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2 Domain Signals Rapid Fas-Independent Cell Death: A Direct Pathway for T Cell-Mediated Killing of Target Cells?1
Departments of
*
Pediatric Research and
Pediatrics, The National Hospital, and
Section for Immune Therapy, The Norwegian Radium Hospital, Oslo, Norway
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 domain induces apoptotic cell death. mAbs to
this region rapidly induced apoptosis of HLA-A2-expressing Jurkat E11
cells, as determined by morphologic changes, phosphatidylserine
exposure on the cell surface, and propidium iodide uptake. In contrast,
apoptosis was not induced following culture with mAbs directed to other
regions of the class I molecule. Death signaling by class I molecules
is apparently dependent on coreceptor activation, as apoptosis is also
signaled by HLA-A2 molecules, where the intracytoplasmic residues were
deleted. HLA class I 2-mediated cell death appeared to
proceed independent of the Fas pathway. Compared with apoptotic
signaling by Fas ligation, HLA class I 2-mediated
responses displayed a faster time course and could be observed within
30 min. Furthermore, class I 2-induced cell death did
not involve observable DNA fragmentation. The apoptotic response was
not affected significantly by peptide inhibitors of IL-1ß converting
enzyme (ICE)-like proteases and CPP32. Taken together, activation of
the TCR-binding domain of the class I 2 helix may result
in apoptotic signaling apparently dependent on a novel death pathway.
Thus, target HLA class I molecules may directly signal apoptotic cell
death following proper ligation by the TCR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recognition of a target cell induces a complex array of responses in
the CTL (6, 7). TCR binding to peptide ligand and allele-specific
1 and 2 residues of the class I
molecule initiates signaling through the TCR/CD3 complex. These signals
partly activate CD8 coreceptor binding to the 3 domain
of the same MHC class I molecule, thereby increasing TCR-ligand
interactions (8, 9, 10, 11, 12). Furthermore, other auxiliary molecules also seem
to coregulate CTL adhesion to target cells (13, 14).
Following proper activation of the CTL, the effector or cytolytic phase is activated to eliminate target cells (15). In this context, perforin- and Fas-mediated pathways have been recognized as the two major complementary cytotoxic pathways used by CTLs (16, 17). The Ca2+-dependent perforin pathway has been implicated in virtually all forms of cell-mediated cytotoxicity (15, 16, 17, 18, 19). Most effector CTL have perforin and granzymes stored in cytoplasmic granules that are released upon TCR signaling (6, 20). Perforin forms channels in the target cell, and it is believed that transmitted perforin and granzymes (mainly granzyme B) in concert induce apoptotic cell death (21, 22). NK cells seem to mainly, but not entirely, rely on the perforin-mediated pathway to eliminate class I-deficient target cells (18, 19, 23). In addition, highly potent peritoneal exudate lymphocytes seem to exert their cytotoxic function independent of the perforin pathway, as they contain no lytic granules (24).
Transmembrane signaling by Fas molecules is the other recognized major
pathway in CTL-mediated induction of apoptotic target cell death (16, 17). Activation of CTL rapidly induces CD95 ligand expression, and thus
enables these cells to induce cell death in Fas-expressing and
sensitive target cells (25). In contrast to the perforin pathway,
Fas-mediated apoptosis is Ca2+ independent (26).
Furthermore, in some situations CTL may also use TNF- to kill target
cells (27). However, this pathway is slower than Fas- and
perforin-dependent pathways (15).
Recent studies demonstrate that HLA class I molecules may have other
biologic functions in addition to Ag presentation, as class I molecules
may transduce regulatory signals. Thus, activation of class I molecules
may apparently result in cell activation (28, 29, 30), growth inhibition
(31, 32, 33, 34, 35, 36, 37), and cell aggregation (37, 38, 39). Furthermore, engagement of
class I 3 residues may cosignal or directly induce
apoptosis (40, 41). We have reported recently that the TCR-accessible
region of the HLA class I 2 domain may have a unique
function in class I-mediated signaling (37). It is believed that the
perforin/granzyme and Fas pathways account for all acute cytolytic
activity of CTL, and that there is no distinction between autologous
and allogeneic CD8+CTL-mediated cytotoxicity (16, 17).
There is presently no direct evidence for alternative pathways of
CTL-mediated target cell elimination. However, studies with
perforin-less mice show that these are still able to reject allogeneic
CD95- tumor cells injected i.p. as efficiently as perforin
normal mice (15). Furthermore, murine CD8+ T cells
apparently clear rotavirus infection independent of perforin and Fas
(42). Thus, these and other observations may suggest additional
cytotoxic pathway(s).
We asked whether direct activation of epitopes encompassing TCR contact
residues on HLA molecules could represent an alternative death pathway.
In this study, we demonstrate that HLA class I molecules can signal
Fas-independent cell death following specific engagement of the
TCR-binding region of the class I 2 domain.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hybridomas MA2.1 (anti-A2, B17, IgG1) (43), PA2.1 (anti-A2, w69, IgG1) (44), BB7.2 (anti-A2, w69, IgG2b) (45), W6/32 (anti-monomorphic HLA, IgG2a) (46) and OKT3(anti-CD3, IgG2a) were obtained from American Type Culture Collection (Rockville, MD). The CR11-351 hybridoma (anti-A2, 28, IgG1) (47) was generously provided by Dr. Carlo Russo (Cornell University, New York, NY). The supertypic anti-HLA-A mAb RG1 (IgG1) has been described previously (48). Anti-CD95 (phycoerythrin-conjugated DX2, IgG1) was obtained from PharMingen (San Diego, CA). Apoptosis-inducing anti-CD95 (CH11, IgM) and apoptosis-inhibitory anti-CD95 (ZB4, IgG1) were from Immunotech (Marseseille Cedex, France). Murine IgG1 (MOPC-21) control was purchased from Sigma Chemical (St. Louis, MO), and murine IgM control (TEPC 183) was obtained from BiosPacific (Emeryville, CA). IgG1-phycoerythrin control was purchased from Becton Dickinson (San Jose, CA). Human rFas-Fc chimera was obtained from R&D Systems (Abingdon, U.K.). IL-1ß-converting enzyme (ICE)3 inhibitor II Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-CMK) was from Calbiochem (La Jolla, CA). ICE inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK) and CPP32 inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) were purchased from BACHEM Feinchemikalien (Bubendorf, Switzerland). Streptavidin-FITC was from Dako (Glostrup, Denmark).
Cell culture
Hybridomas were maintained in DMEM supplemented with 17% FCS (Life Technologies, Gaithersburg, MD), 100 U/ml penicillin, and 100 µg/ml streptomycin. Human cells were cultured in RPMI 1640 supplemented with 10% FCS, 1.5 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Jurkat cells transfected with either native (Jurkat E11) or truncated HLA-A2 (Jurkat E13) genes or native (Jurkat E10) or truncated (Jurkat E12) HLA-B27 genes (49) were kindly provided by Dr. Peter E. Lipsky (The University of Texas Southwestern Medical Center at Dallas, TX). Jurkat cells were subcultured routinely every 2 to 3 days to maintain cell densities between 1 x 105 and 1 x 106 cells/ml. Experiments were performed on 5 x 105 cells/ml, unless otherwise indicated.
Human PBMC were isolated by Lymphoprep (Nycomed Pharma, Oslo, Norway) centrifugation. All cultures were grown at 37°C in a humidified 5% CO2 atmosphere.
Flow cytometry
Ag expression was determined with flow cytometry, as described (37). Cells were finally washed twice in PBS and fixed with 1% paraformaldehyde in PBS. Samples were analyzed using a FACScan (Becton Dickinson), and data were collected on 10,000 cells.
Flow-cytometric determination of apoptosis and cell death
Apoptosis was determined by monitoring changes in cell size and granularity by flow cytometry and assessment of phosphatidylserine exposure by Annexin V-FITC binding using the ApoAlert Annexin V apoptosis kit (Clontech Laboratories, Palo Alto, CA). DNA fragmentation was determined with the TUNEL assay kit from Boehringer Mannheim (Mannheim, Germany), according to the manufacturer’s instructions. Cell membrane permeability was assessed by determining uptake of the DNA-binding fluorescent dye propidium iodide (PI; 2.5 µg/ml) after incubation for 10 min.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2
epitopes signals apoptosis
CTL recognition of target cells involves TCR interactions with
peptides and specific HLA class I residues (2). X-ray crystallographic
data have recently identified amino acid residues within the class I
2 domain that make contact with the TCR (50). These
residues may be responsible for TCR/MHC class I cross-talk and
conveying signals to the target cell. Activation of class I
2 residues that interact with the TCR may initiate
target cell signaling, as evidenced by cell aggregation of most normal
and leukemic cells and growth inhibition of mitogen-stimulated T cells
following mAb stimulation of this class I region (37). In this study,
we addressed the involvement of the TCR-binding region of the HLA class
I 2 domain in signaling cell death. HLA-A2-expressing
Jurkat E11 cells (49) were used as a model system, as Jurkat
cells can be induced to undergo apoptosis by different pathways
(51, 52, 53, 54) and rapidly respond with cell signaling (i.e., cell
aggregation) following engagement of residues within the TCR contact
region of HLA-A2 molecules (data not shown).
CTL targets are believed to die from programmed cell death or apoptosis
(20). This is an active process with characteristic morphologic
changes, including cell shrinkage and nuclear condensation (55). Thus,
to examine whether apoptosis could be signaled by activation of the
class I 2 domain, we first examined the impact of
anti-HLA class I on cell size and granularity using flow cytometry.
Jurkat E11 cells were cultured with mAbs to positions within (RG1 and
CR11-351) (37, 48, 56, 57) the TCR-accessible 2 domain
of HLA-A2 molecules (2, 50) or other distant class I epitopes (MA2.1,
PA2.1, BB7.2, and W6/32) (57). These studies clearly demonstrated that
both RG1 (epitope involves residues 144 and 151) (37, 48) and CR11-351
(epitope involves residues 142, 145, and 149) (56, 57) induced a major
impact on cell morphology, as evidenced by a shift in cell size and
granularity (Fig. 1
). Within 3 h, up
to 50% of the cells could be recognized as a second population of
smaller cells compared with control cultures (Fig. 1A). In contrast, no significant influence on cell
size or granularity was observed with the anti-HLA class I mAbs
MA2.1 (involving position 62) (57, 58), PA2.1, BB7.2 (both specific for
positions 107 and 161) (57, 59, 60, 61), W6/32 (recognizing a
conformational determinant involving conserved residues on the
1 and 2 domains) (62), or isotype control
mAb MOPC-21 after 2, 3, or 5 h of cell culture (Fig. 1 and data
not shown). The effect of CR11-351 was readily observable within 60 min
(Fig. 1B). For both RG1 and CR11-351, the effect was
stronger after prolonged culture, reaching a maximum after 5 to 6
h (Fig. 1B and data not shown). Thus, engagement of
TCR-accessible epitopes on the class I 2 domain rapidly
leads to cell shrinkage and changes in granularity, as observed with
cells undergoing apoptosis.
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. Furthermore, determination of PI
uptake disclosed an increase in both the number of dead (Annexin
V-positive/PI-positive) and apoptotic (Annexin V-positive/PI-negative)
cells, as compared with control cultures (Fig. 2), indicating a rapid
progress through the apoptotic stage. In contrast, no
phosphatidylserine exposure, nor changes in cell distribution or PI
uptake were observed with the anti-HLA class I mAbs PA2.1, MA2.1,
BB7.2 or W6/32 after 2, 3, of 5 h of cell culture (Fig. 2 and data
not shown). In addition, in experiments using the HLA-A2-negative
Jurkat E10 cell line as control, no RG1 or CR11-351 effects were
observed (Fig. 2). Thus, class I molecules may apparently transduce
apoptotic signals following specific engagement of the TCR-accessible
2 region of HLA class I molecules.
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The role of the cytoplasmic domain of class I molecules in
signaling apoptotic responses has not been determined. To examine a
possible involvement of this domain in signaling programmed cell death,
RG1 and CR11-351 responses were assessed using Jurkat E13 cells
expressing truncated HLA-A2 (A21
312) (49)
molecules. These experiments showed that Jurkat E13 cells rapidly
responded to RG1 and CR11-351 incubation with both phosphatidylserine
exposure and a shift in the cell population with reduction of cell size
(Fig. 3 and data not shown). However, no
responses were observed in control cultures with MOPC-21 or PA2.1, nor
with the HLA-A2-negative Jurkat cell line E12 (Fig. 3). Thus, mAbs RG1
and CR11-351 induce apoptosis by activating an HLA class I signaling
pathway that seems to be independent of the cytoplasmic region of class
I molecules.
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2-induced apoptosis is independent of
CD95 signaling
CD95 and perforin are the two recognized major pathways for
CTL-induced target cell destruction (16, 17). To assess whether CD95
was involved in HLA class I-mediated apoptosis, we first examined the
expression of CD95 on Jurkat E11 cells using flow cytometry. These
studies revealed that Jurkat E11 constitutively expressed CD95 (data
not shown). Furthermore, to address a possible apoptotic response due
to CD95-CD95L interactions, the impact of RG1 and CR11-351 was examined
on Jurkat E11 cells preincubated with control mAb MOPC-21, neutralizing
anti-Fas mAb ZB4, or human rFas-Fc chimera. Control cultures with
CH11 (anti-Fas mAb-inducing apoptosis) were included. These
experiments clearly demonstrated that whereas ZB4 and human rFas-Fc
chimera blocked CH11 responses, they had no effect on the ability of
RG1 or CR11-351 to induce apoptosis (Fig. 4). As target cells are believed to die
rapidly following recognition by CTLs, we also compared the kinetics of
CR11-351- and CH11-induced cell death to evaluate the relative
importance of the two pathways. These assessments demonstrated that
CR11-351 induced the most rapid and pronounced induction of
phosphatidylserine exposure and cell death (Fig. 5). Thus, after 1 h, the levels of
Annexin V binding and cell death were similar to control cultures with
CH11 compared with strong induction of Annexin V binding and cell death
with CR11-351. Interestingly, a relative constant proportion of cells
(23–27%) was observed in the early/intermediate (transit) apoptotic
stage during the 2- to 4-h period. In contrast, a steady accumulation
of dead cells (27–41%) was observed in the same period. During the
same time interval, an apoptotic picture emerged with Fas activation.
In this study, cells accumulated in the early/intermediate (transit)
apoptotic stage with no significant increase in the number of dead
cells. The data in Figure 5 indicate that the class I pathway may be a
major pathway in CTL-mediated target cell killing.
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, 2, and 5, and data not shown). To further compare Fas and HLA class
I-induced apoptosis of Jurkat E11 cells, the cells were stimulated with
CH11, RG1, CR11-351, or isotype control mAbs and analyzed for DNA nicks
with the TUNEL assay. Due to a more rapid effect of RG1 and CR11-351
compared with CH11, cultures with anti-class I mAbs were analyzed
after 1, 2, 3, and 4 h, while the effect of CH11 was determined
after 3 and 4 h. These studies showed that whereas CH11 clearly
induced DNA fragmentation, only a minor increase in fluorescence
staining was observed with the anti-class I mAbs RG1 and CR11-351
(Fig. 6 and data not shown). Taken
together, these data suggest that activation of the TCR-binding
2 region of HLA class I molecules rapidly signals
apoptosis independent of the CD95 pathway. This class I pathway may
contribute significantly to target cell killing by CTL.
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ICE and CPP32 are proteases that are rapidly activated in cells
induced by different stimuli to undergo apoptosis. Following Fas
ligation, ICE-like and CPP32-like proteases are sequentially activated
(64). Fas-mediated signaling in Jurkat cells and cytoplasts, as
measured by phosphatidylserine exposure, is inhibited by peptide
inhibitors of ICE activity (65). To determine the role of ICE and
CPP32 activity in signaling by the class I 2
domain, we assessed the impact of specific ICE and CPP32 peptide
inhibitors on RG1- and CR11-351-induced responses. Control cultures
with CH11 were also included. Jurkat E11 cells were preincubated for
2 h with the ICE inhibitors Ac-Tyr-Val-Ala-Asp-chloromethyl ketone
or Z-Val-Ala-DL-Asp-fluoromethylketone or the CPP32 inhibitor
Ac-Asp-Glu-Val-Asp-aldehyde before addition of RG1, CR11-351, CH11, or
isotype control mAb, and further cultured for 4 h. Apoptosis was
determined by assessment of Annexin V-FITC binding with flow cytometry.
These experiments showed that whereas peptide inhibitors of ICE and
CPP32 profoundly inhibited Fas-mediated apoptosis, the apoptotic
responses induced by RG1 and CR11-351 were not influenced significantly
by blocking ICE or CPP32 activity (Table I). Thus, HLA class 2
mediated death signaling proceeds apparently independent of ICE and
CPP32 involvement.
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The experiments reported above have all been performed on
transformed cells. To establish a model system for normal cells, we
investigated the impact of HLA signaling on resting and activated
normal T cells. We have demonstrated previously that mAbs RG1 and
CR11-351 inhibit the proliferation of mitogen-stimulated lymphocytes,
indicating some form of negative signaling (37). To determine whether
negative signaling by the TCR-binding region of the class I
2 domain required cell proliferation, the impact of
RG1 and CR11-351 on unstimulated and mAb OKT3-stimulated PBMC was
examined. Cell size and granularity (forward scatter vs side scatter)
were examined with flow cytometry, and regions representing living (R1)
and dead or dying lymphocytes (R2) were defined based on Annexin V-FITC
binding and PI uptake (data not shown). The effects of RG1 and CR11-351
were assessed based on the increase in the number of lymphocytes in R2
compared with cultures with control mAb MOPC-21.
In three different experiments, no significant increase in cell numbers
was observed in region R2 when resting PBMC were incubated with RG1 or
CR11-351 for 24 h (Fig. 7 and data
not shown). In contrast, with cells prestimulated for 18 h with
OKT3 and then further activated for 6 h with RG1 or CR11-351, we
observed an increasing number of dead and dying T lymphocytes compared
with similar cultures with isotype control mAb (Fig. 7 and data not
shown). On the average, we found an increase in R2 of 43.5% (range,
37.3–51.2%) with RG1, 46.5% (range, 44.8–49.2%) with CR11-351, and
10.7% (range, 7.1–15.4%) with PA2.1 relative to MOPC-21. Taken
together, the cell’s state of activation appears to determine
sensitivity or resistance to death signaling by the TCR-binding region
of the class I 2 domain.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 domain can rapidly induce
morphologic changes, cell surface phosphatidylserine exposure, and cell
death independent of the Fas signaling pathway. These data support a
unique functional role of this class I region, and furthermore, suggest
that TCRs interacting with MHC class I/peptide ligands may directly
impose negative signaling in the target cell. This may represent a
novel pathway to target cell killing by CTL.
CTL activation and effector functions are subjected to a complex array
of control mechanisms and signaling events that only partly have been
determined. In the initial phase, CTL contact with the APC is
determined by both TCR affinity for peptide and the level of presented
foreign peptide/MHC complexes (6, 7, 66). Polymorphic residues on the
1 and 2 domains in adjacent positions to
the peptide-binding groove will also contribute to determine the
overall binding capacity of the TCR (2).
Sufficient TCR-MHC/peptide interaction signals activation and adhesion
of CD8 to a conserved region on the 3 domain of class I
molecules. Thus, CD8 contributes to increase the overall TCR avidity to
MHC/peptide and also acts as a cosignaling receptor (6, 7, 8, 9, 10, 11, 12). Other
costimulatory and adhesion molecules are also required for CTL
activation and cytolytic activity (6, 7, 13, 14). In this context, we
have shown recently that engagement of TCR-accessible epitopes on the
class I 2 domain induces profound cell aggregation
responses independent of LFA-1/ICAM interactions (37). Unidentified
adhesion molecules may therefore further participate in coregulation of
CTL responses.
We have focused on the role of HLA class I molecules in delivering
death signals to a potential target cell. In this context, the
signaling capacity of the TCR-binding region of the class I
2 domain has been further assessed. MHC class I
molecules have been implicated both in positive and negative signaling
(28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 41). Sambahra and Miller (40) showed that the
3 domain cosignals induction of apoptosis upon TCR
stimulation of immature T cells. Moreover, class I 3
domain activation alone may also be sufficient to induce apoptotic cell
death (41).
The mAbs RG1 and CR11-351 bind epitopes in the class I 2
region encompassing amino acids 149–151 (37, 48, 56, 57). These
residues have recently been demonstrated to be directly involved in TCR
contact with HLA-A2 using x-ray crystallography (50). We have shown
previously that this region of class I molecules may be unique in cell
signaling (37). Data presented in this work show that activation of
HLA-A2 molecules with mAbs RG1 and CR11-351 directly signals rapid
morphologic changes, phosphatidylserine exposure, and cell death in
Jurkat cells expressing HLA-A2 molecules. In contrast, none of these
effects was observed with mAbs against distant class I 1
or 2 epitopes. Thus, these results and previous
observations (35, 37) clearly demonstrate a unique ability of the
TCR-accessible region of the class I 2 domain to
transduce regulatory signals. Importantly, this strongly suggests that
the TCR can impose regulatory signals and induce cell death upon
binding the target MHC class I/peptide complex.
Previously we observed that all normal PBMC and most, but not all,
leukemic hemopoietic cell lines expressing HLA-A2 specifically
responded to RG1 and CR11-351 with profound cell aggregation.
Furthermore, mitogen-stimulated T cells responded significantly to
these mAbs with growth inhibition. In contrast, the majority of tested
leukemic cell lines were unaffected (37 and data not shown). Thus,
activation of the TCR-binding class I 2 region gave
different responses in normal and leukemic cells. The Jurkat E11 T cell
line expressing HLA-A2 molecules was found to respond very rapidly to
RG1 and CR11-351 activation with cell aggregation and morphologic
changes, thus representing a model system for class I 2
signaling.
Assessments with RG1 and CR11-351 showed a rapid and profound induction
of phosphatidylserine exposure in HLA-A2-transfected Jurkat cells, as
determined by Annexin V-FITC binding. These studies were in agreement
with the observed changes in cell size and granularity. Thus,
activation of the TCR-binding region of the HLA class I
2 domain imposed responses in the Jurkat transfectants
characteristic of apoptotic cell death.
The role of the cytoplasmic class I region in signaling apoptosis has
not been determined. We have shown previously that RG1 and CR11-351
induce cell aggregation in Jurkat E13 transfectants expressing HLA-A2
where the cytoplasmic residues are deleted (37). Similarly, we now
describe that RG1 and CR11-351 induce rapid morphologic changes and
phosphatidylserine exposure in Jurkat E13 cells. Thus, class I
2-mediated cell aggregation responses and apoptotic
responses are apparently signaled independent of the cytoplasmic class
I domain. Importantly, these data strongly indicate that class I
2-mediated signaling requires engagement of a
membrane-bound coreceptor.
Fas activation is implicated in controlling the level of activated T
cells and as a major pathway in mediating CD8+CTL target
cell lysis. We showed that Jurkat E11 cells constitutively expressed
CD95, and it was therefore of interest to determine whether the CD95
pathway was involved in class I signaling and also to compare the
effects of the anti-Fas mAb CH11 with RG1 and CR11-351. Blocking of
possible CD95-CD95L interactions with neutralizing anti-Fas mAb or
human rFas-Fc chimera did not influence the ability of RG1 or CR11-351
to induce apoptosis. Furthermore, we found that class I activation more
rapidly induced changes in cell morphology and phosphatidylserine
exposure compared with CD95 activation. We also assessed the mAb’s
potential to induce DNA fragmentation in Jurkat E11 with the TUNEL
assay. Interestingly, whereas CH11 induced a significant response
within 4 h, only a minor shift in staining intensity was observed
following RG1 and CR11-351 stimulation. Thus, a direct or indirect
activation of the CD95 pathway appeared not to be involved in HLA class
I 2-mediated signaling of cell death. Furthermore, in
contrast to Fas- and perforin-mediated pathways, MHC class I
2-mediated signaling of cell death appears to proceed
independent of DNA fragmentation into oligonucleosomes. Triggering of
programmed cell death without observable DNA fragmentation into
oligonucleosomes has also been observed following activation of other
cell surface Ags (52, 53). Ligation of CD45 on T and B lymphocytes
rapidly induces this kind of apoptosis (52), and it would therefore be
of great interest to determine whether CD45 is activated in HLA class I
2-induced apoptosis.
The basis for the fundamental differences in apoptotic responses is not
known. The Fas-mediated pathway involves ICE and CPP32 activation, as
demonstrated by assessments with inhibitory peptides to these enzymes
(64, 65). In contrast, we found no requirements for these proteases in
class I 2-mediated signaling of apoptosis. Thus, CD95
and HLA class I 2-mediated death signaling clearly
depend on different signaling pathways.
It has been demonstrated that susceptibility to CTL-induced apoptosis
is a function of the proliferative status of the target (67). The cell
cycle status may also be important in relation to responses to HLA
class I 2 activation. Whereas anti-class I
2 activation rapidly induced apoptosis in more than 60%
of Jurkat E11 cells, a resistant subpopulation was always observed.
Interestingly, with unstimulated PBMC we observed no mAb RG1 or
CR11-351 impact on cell morphology or viability. However, the mAbs
clearly induced cell aggregation (data not shown). In contrast, cells
preactivated with OKT3 were clearly sensitive to RG1 and CR11-351.
Thus, normal nonproliferating PBMC may signal cell aggregation, but
apparently not cell death upon activation of the TCR-accessible region
of the class I 2 domain. This may favor a model in which
the TCR participate directly on two levels: 1) to activate new adhesion
molecules and 2) to deliver a lethal hit to transformed (i.e.,
proliferating) cells. Thus, resting cells with latent virus infections
may potentially escape this type of death response.
Recent studies have implicated a functional role of the class I
3 domain in signaling apoptosis (40, 41). In the case of
Jurkat cells, these were shown insensitive to apoptotic responses
induced by class I 3 activation alone (41), indicating
distinct roles for the 2 and 3 domains in
signaling. It is therefore possible that class I 2 and
3 activation by the respective ligands TCR and CD8 may
coregulate apoptotic responses in a target cell.
In conclusion, our data suggest that peptide-directed TCR interaction with target cell MHC class I molecules may directly induce phosphatidylserine exposure for target clearance by phagocytes, and furthermore, effect cell disintegration by programmed cell death. Thus, MHC class I-mediated signaling responses may in some target cells directly determine sensitivity or resistance to CD8+CTL-mediated lysis.
Our results suggest a model in which the physical binding of CTL TCR with target peptide/MHC class I complex per se represents the first line of cytotoxic defense against virus-infected or transformed cells. The success of this strategy will depend on the ability of the target cell to respond with sufficient activation of the HLA class I adhesion and death-related signaling pathways.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Rolf D. Pettersen, Department of Pediatric Research, Rikshospitalet, The National Hospital, N-0027 Oslo, Norway.
3 Abbreviations used in this paper: ICE, IL-1ß-converting enzyme; CD95L, CD95 ligand; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling.
Received for publication August 18, 1997. Accepted for publication January 7, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain of HLA-A2. Nature 345:41.[Medline]
3 domain of human MHC class I molecules. J. Immunol. 153:1054.[Abstract]
2 domain in regulation of cell adhesion and proliferation. J. Immunol. 156:1415.[Abstract]
3 domain of class I MHC. Science 252:1424.3 domain. J. Immunol. 148:3202.[Abstract]
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 B. Murphy, J. Yu, Q. Jiao, M. Lin, T. Chitnis, and M. H. Sayegh A Novel Mechanism for the Immunomodulatory Functions of Class II MHC-Derived Peptides J. Am. Soc. Nephrol., April 1, 2003; 14(4): 1053 - 1065. [Abstract] [Full Text] [PDF]
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 R. D. Pettersen, G. Bernard, M. K. Olafsen, M. Pourtein, and S. O. Lie CD99 Signals Caspase-Independent T Cell Death J. Immunol., April 15, 2001; 166(8): 4931 - 4942. [Abstract] [Full Text] [PDF]
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 M. T. Eby, A. Jasmin, A. Kumar, K. Sharma, and P. M. Chaudhary TAJ, a Novel Member of the Tumor Necrosis Factor Receptor Family, Activates the c-Jun N-terminal Kinase Pathway and Mediates Caspase-independent Cell Death J. Biol. Chem., May 12, 2000; 275(20): 15336 - 15342. [Abstract] [Full Text] [PDF]
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V. Loyer, P. Fontaine, S. Pion, F. Hetu, D.-C. Roy, and C. Perreault The In Vivo Fate of APCs Displaying Minor H Antigen and/or MHC Differences Is Regulated by CTLs Specific for Immunodominant Class I-Associated Epitopes J. Immunol., December 15, 1999; 163(12): 6462 - 6467. [Abstract] [Full Text] [PDF]
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M. Mori, Y. Terui, M. Ikeda, H. Tomizuka, M. Uwai, T. Kasahara, N. Kubota, T. Itoh, Y. Mishima, M. Douzono-Tanaka, et al. beta 2-Microglobulin Identified as an Apoptosis-Inducing Factor and Its Characterization Blood, October 15, 1999; 94(8): 2744 - 2753. [Abstract] [Full Text] [PDF]
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M. A. Maldonado, G. C. MacDonald, V. N. Kakkanaiah, K. Fecho, M. Dransfield, D. Sekiguchi, P. L. Cohen, and R. A. Eisenberg Differential Control of Autoantibodies and Lymphoproliferation by Fas Ligand Expression on CD4+ and CD8+ T Cells In Vivo J. Immunol., September 15, 1999; 163(6): 3138 - 3142. [Abstract] [Full Text] [PDF]
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R. D. Pettersen, K. Hestdal, M. K. Olafsen, S. O. Lie, and F. P. Lindberg CD47 Signals T Cell Death J. Immunol., June 15, 1999; 162(12): 7031 - 7040. [Abstract] [Full Text] [PDF]
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G. Kovalev, K. Duus, L. Wang, R. Lee, M. Bonyhadi, D. Ho, J. M. McCune, H. Kaneshima, and L. Su Induction of MHC Class I Expression on Immature Thymocytes in HIV-1-Infected SCID-hu Thy/Liv Mice: Evidence of Indirect Mechanisms J. Immunol., June 15, 1999; 162(12): 7555 - 7562. [Abstract] [Full Text] [PDF]
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