshold for
CD3 activation, but did not substitute for CD161 costimulation, implying that the latter activity was not solely protein kinase C-dependent. Furthermore, unlike with murine NK1+ T cells, CD161 ligation did not alter the pattern
of cytokines produced by V
24invt T cells. Antibody-mediated blocking showed that CD161 costimulation was necessary for CD1d-dependent V
24invt T cell proliferation
and cytokine secretion, as both of these activities were specifically inhibited by all three CD161 mAbs tested. Thus, a
direct role was demonstrated for CD161 in the response of
V
24invt T cells to a physiological ligand, CD1d.
Other NK locus-encoded C-type lectin molecules, CD69
and CD94, were also expressed by most but not all (in the
case of CD94) of the V24invt CD1d-reactive T cell clones
derived from two individual donors. CD94 expression by
V
24invt T cells appears to be variable between donors, and
may be relatively uncommon in vivo (18). The V
24invt T
cell clones in this study retained expression of CD69 up to 4 mo after stimulation. CD69 expression of freshly isolated
V
24invt T cells from several donors was low (18), and
might also be elevated by in vitro culture. Alternatively,
expression may vary between different donors, since multiple independently raised V
24invt T cell clones from additional donors were also constitutively CD69+ and variable
with respect to CD94 expression (S.B. Wilson, personal communication). Conversely, another NK cell marker,
CD56, may be expressed on V
24invt T cells in situ (20),
although it is absent from the established clones we have
studied (19).
Those V24invt T cells that expressed CD94 showed costimulation with CD94 mAb. An mAb against a third recently cloned molecule, p40 (45, 50), was mildly costimulatory in the absence of PMA for both V
24invt and control
T cells expressing this antigen. Similarly, CD28, the classic
costimulatory molecule of conventional T cells, was consistently only weakly costimulatory on CD28+ V
24invt T
cells in the absence of PMA, and had no detectable effect in the presence of PMA. In contrast to the results with
CD161, neither CD94, p40, nor several other candidate
accessory molecules tested appeared central to V
24invt T
cell activation in response to CD1d. None of the three
CD94 mAbs tested had consistent effects on CD1d-dependent V
24invt T cell proliferation or cytokine secretion.
CD28 mAb did not block CD1d-dependent V
24invt T
cell responses, even of those clones that expressed significant levels of this molecule. Furthermore, CTLA4-Ig fusion protein did not block CD1d-dependent T cell activation (S.B. Wilson, personal communication). However,
the requirement for PMA in the CD1d recognition assay
suggests that additional costimulatory signals must be provided concurrently with CD161 ligation for activation of
resting V
24invt T cells in response to CD1d. It is known
that human CD1b- and CD1c-restricted T cells use a
CD28-independent costimulatory pathway (51), and this
appears to be independent of CD161 (M. Exley and S. Porcelli, unpublished observations).
The primary effector function associated with V24invt T
cells has been production of Th1 and Th2 cytokines. Murine hepatic NK1+ T cells have also been shown to have
NK-like cytolytic activity (29), but whether they can mediate CD1d-restricted cytolysis has not been determined.
Furthermore, murine NK1+ T cells directly mediate antitumor effects through a cytotoxic mechanism that appears
to be CD1d-independent (30). This report demonstrates
that an additional effector function for human V
24invt T
cells is direct CD1d-restricted cytolysis. The major mechanism of this effector activity appears to be cytolytic granule
release, based on Ca2+ dependence. Significantly, this activity was PMA-independent and was not affected by
CD161 mAb. These results likely reflect the less stringent
requirements for triggering of the cytolytic effector function of activated T cells than for full activation of resting
cells. The inability of CD161 mAb to block cytolytic activity provides evidence against CD161 functioning as a coreceptor for CD1d recognition, analogous to the role of CD4
and CD8, since CD8 mAb routinely inhibits conventional
cytolytic T cells. In contrast, this suggests a parallel with
other costimulatory molecules such as CD28, which are
not required for cytotoxic T cell lysis of target cells. Alternatively, the V
24invt TCR could have very high affinity
for CD1d, which can eliminate the need for coreceptor ligation for cytolysis, as has been described for some CD8-independent cytolytic T cells (52).
To further assess how CD161 contributes to activation
of V24invt T cells, association between CD161 and p56Lck
was assessed. Rodent NKR-P1C (NK1) is directly stimulatory for both NK cells and NK1+ T cells (3, 13, 39, 40)
and can associate with p56Lck via a cytoplasmic tail motif
CXCP/S/T (47), as used by CD4 and CD8 (48). However, human CD161 does not contain this motif, and mAbs
against this molecule do not directly activate nor do they
block classical human NK cell cytolysis (36). Human
V
24invt T cell CD161 did not detectably associate with
p56Lck using detergent conditions (1% Triton X-100),
which readily confirmed the murine NK1.1-p56Lck interaction. Based on lack of association of human CD161 with V
24invt T cell p56Lck, and by functional analogy with the
classical costimulatory molecule CD28, we propose that
human CD161 ligation results in activation of another signaling molecule. Interestingly, the response of V
24invt T
cells to CD1d transfectants in vitro is PMA-dependent, and CD161 can still provide a costimulatory signal in the presence of PMA, suggesting that the CD161 costimulatory
signal does not depend solely on classical protein kinase C
molecules. Murine NK1 also associates with the FcR
chain in both NK cells and NK1+ T cells (40), providing
an alternate mechanism for recruitment of signal transducing complexes. Further characterization of CD161-associated signal-transducing molecules should provide a molecular mechanism for the involvement of CD161 in positive
regulation of human V
24invt T cell responses to CD1d.
The blocking of V24invt T cell responses to CD1d+ target cells by CD161 antibodies indicates that these target
cells and their physiological CD1d+ counterparts in vivo can
express CD161 ligand(s). Although CD161 is a member of
the C-type lectin superfamily, it is not clear that carbohydrate
alone can be the ligand. As discussed above, one possible
CD161 ligand is CD1d itself. In this model, CD161 contributes to CD1d recognition directly as a coreceptor (8), as
CD4 and CD8 bind MHC class II and I molecules, respectively. An alternative suggested above is that human CD161
acts like CD28 and binds a true costimulatory ligand on
physiological CD1d+ target cells. CD1d on CHO cells is
insufficient to activate V
24invt T cells without mild glutaraldehyde fixation (19), which has been found in other
systems to artificially substitute for costimulatory signals
(51, 53). Similarly, fixation markedly increases V
24invt T
cell response to CD1d+ Hela transfectants, but not to the B
lymphoblastoid cells used in this study (M. Exley, unpublished observations). Therefore, such a CD161 costimulatory ligand may only be expressed by certain cell types.
In summary, we have found that human CD161 functions
not as a direct stimulatory structure but as a costimulatory
molecule for human V24invt T cell responses to their
physiological ligand, CD1d. Activation of resting CD161+
V
24invt T cells via the TCR in combination with signals
mediated by CD161 ligation led to proliferation, and both
Th1- and Th2-type cytokine secretion. However, the potent granule-mediated CD1d-restricted cytotoxic activity
of preactivated V
24invt T cells was CD161-independent.
The costimulation of CD1d recognition through CD161
appears to reflect a different mechanism for activation of
human V
24invt T cells compared with rodent NK cells
and NK1+ T cells, for both of which NKR-P1 is directly
stimulatory and associated with p56Lck (3, 4, 37, 38, 48).
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Footnotes |
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Address correspondence to M. Exley, Cancer Biology, Hematology/Oncology, HIM 1047, Beth Israel-Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Phone: 617-667-0982; Fax: 617-667-0610; E-mail: mexley{at}bidmc.harvard.edu
Received for publication 8 April 1998 and in revised form 12 June 1998.
For antibody and/or cell reagents, we wish to thank Drs. A. Bendelac, J. Hansen, R. Kurrle, L. Lanier, A. Lanzavecchia, M. Lopez-Botet, D. Olive, A. Poggi, E. Reinherz, M. Robertson, and J. Ritz. We would also like to thank Drs. S.B. Wilson, S. Kent, R. Blumberg, and our colleagues in the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, especially J. Gumperz and D.B. Moody, for unpublished results, advice, or comments on the manuscript.This work was supported by National Institutes of Health grants AI-40135 (to S. Porcelli) and AI-33911 (to S. Balk), and by grants from the Arthritis Foundation (Investigator Award, to S. Porcelli) and the American Cancer Society (to S. Porcelli).
Abbreviations used in this paper
CHO, Chinese hamster ovary;
DN, CD4/
CD8 double negative;
V24invt, V
24J
Q TCR-expressing.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Giorda, R.,
E. Weisberg,
T. Ip, and
M. Trucco.
1992.
Genomic structure and strain-specific expression of the natural
killer cell receptor NKR-P1.
J. Immunol.
149:
1957-1963
|
2. |
Ryan, J.,
E. Turck,
E. Niemi,
W. Yokayama, and
W. Seaman.
1992.
Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation
markers.
J. Immunol.
149:
1631-1638
|
3. | Ryan, J., E. Niemi, M. Nakamura, and W. Seaman. 1995. NKR-P1A is a target-specific receptor that activates NK cell cytotoxicity. J. Exp. Med. 181: 1911-1915 [Abstract]. |
4. | Raulet, D., and W. Held. 1995. Natural killer cell receptors: the offs and ons of NK cell recognition. Cell. 82: 697-700 [Medline]. |
5. | Bendelac, A., N. Killeen, D. Littman, and R. Schwartz. 1994. A subset of CD4+ thymocytes selected by MHC class I molecules. Science. 263: 1774-1778 [Medline]. |
6. |
Ohteki, T., and
H. MacDonald.
1994.
Major histocompatibility complex class I related molecules control the development of CD4+8![]() ![]() ![]() ![]() ![]() |
7. |
Lantz, O., and
A. Bendelac.
1994.
An invariant T cell receptor ![]() ![]() ![]() |
8. |
MacDonald, H..
1995.
NK1.1+ T cell receptor-![]() ![]() |
9. | Bendelac, A., M. Rivera, S. Park, and J. Roark. 1997. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu. Rev. Immunol. 15: 535-562 [Medline]. |
10. |
Masuda, K.,
Y. Makino,
J. Cui,
T. Ito,
T. Tokuhisa,
Y. Takahama,
H. Koseki,
K. Tsuchida,
T. Koike,
H. Moriya, et al
.
1997.
Phenotype and invariant ![]() ![]() ![]() |
11. | Bendelac, A., O. Lantz, M. Quimby, J. Yewdell, J. Bennink, and R. Brutkiewicz. 1995. CD1 recognition by mouse NK1+ T lymphocytes. Science. 268: 863-865 [Medline]. |
12. | Bix, M., and R. Locksley. 1995. Natural T cells. Cells that co-express NKRP-1 and TCR. J. Immunol. 155: 1020-1025 [Medline]. |
13. |
Chen, H., and
W. Paul.
1997.
Cultured NK1+ CD4+ T
cells produce large amounts of IL-4 and IFN-![]() |
14. |
Porcelli, S.,
C. Yockey,
M. Brenner, and
S. Balk.
1993.
Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD4![]() ![]() ![]() ![]() ![]() ![]() |
15. |
Dellabona, P.,
G. Casorati,
B. Freidli,
L. Angman,
F. Sallusto,
A. Tunnacliffe,
E. Roosneek, and
A. Lanzavecchia.
1993.
In
vivo persistence of expanded clones specific for bacterial antigens within the human T cell receptor ![]() ![]() ![]() ![]() |
16. |
Dellabona, P.,
E. Padovan,
G. Casorati,
M. Brockhaus, and
A. Lanzavecchia.
1994.
An invariant V![]() ![]() ![]() ![]() ![]() |
17. |
Porcelli, S.,
D. Gerdes,
A. Fertig, and
S. Balk.
1996.
Human
T cells expressing an invariant V![]() ![]() ![]() ![]() |
18. |
Exley, M.,
J. Garcia,
S. Balk, and
S. Porcelli.
1997.
Requirements for CD1d recognition by human invariant V![]() ![]() |
19. |
Davodeau, F.,
M. Peyrat,
A. Necker,
R. Dominici,
F. Blanchard,
C. Leget,
J. Gaschet,
P. Costa,
Y. Jacques,
A. Godard, et al
.
1997.
Close phenotypic and functional similarities between human and murine ![]() ![]() ![]() |
20. |
Prussin, C., and
B. Foster.
1997.
TCR V![]() ![]() |
21. | Yoshimoto, T., A. Bendelac, C. Watson, J. Hu-Li, and W. Paul. 1995. Role of NK1.1+ T cells in a Th2 response and in immunoglobulin E production. Science. 270: 1845-1847 [Abstract]. |
22. | Bendelac, A.. 1995. Mouse NK1.1+ T cells. Curr. Opin. Immunol. 7: 367-374 [Medline]. |
23. |
Brown, D.,
D. Fowell,
D. Corry,
T. Wynn,
N. Moskowitz,
A. Cheever,
R. Locksley, and
S. Reiner.
1996.
![]() |
24. |
Zhang, Y.,
K. Rogers, and
D. Lewis.
1996.
![]() |
25. |
Smiley, S.,
M. Kaplan, and
M. Grusby.
1997.
Immunoglobulin E production in the absence of IL-4-secreting CD1-
dependent cells.
Science.
275:
977-980
|
26. | Chen, Y., N. Chiu, M. Mandal, N. Wang, and C. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity. 6: 459-468 [Medline]. |
27. | Mendiratta, S., W. Martin, S. Hong, A. Boesteanu, S. Joyce, and L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity. 6: 469-477 [Medline]. |
28. | Takeda, K., and D. Lennert. 1993. The development of autoimmunity in C57BL/6 lpr mice correlates with the disappearance of NK1+ cells: evidence for their suppressive action on bone marrow stem cell proliferation, B cell immunoglobulin secretion, and autoimmune symptoms. J. Exp. Med. 177: 155-164 [Abstract]. |
29. |
Takahashi, M.,
K. Ogasawara,
K. Takeda,
W. Hashimoto,
H. Sakihara,
K. Kumagai,
R. Anzai,
M. Satoh, and
S. Seki.
1996.
LPS induces NK1.1+ ![]() ![]() |
30. |
Cui, J.,
T. Shin,
T. Kawano,
H. Sato,
E. Kondo,
I. Toura,
Y. Kaneko,
H. Koseki,
M. Kanno, and
M. Taniguchi.
1997.
Requirement for V![]() |
31. |
Sumida, T.,
A. Sakamoto,
H. Murata,
Y. Makino,
H. Takahashi,
S. Yoshida,
K. Nishioka,
I. Iwamoto, and
M. Taniguchi.
1995.
Selective reduction of T cells bearing invariant
V![]() ![]() |
32. |
Mieza, M.,
T. Itoh,
J. Cui,
Y. Makino,
T. Kawano,
K. Tsuchida,
T. Koike,
T. Shirai,
H. Yagita,
A. Matsuzawa, et al
.
1996.
Selective reduction of V![]() |
33. | Gombert, J., A. Herbelin, E. Tancrede-Bohin, M. Dy, C. Carnaud, and J. Bach. 1996. Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse. Eur. J. Immunol. 26: 2989-2998 [Medline]. |
34. |
Baxter, A.,
S. Kinder,
K. Hammond,
R. Scollay, and
D. Godfrey.
1997.
Association between ![]() ![]() ![]() ![]() |
35. |
Wilson, S.B.,
S. Kent,
K. Patton,
T. Orban,
R. Jackson,
M. Exley,
S. Porcelli,
D. Schatz,
M. Atkinson,
S. Balk, et al
.
1998.
Extreme Th1 bias of regulatory V![]() ![]() |
36. |
Lanier, L.,
C. Chang, and
J. Phillips.
1994.
Human NKR-P1A. A disulfide-linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes.
J.
Immunol.
153:
2417-2428
|
37. | Lanier, L.. 1997. Natural killer cells: from no receptors to too many. Immunity. 6: 371-378 [Medline]. |
38. | Long, E., and W. Seaman. 1997. Natural killer cell receptors. Curr. Opin. Immunol. 9: 344-350 [Medline]. |
39. |
Arase, H.,
N. Arase, and
T. Saito.
1996.
Interferon ![]() |
40. |
Arase, N.,
H. Arase,
S. Park,
H. Ohno,
C. Ra, and
T. Saito.
1997.
Association with FcR![]() |
41. |
Zemmour, J.,
A. Little,
D. Schendel, and
P. Parham.
1992.
The HLA-A, -B negative mutant cell line C1R expresses a
novel HLA-B35 allele which also has a point mutation in the
translation initiation codon.
J. Immunol.
148:
1941-1948
|
42. |
Porcelli, S.,
M. Brenner,
J. Greenstein,
S. Balk,
C. Terhorst, and
P. Bleicher.
1989.
Recognition of cluster of differentiation 1 antigens by human CD4![]() ![]() |
43. |
Porcelli, S.,
C. Morita, and
M. Brenner.
1992.
CD1b restricts
the response of human CD4![]() ![]() |
44. |
Exley, M.,
L. Varticovski,
M. Peter,
J. Sancho, and
C. Terhorst.
1994.
Association of phosphatidylinositol 3-kinase with
a specific sequence of the T cell receptor ![]() |
45. | Poggi, A., N. Pella, L. Morelli, F. Spada, V. Revello, S. Sivori, R. Augugliaro, L. Moretta, and A. Moretta. 1995. p40, a novel surface molecule involved in the regulation of non-major histocompatibility complex-restricted cytolytic activity in humans. Eur. J. Immunol. 25: 369-376 [Medline]. |
46. | Valiente, N., and G. Trinchieri. 1993. Identification of a novel signal transduction surface molecule on human cytotoxic lymphocytes. J. Exp. Med. 178: 1397-1406 [Abstract]. |
47. | Poggi, A., A. Rubartelli, L. Moretta, and M.R. Zocchi. 1997. Expression and function of NKRP1A molecule on human monocytes and dendritic cells. Eur. J. Immunol 27: 2965-2970 [Medline]. |
48. | Campbell, K., and R. Giorda. 1997. The cytoplasmic tail of rat NKR-P1 receptor interacts with the N-terminal domain of p56Lck via cysteine residues. Eur. J. Immunol. 27: 72-77 [Medline]. |
49. | Turner, J., M. Brodsky, B. Irving, S. Levin, R. Perlmutter, and D. Littman. 1990. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell. 60: 755-765 [Medline]. |
50. | Meyaard, L., G.J. Adema, C. Chang, E. Woollatt, G.R. Sutherland, L.L. Lanier, and J.H. Phillips. 1997. LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes. Immunity 7: 283-290 [Medline]. |
51. |
Behar, S.,
S. Porcelli,
E. Beckman, and
M. Brenner.
1995.
A
pathway of costimulation that prevents anergy in CD28![]() |
52. | Eshima, K., T. Suzuki, H. Yamazaki, and S. Shinohara. 1997. Co-receptor-independent signal transduction in a mismatched CD8+ major histocompatibility complex class II-specific allogeneic cytotoxic T lymphocyte. Eur. J. Immunol. 27: 55-61 [Medline]. |
53. | Rhodes, J., H. Chen, S. Hall, J. Beesley, D. Jenkins, P. Collins, and B. Zheng. 1995. Therapeutic potentiation of the immune system by co-stimulatory Schiff-base-forming drugs. Nature. 377: 71-75 [Medline]. |