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*
Sección de Inmunología, Hospital de la Princesa, Madrid, Spain;
Departamento de Bioquímica B e Inmunología, Universidad de Murcia, Murcia, Spain; and
Sección de Inmunología, Hospital Vírgen de la Arrixaca, Murcia, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß or 
T cell clones. Most of the
CD94+NKG2A- T cells have a low to intermediate
expression of CD94 Ag. The cross-linking of the CD94/NKG2 heterodimer
in one of these CD8 ß CD94+NKG2A- T cell
clones (K14B06) was able to: 1) increase the intracellular
concentration of Ca2+, 2) induce the up-regulation of CD25
Ag expression and the secretion of IFN-, and 3) trigger redirected
cytotoxicity in a TCR-independent manner. This activatory property was
not shared by any other costimulatory molecule expressed by the K14B06
T cell clone, including CD8, CD28, CD45, CD69, or CD2 Ags. The
immunoprecipitation of CD94 heterodimer showed a 39-kDa band
with a similar m.w. to the activatory heterodimer found on some NK
clones. A novel form of the NKG2 family (NKG2H) was identified in
K14B06. NKG2H protein represents an alternative spliced form of the
NKG2E gene, displaying a charged residue in the transmembrane portion
and a cytoplasmic tail that lacks immunoreceptor tyrosine-based
inhibitory motifs. The expression of NKG2H in the cell membrane
through its association to CD94 and DAP-12 molecules supports that it
could form part of the activatory CD94/Kp39 heterodimer present on
K14B06 cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain (7). The second family of receptors
for MHC class I molecules is composed of type II proteins with a C-type
lectin domain. CD94 glycoprotein belongs to this group and is able
to covalently assemble to distinct C-type lectins of the NKG2 family
(8). Whereas the CD94/NKG2A heterodimer constitutes an inhibitory
receptor (9, 10), the association of CD94 with the NKG2C protein,
highly homologous to NKG2A but lacking ITIM motifs in the intracellular
domain, displays a triggering role (11). Both CD94/NKG2A and CD94/NKG2C
heterodimers recognize the nonclassical HLA-E class I molecule,
although the HLA-E-bound peptides influence the fine recognition
of the heterodimers (12, 13, 14). A subset of activated T lymphocytes expresses NK class I-specific receptors that are capable of either inhibiting or costimulating T cell activation triggered via CD3/TCR complex (15, 16, 17, 18). In contrast to NK cells, the engagement of Ig-SF NK-type activatory receptors present on T cells does not trigger redirected cytotoxicity, although it alters the strength of the T cell response at a given Ag dose (16, 17, 18).
In this paper, we have studied the functional consequences of the
aggregation of CD94 heterodimers on T lymphocytes. We identified one
ß CD8+ T cell clone (TCC; K14B06) that expresses an
activatory CD94 heterodimer that triggers redirected lysis in a
TCR-independent manner. In addition, the cross-linking of this
activatory receptor with anti-CD94 mAb increased the intracellular
concentration of Ca2+ and induced the up-regulation of CD25
Ag expression, as well as the secretion of IFN-. The
immunoprecipitation of the heterodimer showed that CD94 was covalently
linked to a 39-kDa glycoprotein. The specificity of this activating
CD94-heterodimer does not seem to coincide with that of CD94/NKG2C
present on NK cells since RMA-S cells stably transfected with HLA-E,
which was stabilized in the cell surface with an appropriate
nonapeptide, were not killed by K14B06 TCC. The identification of a
novel NKG2 molecule, named NKG2H, that is transported to the membrane
together with CD94 and DAP-12 suggests that the CD94/NKG2H heterodimer
could be an activatory receptor present on the surface of K14B06 with a
still unidentified ligand specificity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture medium was RPMI 1640 (Life Technologies Laboratories, Grand Island, NY) supplemented with 2 mM L-glutamine (Life Technologies), 10% FCS (Life Technologies), and antibiotics. Recombinant IL-2 (gift of Hoffmann-La Roche, Nutley, NJ) was used in a range between 5 and 25 U/ml. The following murine mAb to human Ags were used: anti-human IL-2R (anti-CD25) from Becton Dickinson (Berenbodegem, Belgium); T3b (19) (anti-CD3), TS2/18 (anti-CD2), TP1/40 (anti-CD11a), TP 1/36 (anti-CD43), HP2/9 (anti-CD44), HP2/19 (anti-CD50), D3/9 (anti-CD45), and TP 55.3 (anti-CD69) were a gift from Dr. F. Sánchez-Madrid (Hospital de la Princesa, Madrid, Spain) (20, 21); 3B1 (anti-CD94) was previously described (22); Z199 was kindly provided by Dr. Moretta (Università di Brescia, Brescia, Italy) (23); HP-F1 (anti-ILT2) was previously described (24); 9.3 mAb (anti-CD28) was kindly donated by Bristol-Myers (Seattle, WA); 3A1 (anti-CD7) was obtained from the American Type Culture Collection (ATCC, Manassas, VA); IOL48 (anti-CD48) and IOL54 (anti-CD54) were from Immunotech (Luminy, France); and PE-conjugated anti-mouse F(ab')2 IgG was from Caltag (San Francisco, CA).
Cell cultures
Human lymphoblastoid cell lines GUS, LG-2, and LG-15
(EBV-transformed normal human B cells), RMA-S cell line stably
transfected with HLA-E (RMA-S/HLA-E) (generous gift from Dr. E. Weiss,
Universitat Monehen, Munich, Germany), and P815 (murine cell
lines) were grown in RPMI 1640 medium supplemented with 10% FCS, 2 mM
L-glutamine, and 1% penicillin-streptomycin. MLC and
cloning were performed according to a method described elsewhere (20, 21). In brief, 106 responder cells were stimulated with
5 x 104 irradiated (6000 rad) lymphoblastoid cell
lines (LCL) and 106 irradiated (4000 rad) spleen cells as
feeder cells. Ten days after stimulation, responding cells were cloned
by limited dilution. Cells were seeded at 5, 2.5, 1.2, 0.6, and 0.3
cells per well in round-bottomed or Terasaki microwells containing
2 x 104 irradiated LCL and 2 x 104
irradiated spleen cells in the presence of 100 U/ml of IL-2.
Proliferating clones derived from wells seeded with a number of cells
that resulted in 
63% growing wells were further expanded by seeding
104 cloned cells in round-bottom wells containing feeder
cells and IL-2. KK2, H6.6, and K14B06 TCC were derived from PBMC; B107A
and HP10.21 TCC were derived from spleen cells; and P72C, P96D, and 53B
TCC were derived from fetal thymus. In some experiments, cells were
used 2–4 wk from the last stimulation (CD25low, quiescent
cells) (20).
Immobilized or soluble Ab stimulation assay
To immobilize stimulating Ab, 96-well flat-bottom plates were precoated with sheep anti-mouse IgG (7.5 µg/ml in 0.1 M Tris-HCl buffer (pH 8.2), 40 µl/well) (Sigma, St. Louis, MO) by overnight incubation at 4°C. Unbound anti-mouse Ab was removed by washing the plate three times with saline solution (SS). Afterward, the appropriate Ab was allowed to bind by adding 50 µl of Ab-containing culture supernatants or Ab solutions. Abs were incubated for 1 h at room temperature, and the plates were washed three times with SS. Once plates were coated with stimulating mAb, TCCs were added in medium containing IL-2 (10 U/ml) at 0.5–1 x 106 cells/well.
Immunofluorescence analysis
TCC were analyzed by one- or two-color immunofluorescence after staining with appropriate mAbs. Cells were analyzed on a FACS cytofluorometer (Becton Dickinson) by using LYSIS II program (Becton Dickinson).
Cytotoxicity assays
51Cr-release cytolytic assays were performed in V-bottom microtiter plates as described (25). Briefly, 5 x 103 51Cr-labeled target cells were incubated with varying amounts of effector cells for 4 h at 37°C. The percentage of specific release (SR) was calculated as follows: SR = 100 x [(E - C)/(T - C)], in which Eis cpm release by incubating targets with T cells, C is cpm release from targets incubated with medium alone, and Tis the total cpm releasable from targets with 0.05 M HCl. Redirected lysis assays were performed as above, except that P815 cells were preincubated for 30 min at room temperature with particular mAbs.
Peptide binding to HLA-E
Peptide loading of HLA-E molecules on RMA-S cells was conducted as described (14). Briefly, RMA-S/HLA-E cells were incubated for 18 h at 26°C in the presence of 100 µCi/ml Na251CrO4, and 100 µM of synthetic peptides solubilized in DMSO. Samples were used as targets in cytotoxic assays (see above). In parallel, cells were washed, stained with HLA-E-specific (3D12) mAb, and analyzed by flow cytometry.
IL-4 and IFN- secretion
IL-4 and IFN- were measured in culture supernatants by
sandwich ELISA method following the manufacturer’s instructions
(PharMingen, San Diego, CA.), using recombinant human ILs as standards,
purified anti-IL-4 (8D4-8) and anti-IFN- (N1B42) as coating
mAbs, and biotinylated anti-IL-4 (MP4-25D2) and anti-IFN-
(4S.B3) as detection mAbs. Briefly, serial cytokine standards dilution
in 3% BSA-PBS and undiluted supernatants harvested from T cell culture
after 24 h of stimulation were incubated overnight with constant
shaking at 4°C on mAb-coated plates. A 45-min incubation period with
biotinylated mAb (0.5 µg/ml) in 3% BSA-PBS and a subsequent 30-min
incubation with avidin-peroxidase (Sigma) were used for detection at
405 nm, using 2,2'-azino-bis(ethylbenzthiazoline-6-sulfonic-acid)
substrate (ABTS; Sigma). Statistical analysis was conducted using the
Kruskal-Wallis test to evaluate the homogeneity of means between
groups.
Measurement of the (Ca2+)i
(Ca2+)i was determined with the fluorescent Ca2+ indicator Quin 2-AM (Calbiochem, La Jolla, CA). Briefly, 107 T cells/ml were incubated for 45 min at 37°C in RPMI 1640 with 10% FCS and 25 µM Quin 2-AM. After this incubation, cells were washed twice and resuspended in RPMI 1640 (5 x 107 cel/ml). T cells (107) were added to 1.3 ml of an electrolitic solution (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.9 mM MgCl2, 25 mM glucose, 16 mM HEPES, and 6 mM Tris (pH 7.2)) and transferred to a fluorometer quartz cuvette, stirred, and heated at 37°C. Fluorescence was monitored in a Perkin-Elmer (Norwalk, CT) LS-5 Luminiscence spectrometer with an excitation wavelength of 339 nm and an emission wavelength of 492 nm. (Ca2+)i was determined as described (26), by using the formula (Ca2+)i nM = 115 [(F - Fmin)/(Fmax - F)], where F indicates the fluorescence at the unknown (Ca2+)i, Fmax is the fluorescence after the Quin 2-AM is released by the addition of 0.05% Triton X-100, and Fmin is the remaining fluorescence after chelating Ca2+ with 100 mM EGTA. Triggering of K14B06 TCC was achieved by adding 5 µg/ml of mAb, followed by the addition of 20 µg/ml polyclonal sheep anti-mouse IgG (Sigma).
Radiolabeling and immunoprecipitation
Cells were surface-labeled with 125I (Amersham, Buckinghamshire, U.K.) and lysed, and solubilized proteins were immunoprecipitated as described (9). Samples were analyzed by SDS-PAGE, followed by autoradiography.
RT-PCR
Total RNA was extracted from K14B06 TCC by the acidic phenol method (27). Reverse transcription generating first strand cDNA was performed using 1 µg of total RNA and random hexamers in a total volume of 20 µl. First strand cDNA (5 µl) were used as template for PCR with specific primers. Primers used were: NKG2C (5'-ATGAGTAAACAAAGAGGAACCTTC-3'), which contains the ATG initiation codon; NKG2#3 (5'-CTCTAAAGCTTATGCTTACAATGAT-3'), which spans NKG2C termination codon; and NKG2X3'UT (5'-AAATAACACAATTCATTTTAAGGC-3'). PCR products were subcloned in pCR2.1 vector (Invitrogen, Carlsbad, CA) and sequenced. To construct the chimeric pEGFP-NKG2H vector, NKG2H cDNA was excised from pCR2.1 with BamHI and EcoRV restriction enzymes and subcloned into BglII and SmaI sites of pEGFP-C1 polycloning site (Clontech, Palo Alto, CA). DAP-12 cDNA was PCR amplified from NK cells according to published sequence (7) and subcloned into XbaI and EcoRI sites of pJFE14 vector.
For fluorescence experiments COS-7 cells were seeded onto glass coverslips and transfected using the DEAE-Dextran method as described (9). After 48 h posttransfection, cells were fixed, incubated with specific mAb, washed, stained with a Cy-3-tagged goat anti-mouse IgG (Amersham), and fluorescence analyzed. Images were acquired with a COHU high performance CCD camera (Chou, Tokyo, Japan) coupled to the microscope and connected to a Leica (Cambridge, U.K.) Q550CW workstation. Images were visualized, processed, and stored by using Leica QFISH software version VI-01.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Redirected lysis assays have been extensively used to show that
the aggregation of the CD94/NKG2A heterodimer (recognized by the mAb
Z199 (23)) inhibits the redirected cytotoxicity of NK clones, whereas
the cross-linking of CD94 stimulates the lysis of target cells by
CD94+Z199- NK clones (28). We studied the
functional consequences of these procedures on CD94+ ß
and TCC. In accordance with previous data (15, 29), the addition
of anti-CD94 did not trigger redirected lysis in the absence of
anti-CD3 or PHA on the TCC bearing the inhibitory CD94/NKG2A
heterodimer (CD94+Z199+ TCC), although it was
able to inhibit the redirected lysis of P815 in the presence of
anti-CD3 or PHA in some of these clones (3 TCC of 15 ß
and TCC tested (data not shown)). In contrast, one in eight
CD94+Z199- TCC tested (named K14B06) killed
the P815 cell line in the presence of anti-CD94 mAb (Fig. 1
A). The addition of
anti-CD94 mAb did not modify the mitogen or Ab-redirected lysis of
P815 by the other seven CD94+Z199- TCC tested.
It should be noted that the surface expression of CD94 on the K14B06
TCC was a stable phenotype during a prolonged culture period. To study
whether the triggering of cytotoxicity on K14B06 TCC was restricted to
the engagement of CD94 or if it was a property also shared by other
membrane molecules, the redirected lysis of P815 by K14B06 was tested
in the presence of different mAbs. As shown in Fig. 1B, none
of the mAb tested, which were specific for a wide range of membrane
molecules, was able to stimulate the activation of the TCC, suggesting
that the signals induced by the aggregation of CD94 are different
from those transduced by other costimulatory and/or accessory
molecules.
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We tested whether the aggregation of CD94 on K14B06 increased the
intracellular concentration of calcium, as shown in Fig. 1
C.
The addition of anti-mouse Abs to anti-CD94 preincubated K14B06
induced a 3-fold increase in intracellular calcium levels, attaining a
calcium increase similar to that observed upon anti-CD3
cross-linking (Fig. 1D). The addition of anti-mouse Abs
to either nontreated or to anti-CD2 preincubated K14B06 had no
effect on the intracellular concentration of Ca2+ (data not
shown).
To clarify whether the aggregation of CD94 on the K14B06 TCC had other
physiological effects besides the triggering of cytotoxicity, we added
"quiescent" CD25dim/- K14B06 cells (see
Materials and Methods) to wells with immobilized mAb. As
shown in Fig. 2A, the
aggregation of CD94 heterodimer, but not that of CD45 Ag, not only
up-regulated the expression of CD25 (Fig. 2A) but also
induced IFN- secretion (Fig. 2B). Twenty-four-hour
supernatants of anti-CD94-stimulated K14B06 TCC contained
significantly more IFN- (98.2 ± 39.1 pg/ml) than either
nonstimulated cultures (25.1 ± 22.33 pg/ml, p <
0.005) or anti-CD45-stimulated cells (22.5 ± 9.8 pg/ml,
p < 0.001), although the cytokine concentration was
significantly lower than that observed upon optimal anti-CD3
activation (241.8 ± 30.7 pg/ml, p < 0.001). None
of the stimuli tested were able to induce IL-4 secretion (data not
shown). In contrast, none of the other mAbs tested in Fig. 1B (only the anti-CD45 mAb is represented here) were
able to modify CD25 expression (Fig. 2A) or induce IFN-
production, although some of them transduced costimulatory signals. As
shown in Fig. 2A, coimmobilized anti-CD45 increased the
IFN- production (425 ± 114.59 pg/ml; Fig. 2B)
triggered by optimal concentration of plastic-bound anti-CD3
(241.8 ± 30.7 pg/ml, p < 0.005) and increased
the CD25 up-regulation induced by suboptimal concentrations of
anti-CD3 mAb (data not shown).
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It has been reported that the activatory CD94/NKG2C heterodimer
expressed on NK cells recognizes HLA-E molecules loaded with
nonapeptides derived from HLA class I signal sequences (12, 14). To
test whether the activatory CD94 heterodimer expressed by K14B06 T
cells was also able to recognize HLA-E molecules, we used the
TAP-deficient mouse cell line RMA-S transfected with HLA-E cDNA (30).
Culture of these cells in the presence of exogenous peptides derived
from certain HLA-leader sequences is able to stabilize HLA-E on the
cell surface (see Materials and Methods). Two NK clones,
which have been previously described as expressing CD94/NKG2C
activatory receptor and as recognizing HLA-E molecules loaded with
peptides derived from HLA-G1 signal sequences (14), were compared with
K14B06 TCC for their ability to recognize HLA-E on the surface of RMA-S
cells in a 51Cr-release cytotoxic assay. As shown in Fig. 3, both NK cell clones were capable of
killing cells cultured in the presence of a peptide that stabilizes
HLA-E on the cell surface (HLA-G1), but not in the presence of
B2705-derived peptide as previously described (14). In contrast, K14B06
TCC cytotoxic activity was not triggered under such experimental
conditions.
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B,
the immunoprecipitation with anti-CD94 mAb resulted in the
isolation of a 68-kDa complex in nonreducing conditions. Under reducing
conditions (Fig. 4A) a single 39-kDa band is detected,
similar to that described for the activatory form of CD94/NKG2 complex
on NK cells (28). We studied the presence of mRNA for NKG2A, NKG2C, and
NKG2E by reverse PCR in K14B06 TCC. Primers corresponding to NKG2C cDNA
sequence amplified a specific band of the expected size from K14B06 TCC
RNA. However, sequencing of this PCR product yielded a nucleotide
sequence coding a NKG2 protein that also lacked ITIM motifs in the
cytoplasmic tail, but that showed some nucleotide differences with
NKG2C and did not contain a STOP codon. This new sequence was highly
homologous to the sequence contained in a clone that had been
previously isolated in the laboratory from an NK cDNA library that was
lacking the 5' sequences coding the NKG2 cytoplasmic region. Using a 5'
primer corresponding to NKG2C cDNA spanning the start codon sequence
and a primer corresponding to the putative 3' untranslated region
sequence contained in the cDNA library clone, we were able to PCR
amplify a full-length cDNA from K14B06 TCC. The open reading frame in
this cDNA codes for a protein that we called NKG2H. Alignment of NKG2H
peptide sequence to other members of the NKG2 family is shown in Fig. 5. Comparison of NKG2H cDNA nucleotide
sequence with NKG2 genomic sequences revealed that NKG2H is, in fact,
an alternative spliced form of NKG2E in which intron VI has not been
removed and that does not contain exon VII. In this case, the splice
donor site of exon VI in NKG2E gene is not utilized. Therefore, the end
of the open reading frame and the following 3' untranslated sequences
are identical to exon VI and the beginning of the intron VI of NKG2E
gene (31).
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, D
and F). In cells cotransfected with the three cDNAs, the
GFP-NKG2H chimera protein was localized to the cellular membrane (Fig. 6A). This was corroborated by staining of the cellular
surface with an anti-CD94 mAb and a red fluorochrome (Fig. 6, B and C). Similar results were obtained upon
transfection with a plasmid encoding a FLAG-NKG2H-tagged protein.
Anti-FLAG M2 mAb was able to stain the surface of NKG2H-FLAG plus
CD94-transfected cells only upon cotransfection of a DAP-12 expression
plasmid (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß TCC
triggered by the cross-linking of a CD94/kp39 heterodimer. The
engagement of CD94 heterodimers expressed on K14B06 results in early
activation events, such as Ca2+ mobilization, and in late
effector functions, such as induction of cytolytic activity and IFN-
production. These results are unexpected and differ from those
described with other T cell costimulatory/accessory molecules,
including activatory NK receptors, such as p50 molecules
(KIR2DS) (14, 16, 17) or human NKR-PA1, another type II membrane
glycoprotein of the C-type lectin superfamily (33). In these reports,
KIR2DS proteins are trigger receptors for IFN- secretion but only
acted as coactivatory receptors with regard to T cell proliferation or
killing, being capable of altering the strength of T cell response at a
given Ag dose, but never inducing cytolytic activity. None of the other
membrane molecules studied was able to activate the K14B06 TCC in a
TCR-independent manner. Therefore, the cytotoxicity triggered by the
aggregation of CD94/kp39 heterodimer on K14B06 TCC is a
property that does not seem to be shared by other
costimulatory molecules expressed on T lymphocytes.
The aggregation of the CD94/NKG2 dimers only activated redirected lysis
or up-regulated CD25 Ag in one of the eight
CD94+/Z199- TCC assayed, suggesting a high
heterogeneity either in the molecular structure of the CD94 heterodimer
or in the transduction pathway among T cells. Since we were able to
identify DAP-12 expression by RT-PCR in K14B06, but not in two other
CD94+NKG2A- TCC, the absence of the associated
DAP-12 signal transducing molecule could at least explain the
functional defect upon CD94 engagement in some
CD94+NKG2A- TCC (data not shown). However, it
is also possible that other mechanisms could account for this
functional heterogeneity. We also observed that K14B06 TCC had the
highest membrane expression of CD94 by flow cytometry analysis among
all of the CD94+NKG2A- TCC tested (data not
shown). We were unable to unequivocally identify the activatory CD94
heterodimer expressed on K14B06 TCC. The immunoprecipitation assays
showed a 39-kDa glycoprotein very similar to that found in NK cells
with CD94 activatory phenotype (28). The expression of mRNA encoding
NKG2H, an alternative spliced form of NKG2E gene that lacks the ITIMs
found on NKG2A and killer inhibitory receptors (KIR) and contains a
charged residue in its transmembrane domain, leads us to hypothesize
that this molecule could be part of the activating CD94 heterodimer
expressed on K14B06 T cells. The cotransfection experiments shown in
Fig. 6, together with the 39-kDa band immunoprecipitated in COS cells
also cotransfected with plasmids coding for CD94, DAP-12, and the
FLAG-NKG2H-tagged protein (data not shown), support this hypothesis.
Accordingly, the sequence of several PCR products obtained from K14B06
TCC with the primers used to amplify NKG2C, always yielded NKG2H
sequence. This result suggests that NKG2H mRNA should be more abundant
than NKG2C mRNA. However, we have not formally excluded that NKG2C
could also be present on K14B06 cell membrane and could be responsible
for the CD94-triggered T cell activation. The fact that HLA-E molecule
loaded with HLA-G1 peptide is not recognized by the CD94/kp39
heterodimer expressed on the K14B06 TCC (although it triggers the lysis
of RMA-S cells by two NK clones expressing the CD94/NKG2C heterodimer),
suggests that CD94/NKG2C heterodimer is either not expressed or not
fully functional upon HLA-E recognition in this TCC.
It is possible that the activatory CD94/NKG2H heterodimer expressed on
K14B06 TCC could be part of the innate recognition system, recognizing
nonself structures expressed by transformed or infected cells that do
not need to be related to MHC molecules. The secretion of IFN-, a
cytokine strongly associated with antimicrobial immunity, also suggests
that activatory CD94 heterodimer(s) present on K14B06 could react with
molecular patterns present on different microorganisms or infected
cells rather than with individual structures (34). However, the
structural similarity between NKG2H and other members of the NKG2
family opens the possibility that the ligand specificity of CD94/kp39
on K14B06 was related to that of the CD94/NKG2A or CD94/NKG2C
heterodimers (HLA-E molecules) (11, 14). To preserve self-tolerance,
the activatory CD94 heterodimer should interact either with nonself
MHC-like molecules, such as those described on some viruses, or with
MHC molecules loaded with peptides that are either shared by some
pathogens or that changed the conformation of the class I molecule in
an identical way (35, 36, 37, 38). Alternatively, the activatory CD94
heterodimer could recognize self-MHC molecules that have incorporated
self peptides. However, the TCR-independent cytotoxicity triggered by
the CD94/kp39 heterodimer on K14B06 TCC should require powerful control
mechanisms to avoid autoreactivity. The analysis of NK cells has shown
that the presence of stimulatory antiself receptors does not lead to
lysis of autologous cells if the inhibitory signals transduced by the
KIRs are the dominant controllers of the cytotoxic activity (1, 2, 3, 4).
Although K14B06 TCC express Ig-like transcript 2 (ILT2), the addition
of a mAb specific for this molecule (HP-F1) (24) was not able to
inhibit CD94-redirected lysis (data not shown). Nevertheless, other
still undefined membrane molecules could balance the
CD94/kp39-dependent cytotoxicity. Interestingly, the CD94-triggered
lysis, but not the CD3-mediated redirected cytotoxicity, was
specifically inhibited by anti-CD48 (data not shown), suggesting
that CD94/NKG2-derived activatory signals are not identical to those
triggered by TCR/CD3 engagement and can be specifically regulated.
In summary, we have described for the first time the triggering of
cytotoxicity upon CD94/kp39 heterodimer engagement on cytolytic ß
T cells and the sequence of NKG2H, a novel NKG2 family protein able to
associate to CD94. The activatory CD94/NKG2 heterodimer present on a
subset of CD8+ T cells could be involved in the innate
immunity and may function as a source of the proinflammatory cytokine
IFN-.
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Acknowledgments |
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Footnotes |
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2 These authors contributed equally to this work, and the order of authorship is arbitrary.
3 Address correspondence and reprint request to Dr. Pedro Aparicio, Departamento de Bioquímica B e Inmunología. Facultad de Medicina, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain. E-mail address:
4 Abbreviations used in this paper: Ig-SF, Ig superfamily; TCC, T cell clone; ITIM, immunoreceptor tyrosine-based inhibitory motif; KIR, killer inhibitory receptor; GFP, green fluorescent protein.
Received for publication July 30, 1998. Accepted for publication December 29, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion from T cells and natural killer cells. Proc. Natl. Acad. Sci. USA 95:3798.4ß7 integrin expression on human
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/ stimulated by nonpeptidic ligands. J. Exp. Med. 186:1769. T cells expressing NK cell receptors. J. Immunol. 159:3723.[Abstract]
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 R. Miyashita, N. Tsuchiya, K. Hikami, K. Kuroki, T. Fukazawa, M. Bijl, C. G. M. Kallenberg, H. Hashimoto, T. Yabe, and K. Tokunaga Molecular genetic analyses of human NKG2C (KLRC2) gene deletion Int. Immunol., January 1, 2004; 16(1): 163 - 168. [Abstract] [Full Text] [PDF]
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R. K. Strong, M. A. Holmes, P. Li, L. Braun, N. Lee, and D. E. Geraghty HLA-E Allelic Variants. CORRELATING DIFFERENTIAL EXPRESSION, PEPTIDE AFFINITIES, CRYSTAL STRUCTURES, AND THERMAL STABILITIES J. Biol. Chem., February 7, 2003; 278(7): 5082 - 5090. [Abstract] [Full Text] [PDF]
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N. Dulphy, C. Rabian, C. Douay, O. Flinois, S. Laoussadi, J. Kuipers, R. Tamouza, D. Charron, and A. Toubert Functional modulation of expanded CD8+ synovial fluid T cells by NK cell receptor expression in HLA-B27-associated reactive arthritis Int. Immunol., May 1, 2002; 14(5): 471 - 479. [Abstract] [Full Text] [PDF]
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B. P. Shum, L. R. Flodin, D. G. Muir, R. Rajalingam, S. I. Khakoo, S. Cleland, L. A. Guethlein, M. Uhrberg, and P. Parham Conservation and Variation in Human and Common Chimpanzee CD94 and NKG2 Genes J. Immunol., January 1, 2002; 168(1): 240 - 252. [Abstract] [Full Text] [PDF]
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A. I. Roberts, L. Lee, E. Schwarz, V. Groh, T. Spies, E. C. Ebert, and B. Jabri Cutting Edge: NKG2D Receptors Induced by IL-15 Costimulate CD28-Negative Effector CTL in the Tissue Microenvironment J. Immunol., November 15, 2001; 167(10): 5527 - 5530. [Abstract] [Full Text] [PDF]
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J. Li, I. Goldstein, E. Glickman-Nir, H. Jiang, and L. Chess Induction of TCR V{beta}-Specific CD8+ CTLs by TCR V{beta}-Derived Peptides Bound to HLA-E J. Immunol., October 1, 2001; 167(7): 3800 - 3808. [Abstract] [Full Text] [PDF]
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