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Department of Immunology, Imperial College School of Medicine and
Medical Research Council Clinical Science Center, Hammersmith Hospital, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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treatment of EC resulted in a marked
up-regulation of MHC class I, but no change was observed in the level
of CD80 expression. No CD86 molecules were detectable under either
condition. The ability of peptide-pulsed EC to induce the proliferation
of either the HY-specific, H2-Kk-restricted
CD8+ T cell clone (C6) or C6 TCR-transgenic naive
CD8+ T cells was analyzed. Resting T cells were stimulated
to divide by quiescent peptide-prepulsed EC, while peptide-pulsed,
cytokine-activated EC lost the ability to induce T cell division.
Furthermore, Ag presentation by cytokine-activated EC induced
CD8+ T cell hyporesponsiveness. The immunogenicity of
activated EC could be restored by adding nonsaturating concentrations
of anti-H2-Kk Ab in the presence of an optimal
concentration of cognate peptide. This is consistent with the
suggestion that the ratio of TCR engagement to costimulation determines
the outcome of T cell recognition. In contrast, activated
peptide-pulsed EC were killed more efficiently by fully differentiated
effector CD8+ T cells. Finally, evidence is provided that
Ag recognition of EC can profoundly affect the transendothelial
migration of CD8+ T cells. Taken together, these results
suggest that EC immunogenicity is regulated in a manner that
contributes to peripheral tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of studies analyzing this issue have focused on Ag presentation by EC to CD4+ T cells, and conflicting results have been reported in both the human and the murine system. The lack of proliferative responses following cognate recognition of EC by murine (1) and costimulation-dependent human (2) T cell clones has been reported. Most interestingly, in both systems the failure to induce T cell division was not accompanied by the induction of unresponsiveness. In other studies acetylcholine receptor-specific human T cell lines and murine OVA-specific, TCR-transgenic T cell lines were able to proliferate to Ag presented by EC (3, 4). Many studies have suggested that due to the limited ability of EC to deliver costimulatory signals, the consequences of this interaction are likely to correlate with the stringency of T cell requirements for B7-mediated costimulation (2, 4, 5).
It is likely that cognate interactions between T lymphocytes and EC may modulate T cell functions other than cell division. We have recently provided in vitro evidence that Ag presentation by EC may contribute to the development of the immune response by enhancing the recruitment of Ag-specific CD4+ T cells into the tissue rather than by inducing T cell proliferation (6). Furthermore, these studies suggested that T cell division is incompatible with transendothelial migration.
CD8+ CTL are major effectors of immune responses
and carry out their function by migrating through postcapillary venules
and infiltrating target tissues. The few studies that have analyzed the
functional consequences of cognate interactions between EC and
CD8+ T cells have focused on the development of
proliferative responses, and similar to what has been described for
CD4+ T cells, contradictory conclusions have been
drawn concerning the outcome of such interactions. Some studies have
described that EC can induce human and murine
CD8+ T lymphocyte differentiation
(7, 8, 9), although with a low efficiency (7)
compared with conventional APCs and with impaired ability to secrete
IFN- following antigenic rechallenge (8). In contrast,
other studies concluded that human EC are unable to stimulate a
functional response by freshly purified peripheral blood
CD8+ T cells (10). These
discrepancies may reflect differences in the stage of differentiation
of the CD8+ T cell population analyzed or that
the stimulator EC were not activated by cytokines. No information is
currently available on the effect that cognate recognition of EC may
have on the migratory ability of CD8+ T
cells.
In the present study we have investigated the effects of Ag presentation by primary cultures of murine lung EC to male-specific minor histocompatibility Ag (HY)-specific CD8+ T cells at different stages of differentiation, namely naive, effector, and memory T cells. EC were used either in a resting state or were activated by cytokine treatment. In addition, the effect of cognate recognition of resting or activated EC on the migratory ability of CD8+ T cells was analyzed. The results obtained are consistent with a role for EC Ag presentation in the induction and maintenance of peripheral tolerance and the prevention of autoimmune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C6 TCR Tg mice, specific for the HY peptide epitope TENSGKDI
presented by H2-Kk, were generated using a CD2
cassette (11) encoding the 
8 and ß11 TCR chains from
the HY-specific CD8+ T cell clone C6
(12). CBA/Ca mice were purchased from Olac Harlan
(Bicester, U.K.) and were used at 6–8 wk of age.
Peptides and Abs
The peptide TENSGKDI derived from the Smcy gene product was used as cognate Ag for the C6 TCR. The mAbs used for naive T cell purification as hybridoma supernatant were anti-H2-Ak (10.2.16, TIB93, American Type Culture Collection, Manassas, VA), anti-H2-Ek (14-4-4S, HB, American Type Culture Collection), anti-H2-Ek, d/Ab, d (M5/114, HIB120, American Type Culture Collection), anti-CD4 (YTS 191) (13), and anti-CD44/Ly-24/Pgp-1 (142.5) (14). The following purified mAbs were used in functional assays: anti-CD80/B7-1 (16-10A1, PharMingen, San Diego, CA), anti-CD86/B7-2 (GL-1, PharMingen), and anti-H2-Kk (HB25, American Type Culture Collection). Purified rat IgG, hamster IgG, and mouse IgG (PharMingen) were used as the negative control. The anti-clonotypic TCR Ab KTL2 (15) was used as the purified mAb.
Preparation of Con A supernatant
Murine splenocytes (5 x 106/well) were seeded onto a 24-well plate and incubated with medium containing 2.5 µg/ml Con A lectin for 96 h. At this time the supernatant was harvested, filtered, aliquoted, and stored at -20°C.
EC culture
Murine microvascular EC were purified from lung tissue obtained
from female CBA/Ca mice based on a technique described by Dong and
colleagues (16, 17). Following isolation, EC were serially
subcultured at 37°C with 5% CO2 in DMEM (Life
Technologies, Paisley, U.K.) supplemented with 2 mM glutamine (Life
Technologies), 100 U/ml penicillin (Life Technologies), 100 µg/ml
streptomycin (Life Technologies), 1 mM sodium pyruvate (Life
Technologies), 10 mM HEPES (Life Technologies), 1% nonessential amino
acids (Life Technologies), and 50 µM 2-ME (Life Technologies) with
freshly added 20% heat-inactivated FCS (Globepharm, Esher, U.K.), 150
µg/ml EC growth supplement (Sigma, Poole, U.K.), and 12 U/ml heparin
(CP Pharmaceuticals, Wrexham, U.K.) in 2% gelatin-coated (Sigma)
tissue culture flasks (Greiner Labortechnik, Dursley, U.K.). At
confluence the EC were detached from the culture flasks using
trypsin/EDTA (Life Technologies) and passaged. The EC lineage of the
cell populations obtained was confirmed by positive staining with
anti-CD31 and anti-CD105 Abs (data not shown). For functional
assays the EC were used between passages 4 and 6. To activate the EC,
10% supernatant from Con A-treated splenocytes or 400 U/ml murine
IFN- (PeproTech, London, U.K.) was added to the tissue culture flask
and incubated for 96 h.
Purification of naive TCR-Tg CD8+ T cells
Spleen and pooled lymph node (both s.c. and mesenteric) cells were depleted of adherent cells by two 45-min rounds of adherence to plastic on tissue culture dishes at 37°C. The nonadherent cells were subsequently collected and incubated with a cocktail of purified mAbs (anti-H2-Ak, anti-H2-Ek, anti-H2-Ek,d/Abd, anti-CD4, anti-CD44/Ly-24/Pgp-1) at saturating concentrations for 30 min at 4°C. The cells were then washed twice to remove excess Ab and further enriched by magnetic immunodepletion. Briefly, mAb-treated cells were incubated with magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) coated with goat anti-rat and sheep anti-mouse Ig for 15 min at 4°C, and bead/mAb-coated cells were removed by passage through a magnetic column (miniMAC system, Miltenyi Biotec). The purified cells were resuspended in medium ready for the proliferation assay, and accessory cell contamination was assessed by culture with 2 µg/ml Con A in a 48-h assay.
C6 T cell clone
The C6 T cell clone, specific for HY peptide epitope TENSGKDI presented by H2-Kk, was maintained in culture by stimulation every 2 wk with splenocytes from CBA/Ca male mice and rIL-2 (20 U/ml; Roche, Mannheim, Germany) in RPMI 1640 medium supplemented with 10% FCS (Globepharm), 2 mM glutamine (Life Technologies), 50 IU/ml penicillin (Life Technologies), 50 µg/ml streptomycin (Life Technologies), and 50 µM 2-ME (Life Technologies). For use in experiments, the T cells were purified by isolation on a Lympho-Sep (Harlan-Sera, Crawley, U.K.) gradient 15 days (proliferation and transmigration experiments) or 5 days (cytotoxicity assays) after restimulation and were washed twice by low speed centrifugation (210 x g, 5 min) before use to exclude any contamination by accessory cells.
T cell proliferation assays
Naive TCR-Tg CD8+ T cells (2 x
104 cells/well) or the C6 T cell clone
(104 cells/well) were cultured in the presence of
EC (2 x 104/well) treated with 30 Gy of
-irradiation in round-bottom microtiter plates in a total volume of
200 µl. The stimulator cells were prepulsed overnight with peptide
and then washed to remove any soluble peptide. Wells were pulsed with 1
µCi of [3H]TdR (Amersham, Aylesbury, U.K.),
after 72 h (naive TCR-Tg CD8+ T cells) or
48 h (C6 T cell clone), and the cultures were harvested onto
glass-fiber filters 18 h later. Proliferation was measured as
[3H]TdR incorporation by liquid scintillation
spectroscopy.
Two-stage cultures for tolerance induction
Purified TCR-Tg naive CD8+ T cells
(106/well) were plated out in 24-well plates in
the presence of allogeneic cytokine-treated EC (2 x
105/well) irradiated with 30 Gy of
-irradiation or untreated irradiated EC (2 x
105/well). In some wells the EC had been pulsed
with the cognate Kk/HY peptide overnight. After 3
days T cells were harvested and purified by centrifugation on a
Lympho-Sep (Harlan-Sera) gradient. Recovered cells were then rested for
a further 72 h in RPM1 1640 medium with supplements and 10% FCS
in 24-well plates before being used in a rechallenge proliferation
assay using peptide-prepulsed female CBA/Ca splenocytes (5 x
105/well).
T cell clones (3 x 105) were plated out in 24-well plates in the same conditions as those described for the naive CD8+ T cells in a total volume of 500 µl in a 24-well plate. After overnight incubation in the same conditions as those described for the naive T cells, the cells were harvested, rested for an additional 72 h in RPM1 1640 medium with supplements and 10% FCS, and used in proliferation assays.
Cytokine assays
Triplicate samples of cell-free culture supernatants were
collected after overnight incubation and were stored at -70°C. IL-4
and IFN- were detected using Quantikine cytokine ELISA kits (R&D
Systems, Abingdon, U.K.) according to the manufacturer’s directions.
Plates were read using a THERMOmax ELISA Reader (Molecular Devices,
Abingdon, U.K.). The concentrations of IL-4 and IFN- were calculated
by quadratic curve fitting to known standards using Soft Max Pro 1.2.0
program (Molecular Devices).
Cytotoxicity assays
EC killing by effector C6 T cells was assessed using a protocol described by Pardi and Bender (10). Resting or cytokine-treated EC were seeded onto 2% gelatin-coated flat-bottom 96-well plates (Greiner Labortechnik) at 1–2 x 104/well and incubated with 100 nM Kk/HY peptide or T cell culture medium overnight. EC were then labeled in the wells with 1–2 µCi of 51Cr (Amersham) for 2 h. An Abelson virus-transformed pre-B cell line, derived from a mouse expressing H2-Kk, was incubated with peptide overnight and labeled with 50–100 µCi of 51Cr for 2 h, then washed three times in medium and added to the plates at 104 cells/well. Effector CD8+ T cells were harvested, purified, and added to the plates in the ratio indicated. Spontaneous release was measured from labeled target cells in medium alone, and maximal release was measured from labeled target cells lysed with 1% Triton-X 100. Following incubation at 37°C for 4 h, 100 µl of supernatant was harvested from each well, and 51Cr release was measured using a Wallac gamma counter (Gaithersburg, MD). Specific lysis was calculated using the equation: % specific lysis = [(counts sample - counts spontaneous)/(counts maximal - counts spontaneous)] x 100.
Lymphocyte transmigration assays
The transmigration experiments were conducted using EC monolayers grown on Transwell tissue culture well inserts (diameter, 24.5 mm; Costar, High Wycombe, U.K.) that contained polycarbonate membranes with a 3-µm pore size (Costar) as previously described (6, 18). EC (105) were seeded onto fibronectin-coated (50 µg/ml; Sigma) polycarbonate membranes overnight to form a monolayer. In some well inserts, 100 nM HY peptide was added for 16–18 h before the transmigration assay. Before the assay, the EC monolayers were gently washed with warm medium. Purified C6 CD8+ T cells (7 x 105) in RPMI 1640 supplemented with 10% FCS were added to each insert and left to migrate through the monolayer; the well content was replaced with fresh medium. After 1 h the number of migrated T cells was determined by counting the lymphocytes present in the well medium. This was performed at different time points for the next 6 h. In these experiments results are expressed as a percentage of the value for transmigrated cells.
Flow cytofluorometric analysis
For cytofluorometric analysis, 5 x 105 cells were incubated with the indicated mAb at 4°C for 30 min. After washing twice in PBS with 2.5% FCS, the cells were incubated for an additional 30 min at 4°C with 100 µl of either a 1/100 dilution of fluoresceinated goat anti-rat Ig (PharMingen), a 1/100 dilution of fluoresceinated goat anti-hamster Ig (PharMingen), or a 1/64 dilution of fluoresceinated goat anti-mouse Ig (Sigma). After two additional washes, stained cells were analyzed using an EPICS Profile flow cytometer (Coulter, Luton, U.K.).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Resting EC from primary cultures obtained from murine lung
expressed low levels of H2-Kk molecules (Fig. 1
a) compared with those seen
on primary cultures of fibroblasts or renal cells isolated from the
same animal (data not shown). In addition, murine EC constitutively
expressed low levels of CD80 molecules (Fig. 1b), similar to
what has been observed by others (4). This was not an
organ- or strain-related characteristic, in that a similar pattern of
expression was observed in a murine brain endothelioma line (gift from
E. Dejana and A. Vecchi, Istituto Mario Negri, Milan, Italy), in
primary cultures of EC isolated from murine hearts, and in primary
cultures of lung microvascular EC isolated from BALB/c and C57BL/6
mouse strains (data not shown). The phenotypic changes induced in
murine EC by incubation in medium containing 10% supernatant from Con
A-treated splenocytes or murine IFN- (400 U/ml) for 72 h were
also analyzed. Following this treatment, the expression of
H2-Kk molecules was dramatically up-regulated
(Fig. 1c), while the level of CD80 molecules remained
unchanged (Fig. 1d). MHC class II molecules were also
induced, however at significantly lower levels than those observed for
MHC class I molecules (data not shown). The failure of EC to
up-regulate CD80 molecules was not due to an effect mediated by IL-10
or TGF-ß, as neutralizing Abs against both cytokines did not alter
the pattern of H2-Kk/CD80 expression (data not
shown). Similarly, the Con A lectin used to produce IFN--enriched
supernatant did not affect the expression of endothelial cell surface
molecules. EC were incubated with 2.5 µg/ml Con A lectin for 72
h, as described for the production of splenocyte supernatant, and no
changes were detected in the expression of any of the surface molecules
tested (results not shown). Notably, neither CD86 molecules nor CD40
molecules were expressed by the EC under any circumstances (data not
shown).
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The ability of resting and activated EC to present Ag and induce
proliferation by CD8+ T cells was investigated.
Ag-pulsed, MHC class Idim resting EC were capable
of inducing significant proliferative responses by both the C6 T cell
clone (Fig. 2a) and naive C6
TCR-Tg T cells (Fig. 2d) of a magnitude similar to that
observed following stimulation with peptide-prepulsed splenocytes (Fig. 2, c and f). T cell proliferation could be
partially inhibited by Ab-mediated CD80 blockade, while isotype-matched
control Ab had no effect. Similarly, an anti-CD86 mAb did not
inhibit EC-induced T cell proliferation (data not shown).
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b)
and naive C6 TCR-Tg T cells (Fig. 2e). A proliferative
response could be partly reconstituted by the addition of submitogenic
doses of rIL-2. However, CD8+ T cell
proliferation in response to peptide-pulsed EC could not be restored by
the addition of neutralizing Abs against IL-10 or TGF-ß (data not
shown). The possibility that in the presence of high TCR engagement, a
limiting level of CD80 expression might favor CD152 ligation and thus
inhibit proliferation (19) was ruled out by the failure to
restore proliferation by the addition of an anti-CD152 Ab (data not
shown).
Since it has been reported that proliferative hyporesponsiveness can be
induced in CD8+ T cells following cognate
recognition in association with CD80-mediated costimulation
(20), we set out to investigate the functional
consequences of cognate interaction between CD8+
T cells and EC. While failing to induce T cell division (Fig. 3, a and f),
cognate recognition of either cytokine-treated or untreated EC induced
substantial levels of IFN- production (Fig. 3, b and
g), suggesting that IFN- secretion occurs independently
of B7-mediated signals. These data are consistent with previous
findings by Sancho and colleagues, who observed that IFN- production
by CD8+ T cells depends on interactions between
ICAM-1 molecules on EC and LFA-1 on T cells and is enhanced by IL-15
(21). No IL-4 was detected in these cultures (data not
shown).
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The lack of proliferation following Ag presentation by activated EC was
accompanied by the induction of proliferative hyporesponsiveness to
subsequent antigenic rechallenge in both the C6 T cell clone and naive
C6 TCR-Tg T cells (Fig. 3, c and h, respectively)
that have been cocultured overnight with activated Ag-pulsed EC and
rested for an additional 72 h to allow the recovery of Ag receptor
sensitivity failed to proliferate in response to Ag-pulsed splenocytes.
Both the T cell clone and the naive T cells cocultured with resting
peptide-pulsed EC (Fig. 3, c and h, respectively)
remained capable of responding to an antigenic rechallenge delivered by
Ag-loaded splenocytes. The observation that Ag-hyporesponsive T cells
still proliferated vigorously in response to rIL-2, suggesting that
anergy induction was the predominant mechanism accounting for the loss
of responsiveness (Fig. 3, d and i). Consistent
with this interpretation, no induction of IL-4 secretion was observed
in the rechallenge cultures (data not shown). Interestingly, IFN-
production by the C6 T cell clone (Fig. 3e) upon antigenic
rechallenge appeared unaffected by Ag recognition on cytokine-treated
EC, while it was greatly diminished following cognate interactions
between naive T cells and activated EC (Fig. 3j). Diminished
IFN- secretion has been previously reported by a study analyzing the
functional characteristics of allospecific CTL generated from naive T
cells in the presence of EC as APC and exogenous IL-2 in the human
system (8).
The responsiveness of T cells incubated with EC in the absence of Ag was unaffected (data not shown).
Modulation of signal 1 restores proliferative responses to activated EC
A possible explanation for the results described above is that the
failure to up-regulate CD80 molecules following cytokine treatment
resulted in the delivery of insufficient costimulation relative to the
increase of H2-Kk molecule expression. This, in
turn, may lead to partial activation and the induction of
hyporesponsiveness. To assess this possibility, we analyzed the degree
of modulation of TCR expression following cognate recognition of
resting or activated EC as an indicator of the extent of TCR
engagement. In Fig. 4 TCR expression by
the C6 CD8+ T cell clone 12 h after cognate
recognition of resting and activated EC pulsed with optimal doses of HY
peptide is shown. This time point was chosen based on previous studies
that had established that the C6 TCR was maximally down-regulated by
12 h following antigenic stimulation (11). As a
control, TCR expression by T cells incubated in medium alone is shown.
TCR engagement was greater following Ag presentation by
IFN--treated, H2-Kk-bright EC, as suggested by
the more marked TCR down-regulation (Fig. 4, a and
b).
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c) or
cytokine-activated (Fig. 4d) EC was measured. As previously
observed, despite the higher TCR engagement a minimal T cell response
to peptide presented by IFN--treated EC was detected at a high dose
of peptide compared with that induced by a similar concentration of Ag
presented by resting EC. The addition of a suboptimal dose of
anti-H2-Kk mAb resulted in the partial
inhibition (Fig. 4c) of proliferation induced by the resting
EC, while the same amount of blockade partially reconstituted the
response to activated EC (Fig. 4d, see enhancement part of
the curve). No difference was seen in the presence of an
isotype-matched control mAb (data not shown).
Similarly, a prediction of our interpretation is that decreasing the
amount of peptide bound to MHC class I molecules on the EC should
reconstitute T cell proliferation once an optimal ratio of MHC:peptide
complexes/costimulation is achieved. Indeed, as shown in Fig. 4d (see inhibition part of the curve), increased
proliferation in response to the cytokine-treated EC could be induced
by loading them with low doses of antigenic peptide (between 0.005 and
1 nM, depending on the experiment). These proliferative responses were
lower than those induced by resting EC (Fig. 4c) but were
similarly inhibited by the anti-MHC class I Ab (Fig. 4d,
inhibition part of the curve). Consistent with our hypothesis, these
results suggest that partial blockade of TCR engagement restores a
ratio of signal 1 to costimulation compatible with the induction of T
cell division by cytokine-activated Ag-presenting EC.
The cytotoxic activity of CD8+ T cells is costimulation independent
Although costimulatory signals are required to induce
CD8+ T cell expansion, it is likely that less
stringent conditions are needed once the T cells have differentiated
into cytotoxic effector cells. The ability of effector
CD8+ T lymphocytes to kill resting and activated
EC was investigated using cytotoxicity assays. For these experiments
the C6 T cell clone was used approximately 5 days after restimulation,
when the T cells exhibit their strongest cytotoxic activity.
Peptide-pulsed resting EC were lysed by effector
CD8+ T cells with low efficiency, while
substantial killing of cytokine-treated EC was observed (Fig. 5, a and b). The
addition of an anti-CD80-blocking Ab did not affect cytotoxic
activity under either condition, indicating that CD80-mediated signals
are not required for this effector function.
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We have previously reported that Ag recognition on EC can
profoundly affect transendothelial migration of
CD4+ T cells (6). The effect of
cognate recognition on the migratory ability of the C6
CD8+ T cell clone was investigated using a
transendothelial migration assay. T cells were purified on a Ficoll
gradient and were seeded onto an endothelial cell monolayer grown on a
Transwell insert, and the number of transmigrated T cells was monitored
for the following 6 h. T cells were used at least 2 wk following
antigenic restimulation, when lytic activity against EC was
undetectable (data not shown). As shown in Fig. 6, a few T cells migrated through the
resting EC monolayer (a), while cytokine-treated EC could
support the migration of up to 80% of the total T cell input
(b), probably due to the up-regulation of adhesion
molecules. When antigenic peptide was loaded onto the EC monolayer, T
cell transendothelial migration was profoundly decreased under both
conditions, suggesting that cognate recognition of EC delivers a stop
signal to migrating CD8+ T cells. For comparison,
transendothelial migration of an irrelevant alloreactive
CD4+ T cell clone (2E4, specific for the amino
acid-substituted NOD H2-A molecule NODasp)
through resting and activated EC in the presence or the absence of the
C6 T cell cognate peptide is shown in Fig. 5, c and
d. C6 TCR-transgenic naive T cells did not migrate though EC
monolayers under any circumstances (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. In contrast, lysis of EC by
effector CD8+ T cells was enhanced by
cytokine-mediated activation of the EC. The experiments were performed
using IFN--treated EC because the focus of our study was on the
consequence of recognition of antigenic peptide bound to MHC molecules,
which are up-regulated on EC by IFN-. In addition, all the
experiments reported here were reproduced using EC-treated with
mitogen-induced cytokine pools to resemble more closely physiological
EC stimulation during inflammatory conditions.
The recent definition of human EC as semiprofessional APC
(5) appears to apply also to murine microvascular EC. In
agreement with a recent study we found that murine EC express low
levels of CD80 molecules on their surface (4), unlike
their human counterpart. Endothelial CD80 molecules appear to be
functional, in that the EC can bind CTLA-4-Ig (4) and
induce proliferation by CD8+ T cells following Ag
presentation by quiescent EC. However, in contrast with conventional
APC, such as dendritic cells (DC), CD80 was not up-regulated and was
not accompanied by the expression of CD86 molecules following
cytokine-mediated activation. This may result in an imbalance between
the extent of TCR engagement, which increases, and CD28 engagement,
which remains unchanged, and may lead to the induction of
hyporesponsiveness that we observed following cognate recognition of
cytokine-treated EC. This hypothesis is supported by the observation
that proliferation in response to activated EC could be recovered
either by modulation of TCR engagement by the addition of an
anti-H2-Kk mAb or by titrating down the
amount of antigenic peptide bound to MHC molecules on activated EC,
although it is technically impossible to rule out the interference of
cytokine-induced molecules other than CD80 (e.g., VCAM-1 up-regulation)
in the present system. In support of our interpretation is the
observation that the surface phenotype of cytokine-activated EC may
resemble that of immature DC (high MHC molecule expression and low B7
molecule expression), which have been shown to induce
hyporesponsiveness in vitro (22, 23, 24) and tolerance in vivo
(25, 26). The unfavorable signal 1 to signal 2 ratio
observed on cytokine-activated EC may also explain the recent
observation that murine lung microvascular IFN--treated EC can
sustain proliferative responses by memory CD4+ T
cells, but not naive CD4+ T cells, which have
more stringent costimulation requirements (4, 5).
The functional consequence of this interaction appears to be the
induction of T cell anergy, rather than cytokine-gene reprogramming
leading to immune deviation. Indeed, the proliferative
hyporesponsiveness was accompanied by either unaffected or diminished
IFN- secretion in the absence of induction of other cytokines such
as IL-4, IL-10, or TGF-ß and by increased reactivity to
exogenous IL-2.
The tolerogenic properties of murine EC constitute a major difference from their human counterpart, which, despite being poor APC, do not induce tolerance (1, 2, 8) in responder T cells. These observations may be accounted for by the profound differences in the array of immunogenic molecules displayed on the EC surface, such as CD80, which is absent on human EC (27), and CD40, which is expressed by human EC (28, 29). These differences have to be borne in mind when extrapolating observations made in murine experimental models to the human system, such as the high rate of success of therapeutic strategies aimed at inducing allospecific tolerance to vascularized tissue graft in murine models.
It has been suggested that the induction of anergy in CD4+ T cells in vivo is dependent on the delivery of dominant CD152-mediated signals, and that these conditions arise in the presence of a limiting amount of costimulation (19), particularly when CD80 molecules, which have a slower off-rate from CD152, are the predominant costimulatory molecules (30, 31). However, it is unlikely that CD152-mediated signaling plays a role in our system, because CD152 blockade did not restore activated EC immunogenicity. Finally, an alternative explanation for our observations is that in conditions of high TCR engagement, CD86-mediated signals are required for the induction of CD8+ T cell proliferation. The induction of anergy in CD8+ T cells has been described following transient proliferation in the presence of optimal CD80-mediated costimulation but in the absence of CD86-derived signals (20).
Finally, our data suggest that cognate recognition of EC appears to deliver a stop signal to migrating CD8+ T cells, in line with previous results showing that TCR ligation delivers a stop signal to ICAM-1-triggered T cell locomotion on planar phospholipid bilayers (32). These observations suggest that TCR triggering by the EC may down-modulate migration, although this effect is likely to be combined with that elicited by chemokine gradients in vivo. Further studies are required to estimate which effect may prevail under these circumstances, both in vitro and in vivo.
The results we have obtained in this system may have revealed a novel pattern of regulation of APC function by cytokines and are consistent with an immunoregulatory role of EC as APC. Although EC are immunogenic in their resting state, it is unlikely that they initiate or support proliferative responses by CD8+ T cells, because T cells do not engage in sustained contacts with resting EC due to the high flow rate and the absence of adhesive interactions. In addition, due to the low expression of MHC class I molecules, T cell/EC cognate interactions would require high avidity T cells. This also appears to be an extremely unlikely event, given that MHC class I molecules are generally loaded with self-peptides, and self-reactive peripheral T cells that have escaped thymic selection have low avidity (33, 34). However, if T cell recognition of Ag presented by resting EC were to occur, this would have an inhibitory effect on migration into the underlying tissue, as suggested by our transendothelial migration experiments, and possibly slow down T cell recruitment into the tissue.
Most importantly, EC activation during inflammation would render them tolerogenic to CD8+ T cells by both the induction of nonresponsiveness and the blockade of transendothelial migration. As a consequence, only T cells that do not find their cognate peptide displayed by EC or that have reached their effector stage following activation would be able to cross the endothelial barrier unaffected. It is conceivable that these two criteria selectively apply to CD8+ T cells that are specific for non-self Ags or that have recently been activated in the local lymph nodes and re-enter the circulation once they are fully differentiated into effector CD8+ T cells (35). It has been shown recently that bone marrow-derived APC, such as DC, are required for the initiation of anti-viral immune responses, and that alternative MHC class I pathways that lead to the presentation of peptides derived from exogenous Ag are restricted to these cells (36). Thus, following priming by DC in the local lymph node, recirculating activated virus-specific effector CD8+ T cells are unlikely to find their cognate peptides displayed by EC, so they would not be rendered unresponsive, nor would their migration into the tissue be halted.
EC damage would instead occur when EC are recognized and lysed by DC-primed effector CD8+ T cells, as in the event of vascularized allotransplantation or as a by-product of priming against a viral infection. However, the latter event would normally be self-limiting due to the lack of CD4-mediated help necessary for further CD8+ T cell expansion and differentiation into effector cells, in that self-reactive CD4+ T cells are likely to re-encounter cognate Ag on costimulation-deficient APC and thus be rendered hyporesponsive (31, 37).
The present report supports the idea that the ratio of TCR engagement and delivery of costimulation determines APC immunogenicity and is consistent with a role for EC in the induction and maintenance of peripheral tolerance.
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2 Address correspondence and reprint requests to Dr. Federica Marelli-Berg, Department of Immunology, Imperial College School of Medicine, Hammersmith Hospital, Commonwealth Building, Du Cane Road, London, U.K. W12 0NN.
3 Abbreviations used in this paper: EC, endothelial cells; DC, dendritic cells; HY, male-specific minor histocompatibility Ag.
Received for publication March 15, 2000. Accepted for publication July 18, 2000.
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) or
a low dose of rIL-2 (
) was added. c, For comparison,
proliferation to unpulsed or peptide-pulsed (100 nM) female splenocytes
(5 x 105/well) is shown. d and
e, A similar experiment performed using C6
TCR-transgenic naive T cells is shown. T cells (2 x
104/well) were cultured with 104/well
peptide-prepulsed resting (d, •) or cytokine-treated
(e, •) EC. In some wells an anti-CD80 Ab (
cpm). SEs are shown.
) EC was measured. After overnight
incubation T cells were collected, washed, and rested for an
additional 72 h in medium alone. The T cell clone
(104/well; c) and naive CD8+ T
cells (2 x 104/well; g) previously
cocultured with either peptide-loaded resting (EC NT, 
) or activated
(EC T, 
) EC were then rechallenged with peptide-prepulsed
splenocytes (5 x 105/well) in a proliferation assay.
T cells responsiveness to IL-2 (10 U/ml) was assessed in parallel
(d and i, 
