Download Signal Requirements for the Generation of CD4+ and CD8+ T

1269
Signal Requirements for the Generation of
CD4+ and CD8+ T-Cell Responses to Human
Allogeneic Microvascular Endothelium
Ruggero Pardi and Jeffrey R. Bender
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Microvascular endothelium has been implicated as a major target in the rejection of
vascularized allografts. In an attempt to dissect the stepwise generation of the T-cell-mediated
immune response to microvascular endothelial cells (ECs), we analyzed the requirements for
the two major T-cell subsets, CD4+ and CD8, in the triggering of proliferative and cytotoxic
responses to allogeneic ECs in vitro. Results demonstrate that resting ECs are unable to
stimulate a functional response by purified T, CD4+, and CD8+ cells in the absence of
costimulatory signals. T cells and CD8+ cells develop both proliferative and cytotoxic anti-EC
responses by the addition of as little as 2 units/ml interleukin-2, 10 units/ml interleukin-1, or
irradiated monocytes autologous to the responder lymphocytes, whereas only autologous
monocytes are capable of triggering CD4+ T-cell precursors to proliferate and become
anti-EC-specific effector cytotoxic T lymphocytes. CD8' cell-mediated anti-EC cytotoxicity is
directed toward allogeneic major histocompatibility complex (MHC) class I determinants on
ECs and involves recognition by the CD3/T-cell receptor complex and the CD8 molecule on the
effector T cells. CD4+ cells can be driven to proliferate, produce interleukin-2, and become
anti-EC-specific cytotoxic T lymphocytes despite the lack of detectable membrane MHC class
II determinants on the target cells. Chloroquine inhibition experiments demonstrate that
autologous monocytes/macrophages are required by CD4+ T-cell precursors for the processing
of EC-derived alloantigens and their subsequent presentation in the context of self-MHC
molecules. These results are in agreement with the adoptive transfer experiments in experimental allograft models and suggest that the coordinate engagement of cells of the CD4, CD8,
and monocyte/macrophage series is more effective than the individual cell subsets in the
generation of a functional response to allogeneic nonlymphoid tissues. (Circulation Research
1991;69:1269-1279)
T he continuous exposure of vascular endothelium to circulating lymphocytes, together
with its ability to express pleiotropic major
histocompatibility complex (MHC) antigens in
pathological conditions,1-3 imparts this tissue with
the ability to greatly amplify the immune response
toward genetically incompatible grafts, or self-antigens, in certain autoimmune diseases.4 Accumulating
evidence also supports the notion that microvascular
endothelium can be involved as a primary target of
functional rejection in vascularized allografts. EndoFrom the Scientific Institute S. Raffaele (R.P.), Milan, Italy, and
the Department of Internal Medicine (R.P., J.R.B.), Yale University School of Medicine, New Haven, Conn.
Supported by National Institutes of Health grant HL-43331.
J.R.B. is the recipient of a National Institutes of Health Clinical
Investigator Award (K08 HL-02126/01).
Address for correspondence: Dr. J.R. Bender, Division of
Cardiology, 3-FMP, P.O. Box 3333, Yale University School of
Medicine, 333 Cedar Street, New Haven, CT 06510.
Received October 24, 1990; accepted June 21, 1991.
thelial cell (EC) injury has been demonstrated in
early stages of rejection, with perivascular mononuclear cell infiltrates and interendothelial cell gaps
rapidly progressing to blood vessel lining disruption.5
The immunohistological demonstration of EC involvement in allogeneic as well as self-directed immune response has prompted a number of in vitro
studies focused on the immunogenicity of vascular
endothelium. Our group6 and other groups7 have
shown that cultured ECs, either constitutively or
after exposure to lymphocyte-derived cytokines, express functionally normal MHC determinants, in
addition to polymorphic surface antigens largely restricted to this tissue.8-1' In agreement with these
findings, ECs have been shown to elicit proliferative
and cytotoxic responses by allogeneic lymphocytes,12
to present soluble antigens to T cells,13"4 and to
secrete soluble factors with immunoregulatory activity, such as interleukin (IL)-1,15 IL-6,16 and granulocyte/macrophage colony stimulating factor.'7
1270
Circulation Research Vol 69, No 5 November 1991
In an attempt to investigate the stepwise generation of the T-cell-mediated immune response toward
allogeneic microvascular endothelium, we studied
the signal requirements for the two major T-cell
subpopulations, CD4+ and CD8+, in the induction of
a functional response to ECs in vitro. Results presented here demonstrate that resting microvascular
ECs are poorly immunogenic to purified T cells in
vitro and that a complex interaction between various
T-cell subsets and autologous accessory cells (ACs) is
required to trigger a T-cell-mediated immune response toward this allogeneic tissue. Furthermore,
the data suggest that CD4+ cytotoxic T cells, recognizing processed EC alloantigens, may play a role in
the cascade of events leading to EC damage in vivo in
vascularized allograft rejection.
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Materials and Methods
Microvascular EC Cultures
Stable cultures of human microvascular ECs were
established as previously described.18 Briefly, fresh
discarded preputial skin from randomly selected
anonymous newborns was treated with 0.3% trypsin
and 1.0% EDTA for 1 hour at 37°C, after which the
microvascular ECs were extruded, plated in microvascular EC culture medium,18 and grown to confluence. On reaching confluence, the cultures were
passaged and used for functional assays within the
fifth passage. Cells were identified as ECs by standard light microscopy and immunofluorescence
techniques as previously described18 and discarded
if they contained >1% contaminating cell types.
Experimental ECs were considered in resting conditions based on 1) the lack of surface class II HLA
expression or monolayer reorganization and 2)
background [3H]thymidine incorporation levels.
Stable cultures of autologous fibroblasts were established by plating a small aliquot of cells obtained
in the EC isolation procedure into a 35-mm dish in
the presence of 10% peripartum serum but without
EC selection components.18
Monoclonal Antibodies
Anti-Leu-2a (clone SK1, immunoglobulin [Ig] G1)
binds to the CD8 antigen present on cytotoxic/suppressor T cells; anti-Leu-3a (clone SK.3, IgG1) reacts
with the CD4 antigen on helper/inducer T cells;
anti-Leu-4 (clone SK.7, IgG1) reacts with CD3 complex present on mature T cells; anti-Leu-2a, -3a, and
-4 monoclonal antibodies were purified over Sepharose protein A columns from crude ascites; W6/32, an
IgG2a reacting with a framework-specific determinant
on HLA-A, -B, and -C molecules was provided by Dr.
Peter Parham, Stanford University; CA-141, an IgGI
that reacts with a monomorphic determinant on HLADR, was provided by Dr. H.O. McDevitt, Stanford
University; L243 is an IgG2a that reacts with a different HLA-DR framework-specific epitope than CA141 and was provided by Dr. R. Levy, Stanford
University; anti-lymphocyte function associated
(LFA)-1 antigen (CD11a) (clone TS.1.22) and antiLFA-3 antibodies, both IgGl, react with broadly distributed antigens on hematopoietic tissues (LFA-1)
and on both hematopoietic and nonhematopoietic
tissues (LFA-3) and were provided by Dr. A. Krensky,
Stanford University; anti-Leu-M3 (clone MOP9,
IgG2b) reacts with monocytes/macrophages and was
purchased from Becton Dickinson, Mountain View,
Calif. Anti-Leu-10 (clone SK10, IgGl), which reacts
with a polymorphic determinant of HLA-DQ expressed in linkage disequilibrium with DR-2, -4, and
-6, and anti-HLA-DP (clone B7/21, IgGl), which
reacts with a framework-specific determinant of HLADP, were purchased from Becton Dickinson.
Reagents
Recombinant human interleukin (IL)-la and -1,3
were gifts from S. Gillis, Immunex Corp., Seattle,
Wash. Recombinant human interleukin (IL-2) was
obtained from Cetus Corp., Emeryville, Calif. Chloroquine was purchased from Sigma Chemical Co.,
St. Louis, Mo.
Flow Cytometry
Immunofluorescent staining was performed as described elsewhere in detail.19 All dilutions, washing,
and incubations were performed in cold phosphatebuffered saline (0.1 M, pH 7.3) containing 0.1%
NaN3. Cytofluorographic analysis was performed by
means of an Ortho Cytofluorograph System 50H and
a FACStar (Becton Dickinson), using fluorescein
isothiocyanate-conjugated or phycoerythrin-conjugated monoclonal antibodies.
Isolation of Mononuclear Cell Subpopulations
Leukocyte-rich fractions from healthy Red Cross
blood donors were randomly selected. Peripheral
blood mononuclear cells were isolated by FicollHypaque gradient centrifugation, after which they
were rosetted by a single-step rosetting method20
using neuraminidase-treated sheep erythrocytes to
obtain erythrocyte-rosette-forming cells. Erythrocyte-rosette-forming cells were further depleted of
monocytes and B cells by plastic adherence and
passage over a nylon wool column. This yielded a
T-cell population that was >95% CD3+ and <0.3%
Leu-M3+ when analyzed by flow cytometry. These
T-cell-enriched populations were also functionally
depleted of monocytes, as demonstrated by the lack
of proliferative response to 1 ,ug/ml phytohemoagglutinin (not shown), which effectively induces
[3H]thymidine incorporation if the peripheral blood
mononuclear cells are not monocyte-depleted.
The T cells were used to isolate CD4- (>95%
CD8+) and CD8- (>95% CD4+) subsets by negative
selection using a panning technique.21 Both subsets
contained <0.1% Leu-M3+ cells.
To obtain monocyte-enriched ACs, the nonrosetting fraction from the erythrocyte-rosette-forming
cell isolations was adhered to plastic flasks (60 minutes at 37°C) and recovered with 1% EDTA (10
Pardi and Bender T-Cell Alloreactivity to Microvascular Endothelium
minutes at 4°C) and gentle scraping. The resultant
population was >85% Leu-M3+ and had a characteristic monocyte appearance by light microscopy and
cytofluorographic light scatter.
CD4' Cell Clone Generation
Peripheral blood mononuclear cells obtained from
a donor were rosetted and monocyte-depleted as
described above. CD4+ cells (lx 106) were plated in
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each well of a 24-well plate (Costar Corp., Cambridge, Mass.) containing confluent monolayers of
either the unstimulated MHC class II negative EC30
line or the y-interferon-treated (60 units/ml) MHC
class LI positive EC45 line in medium containing 10%
pooled human serum, 10% lymphoblastoid cell lineconditioned medium, 20 units/ml IL-2, and 2x i0'
irradiated autologous (30 Gy) ACs. The T cells were
refed with fresh medium every 3-4 days and added to
fresh Ta negative or positive EC monolayers at weekly
intervals, at which time they also received fresh,
irradiated autologous ACs. After 28 days, the initial
populations had gone through a 30-40-fold expansion and were >95% CD4+. Cells were then cloned
by limiting dilution into microtiter wells at 0.5 cells/
well and propagated under identical culture conditions. For functional analyses, cloned cells were
"rested" in medium without IL-2 or stimulator cells
for 18-24 hours before the assay.
Proliferation and T-Cell Growth Factor
Production Assays
Lymphocyte proliferation was evaluated on the
basis of [3H]thymidine incorporation. Briefly, 5 x 104
lymphocytes were cultured in microtiter well plates
with or without irradiated (30 Gy) allogeneic stimulator ECs (lx 104/well), irradiated autologous ACs
(1 x 104/well), and IL-1 or IL-2 at the indicated
concentrations. Proliferation was assessed at day 7 by
the addition of 1 ,uCi/well [3H]thymidine 18 hours
before harvest on glass fiber filters. When testing
proliferation of cloned cells, identical cell ratios were
used, including allogeneic ACs in one combination
tested, but the cultures were carried out for 72 hours
and pulsed with [3H]thymidine 12 hours before harvest. All samples were performed in triplicate.
T-cell growth factor activity was quantitated using
[3H]thymidine incorporation during dose-dependent
proliferation of the IL-2-dependent murine T-cell
line HT-2, as described elsewhere.22
T-Cell-Mediated Cytotoxicity Assay
T, CD4+, or CD8+ cells were cultured in 12-well
plates (1 x 106 cells/well) containing confluent EC
monolayers (2x i0' cells/well), with or without irradiated autologous ACs, 2 units/ml IL-2, or 10
units/ml IL-1 in 2 ml medium containing 10% pooled
human serum. In some experiments, culture supernatants from ECs or 72-hour CD4+/EC cocultures
were added at a 1:1 ratio to the indicated coculture
media. After 7 days, the suspension cells, which were
always >90% viable (both CD4+ and CD8+ cells),
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were recovered and used as effectors in 51Cr release
assays. These assays were performed in flat-bottomed
microtiter wells with confluent EC monolayers, either autologous or allogeneic to the stimulating EC
line, which had been labeled with 2 ,Ci/well 5`Cr for
2 hours at 37°C. Spontaneous and maximum release
was determined by incubating labeled targets in
culture medium or 0.7% Triton X-100 in glycerol/
water, respectively. Mean corrected percent lysis in
triplicate culture was calculated as follows: cytotoxicity (%)=(mean experimental cpm-spontaneous
release cpm)/(maximum release cpm-spontaneous
release cpm)x100. Spontaneous release was always
<20% of maximum release.
In some experiments, monoclonal antibodies (10
,ug/ml) were added either directly to the 4-hour assay
or used to pretreat either the effector or target cells
for 30 minutes at 37°C, after which unbound antibody
was washed out and the assay was performed. In
other experiments, ACs were treated with 500 ,uM
chloroquine at the onset of the 7-day stimulation
period. In preliminary experiments, 500 ,uM chloroquine gave the most efficient functional inhibition
without affecting cell viability (not shown) and was
therefore used in subsequent experiments. ACs were
suspended at 1 x 106/ml in RPMI medium containing
500 ,uM chloroquine for 30 minutes at 37°C, after
which they were flooded with 10% fetal calf serum,
washed twice, irradiated (30 Gy), and added to the
responder cells for the 7-day culture. Alternatively,
ACs were pulsed overnight with the relevant ECs at
a 1:10 (AC: EC) ratio and chloroquine-treated the
next day before the initiation of the 7-day culture. In
other experiments, the natural killer (NK)-sensitive
line K562 was 51Cr-labeled (50 LCi/106 cells) and
used as a target in cytotoxicity assays.
Results
Basal Requirements for the Generation of Anti-EC
T-Cell-Mediated Immune Response
Primary sensitization in vitro of highly purified T
cells or T-cell subsets with allogeneic microvascular
ECs is not sufficient to stimulate a detectable anti-EC
cytotoxic response (Figure 1). This is not dependent
on the particular allogeneic combination used or the
time required to generate cytotoxic T lymphocytes
(CTLs), as identical results were obtained using
multiple genetically unrelated EC lines as stimulators
or prolonging the primary lymphocyte/EC coculture
for as long as 3 weeks (not shown).
These results are at variance with the classical
mixed lymphocyte reaction assay, in which mononuclear lymphoid cells are used as stimulators. We
reasoned that either unstimulated ECs did not display immunogenic MHC antigens or that they were
unable to provide additional signals required for
triggering the proliferation and differentiation of
allospecific anti-EC precursor T cells. To address
these points, primary lymphocyte/EC cocultures were
performed in the presence of low concentrations of
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Circulation Research Vol 69, No 5 November 1991
60
45
T cells
60
CD8+ cells
60
CD4+ cells
control
IL-1 10 u/mI
2 u/mI
IL-2
autologous AC
45 -45-
010
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0
0
0
T
cells
or
T-cell
rosetted
purified
subpopulations cocultured with allogeneic endothelial cells for
FIGURE 1. Bar graphs showing
7 days, with or without interleukin (IL)-1, IL-2, or autologous accessory cells (AC), after which the lymphocytes were recovered and
used as effectors in a 4-hour cytotoxicity assay, using the relevant endothelial cell line as a target. Similar results were demonstrated
in four different experiments all of which were performed at an effector to target ratio of 50:1. Error bars represent standard
deviations from the mean of triplicate samples.
recombiant lymphokines or irradiated AC-enriched
populations autologous to the responder T cells. Our
results demonstrate that these additional signals are
indeed capable of driving a functional anti-EC cytotoxic response by T cells (Figure 1). Moreover, they
indicate that the minimal requirements for an anti-EC response by CD8+ and CD4+ T-cell subsets are
distinct, since the former subset can be induced to
become CTLs by the addition to the cultures of 2
units/ml IL-2, 10 units/mi IL-1, or, to a lesser extent,
autologous ACs (Figure 1), whereas only the presence in the primary culture of irradiated autologous
accessory cells can stimulate an anti-EC cytotoxic
response by purified CD4+ T lymphocytes (Figure 1).
As previously shown,23 IL-2 concentrations >5 units/
ml, although more efficient in driving anti-EC CD8+
T-cell-mediated cytotoxicity, would increase the
amount of nonspecific killing, as detected by using
the K562 target cell line (not shown). In contrast,
IL-2 could not generate an anti-EC CD4+ CTL
response even at concentrations as high as 50
units/ml (not shown). EC culture or CD4+/EC coculture supernatants had no effect on the ability to
derive EC-specific CD4+ cytotoxic T cells in the
presence of autologous ACs or EC-specific CD8+
cytotoxic T cells in the presence of IL-2, ruling out
that the inability of CD4+ T cells to respond to ECs
in the absence of ACs was due to the elaboration of
relevant suppression of inhibitory factors in the experimental conditions (not shown).
When the T-cell or T-cell subset proliferation was
evaluated under the same experimental conditions,
similar results were observed (Table 1). IL-2 (2
units/ml) was the most efficient costimulatory signal
for CD8+ lymphocytes, whereas autologous ACs
were essential for driving an anti-EC proliferative
response by purified CD4+ cells (Table 1).
Specificity of the Anti-EC Cytotoxic Response by
CD4+ and CD8+ T Lymphocytes
To further investigate whether the costimulatory
signals required to generate anti-EC cytotoxic T cells
were nonspecifically triggering lytic mechanisms by the
effector cells and whether EC alloantigens were actually involved in the generation of the response, multiple
genetically unrelated EC lines were used as targets in
the final cytotoxicity assay, in addition to the NKsensitive line, K562. The results (Figures 2a and 2b)
demonstrate that the two most efficient pathways for
generating an anti-EC cytotoxic response by T-cell
subsets (i.e., CD8+ cells plus 2 units/ml IL-2 and CD4+
cells in the presence of irradiated autologous ACs)
display a high degree of target-related specificity. This
is further demonstrated by crossover killing experiments, where CD8+ (Figure 2c) or CD4+ (Figure 2d)
cells from another donor were cocultured with a different EC line (EC44) than that shown in Figures 2a and
2b. The absence of reciprocal killing (CD8 or CD4
anti-EC45 lyses EC45 better than EC44, and CD8 or
CD4 anti-EC44 lyses EC44 better than EC45) indicates
that the stimulating EC alloantigens are, in fact, responsible for target specificity and that differential
target sensitivity is not a major component of the noted
phenomenon. Furthermore, fibroblasts syngeneic to
the target EC44 line are also lysed by the CD8+ and
CD4+ effector cells (Figures 2c and 2d), suggesting that
the target antigens are shared by ECs and fibroblasts.
With fibroblast targets, the percent cytotoxicity is lower
at each effector to target cell (EiT) ratio. A greater or
more homogeneous expression of the putative alloge-
Pardi and Bender T-Cell Alloreactivity to Microvascular Endothelium
1273
TABLE 1. Allogeneic Endothelial Cell-Induced Proliferative Response and Interleukin-2 Production by T Cells and
T-Cell Subsets
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Proliferation (cpm)
Responders
HT-2 cell responset (cpm)
Cofactors*
310±40
None
T cells
399+78
T cells
5,310±617
IL-2
9,911 +799
T cells
6,601±+788
IL-1
9,002+786
T cells
Auto ACs
8,356+1,005
19,566±2,100
None
206±46
CD4+ cells
602±90
IL-2
701±109
2,654±769
CD4+ cells
987± 110
1,292±264
IL-1
CD4+ cells
8,166±1,203
20,001±2,504
Auto ACs
CD4+ cells
504±70
320±48
None
CD8+ cells
IL-2
3,899±526
CD8+ cells
8,543+1,320
2,902±456
IL-1
CD8+ cells
3,700+601
ND
ND
Auto ACs
CD8+ cells
Values are mean±SEM of triplicate cultures. HT-2, interleukin (IL)-2-dependent murine T-cell line; Auto ACs,
autologous accessory cells; ND, not done. Purified T cells or CD4+ and CD8+ T cells (1 x 105) were cocultured with
irradiated allogeneic endothelial cells for 6 days, after which 1 ,Ci [3H]thymidine was added, and cultures were
harvested 18 hours later.
*Lymphokine concentrations were 2 units/ml for IL-2 and 10 units/ml for IL-1. Monocyte-enriched populations
autologous to the responder lymphocytes (Auto ACs) were irradiated and added to the cultures at a monocyte to T-cell
ratio of 0.2: 1. T-cell response to Auto ACs, as measured by [3H]thymidine incorporation, never exceeded 2,000 cpm.
tT-cell growth factor activity was assessed with the HT-2 cell bioassay. HT-2 cells (2 x 104) were cultured for 24 hours
in the presence of 100 ,ul of 72-hour culture supernatant from the indicated cell combinations. Samples were labeled
with 1 ,uCi [3H]thymidine 4 hours before harvesting the cells.
neic target molecule(s) may be present on ECs, although it is possible that fibroblasts are relatively more
resistant to T-cell-mediated lysis.
Cell Surface Antigens Involved in T-Cell-Mediated
Anti-EC Cytolysis
To identify the surface molecules involved in the
anti-EC cytotoxic response by T-cell subsets, soluble
monoclonal antibodies directed toward a number of
lymphocyte or EC membrane antigens were added to
the final cytotoxic assay. Figure 3a shows that antibodies recognizing the CD3-T cell receptor complex, the
CD8 antigen, and MHC class I framework determinants were effective, to a variable extent, in blocking
the CD8+ cell-mediated anti-EC cytotoxic activity.
Experiments performed by pretreating either the effector or the target cells with the indicated antibodies
demonstrated that MHC class I determinants on the
surface of ECs, but not of lymphocytes, were involved
in the recognition process leading to EC lysis by CD8+
T cells (not shown).
When the same panel of antibodies was added to
CD4+ cells that had been cultured with ECs, only
antibodies against the CD3iTcR complex or the CD4
molecule could effectively inhibit the effector function
of this cell subset. Anti-class I, anti-HLA-DP, antiHLA-DQ, and two anti-HLA DR antibodies recognizing distinct epitopes of the heterodimer did not block
this activity at E/T ratios as low as 6: 1 (Figure 3b, data
not shown). ECs used as stimulators in the generation
of EC-specific T cells or T-cell subsets were, in fact,
always MHC class II antigen positive at the end of the
7-day coculture period (data not shown). However, to
further confirm the lack of involvement of EC Ia
antigens in the CD4+ T-cell-mediated anti-EC response, resting ECs, in conditions identical to the assay
targets, were stained with antibodies recognizing monomorphic HLA-DP, -DQ, and -DR epitopes. Figure 4
shows that, as previously demonstrated with large vessel-derived ECs,1 microvascular ECs used as targets in
the cytotoxicity assay do not express detectable levels of
membrane MHC class II antigens, whereas surface
MHC class I determinants are constitutively present at
a high density. When induced, EC membrane HLA
class II is detectable on the cell surface (Figure 4d).
Role ofAutologous ACs in the Generation of CD4+
T-Cell-Mediated Anti-EC Response
The above results demonstrate an essential role for
irradiated autologous ACs in the generation of an
anti-EC response by CD4+ lymphocytes, but their precise role is uncertain. Conceivably, these cells produce
soluble factors, distinct from IL-1, that act as costimulatory signals in the expansion and differentiation of
CD4+ allospecific T-cell precursors. Alternatively, ACs
might process and present EC-derived membrane alloantigens in an immunogenic manner to autologous
CD4+ lymphocytes. To explore these possibilities, ACs
were pulsed overnight with the stimulator EC line and
then treated with the weak base chloroquine, an agent
known to block exogenous antigen processing by inhibiting the catabolism of peptides in intracellular acidic
vesicles.24,25 Chloroquine-treated alloantigen-pulsed
ACs were then used as costimulators in the CD4+
lymphocyte/EC primary culture. The results (Figure 5)
demonstrate that treatment of autologous ACs with
chloroquine (500 gM) before the addition of ECs
abrogated the subsequent generation of anti-EC cyto-
Circulation Research Vol 69, No 5 November 1991
1274
a
EC 45
O EC44
0 EC 33
K562
b
451
a
4'
0)
.0
0
15
0
15
50:1 25:1
12.5:1
6:1
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25:1 12.5:1
E/T ratio
EIT ratio
60
60
-d
FIGURE 2. Graphs showing microvascular endothelial cell (EC) line 45 cocultured
for 7 days with CD8+ T cells in the
presence of 2 units/ml interleukin-2 (panel
a) or CD4+ T cells in the presence of
irradiated autologous accessory cells (accessory cell to lymphocyte ratio, 0. 2:1)
6:.1
(panel b). EIT ratio, effector to target cell
ratio. After 1 week, the lymphocytes were
recovered and used as effectors in a 4-hour
cytotoxicity assay, with the relevant or irEC 44
relevant
EC lines as targets, as well as the
Cl EC45
o
5 natural killer target K562. The identical
EC
o
cocultures and cytotoxicity experiments
* K.562 were performed with CD8' T cells (panel
* Fib 44 c) or CD4+ T cells (panel d) obtained
from a different donor than for panels a
and b and stimulated with EC line 44. Fib
44 is syngeneic to EC44.
EC62
45
45
C)
Co
.0
0
,, 30
30
KR
01
--a
1
15
0 50:1
25:1 12.5:1 6:1
E/T ratio
o
N
,v
-a
11
-
-
50:1 25:1 12.5:1
E/T ratio
toxic CD4+ T cells, whereas the drug was ineffective
when used after the ACs had been pulsed overnight
with allogeneic ECs. This strongly suggests that an
intracellular alloantigen processing step, carried out by
ACs, is a prerequisite for the sensitization of anti-EC
allospecific CD4+ precursor T lymphocytes.
Analysis of the CD4' T-Cell-Mediated Anti-EC
Response at the Clonal Level
The restriction elements responsible for the induction of anti-EC-specific, CD4+ CTLs were further
analyzed by generating CD4+ cytotoxic T-cell clones,
under limiting dilution conditions, using MHC class
II negative allogeneic EC lines as stimulators in the
presence of ACs autologous to the responder lymphocytes. Data obtained with one representative
clone, designated AT.30.1, are illustrated in Table 2.
This clone is a CD3+, CD4+, and a1if3 T-cell clone, as
demonstrated by its reactivity with the monoclonal
6:1
antibody WT31, which recognizes an epitope on the
antigen receptor complex of a1lp T cells. Also, the
AT.30.1 clone does not kill K562, nor does it express
classical natural killer cell markers; thus, it is not a
natural killer or natural killer-like T-cell clone (not
shown). The data demonstrate that the same cloned
cell line is capable of EC-specific, self-MHC-restricted proliferation in the presence of autologous
monocytes as a source of ACs and of cytotoxicity to
the relevant EC line. IL-2 also is extremely efficient
in triggering a proliferative response by cloned CD4'
cells. Of note, ECs alone were extremely inefficient
in driving a proliferative response by the AT.30.1
clone, although they could serve as a sensitive target
in the cytotoxic assay.
Three additional clones (out of 36 anti-EC CD4+
clones tested) had functional properties similar to
the AT.30.1 clone; the remainder were not capable of
anti-EC-specific cytotoxic activity (not shown). Anti-
Pardi and Bender T-Cell Alloreactivity to Microvascular Endothelium
0
% specific lysis
25
50
% specific lysis
irrel.Gi
irrel.G23
anti-CD3
anti-HLA-I
anti-HLA-DR
anti-CD8
anti-C D4
a
b
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FIGURE 3. Bar graphs showing microvascular endothelial cells
cocultured for 7 days with CD8+ T cells in the presence of 2
units/ml interleukin-2 (panel a) or CD4+ T cells in the presence
of irradiated autologous accessory cells (accessory cell to lymphocyte ratio, 0.2: 1) (panel b). irrel. G, and G2, irrelevant
immunoglobulin GJ and G2a. After 1 week; the lymphocytes
were recovered and used as effectors in a 4-hour cytotoxicity assay
with the relevant endothelial cell target, in the presence of
saturating concentrations (10-25 pglml) of the indicated monoclonal antibodies. Results are representative of four separate
experiments, allperformed at effector to target cell ratios of 25:1.
1275
body inhibition studies of this clone confirmed the
results obtained at the bulk culture level, demonstrating the involvement of the CD3/T-cell receptor
complex and the CD4 molecule in both the inductive
and effector phases of the anti-EC response by CD4'
lymphocytes (Table 2). Anti-MHC class I and two
anti-HLA-DR antibodies recognizing nonoverlapping epitopes of the DR molecule were again unable
to inhibit the allogeneic EC-specific cytotoxicity mediated by cells of the CD4' sublineage. When y-interferon-treated, MHC class II-expressing ECs were
used as stimulators to generate allospecific CD4+
CTL clones, the recognition pattern of the effector
cells was at variance with the results observed using
MHC class II negative stimulators. Under these
circumstances, the majority of the CTL clones tested
appeared to recognize MHC class II determinants, as
shown by the absence of killing of Ia negative EC
targets and by the inhibition of class IL+ EC target
lysis observed using anti-class II monoclonal antibodies (Table 3).
Discussion
Donor microvascular endothelium is likely to be
involved both as a stimulus and as a target in
FIGURE 4. Cell-sorter immunofluorescent analysis of microvascular
endothelial cells cultured in standard
medium'8 (panels A-C and E) or
medium containing 100 units/ml
yinterferon (panel D), comparing
irrelevant immunoglobulin GJ staining with specific endothelial cell
surface major histocompatibility
complex expression. Monoclonal antibodies used were fluorescein isothiocyante (FITC)-CA-141 (anti-HLADR, panel A), FITC-Leu-10 (antiHLA-DQ, panel B), FITC-anti-DP
(anti-HLA-DP, panel C), FITCCA-141 (panel D), and FITC-W6/32
(anti-HLA class I, panel E). Ten
thousand cells were analyzedfor each
sample.
1276
Circulation Research Vol 69, No 5 November 1991
Exp. 1
60
45
45
k,
Exp. 2
60 r-
2
30
15
15
[-
t
0
50:1
25:1 12.5:1
EIT ratio
1
6:1
0
50:1
25:1
12.5:1
6:1
EIT ratio
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
FIGURE 5. Graphs showing punified CD4+ T cells cocultured for 7 days with allogeneic endothelial cells and autologous accessory cells that had been pretreated with medium
(D), 500 pM chloroquine (o), or 500 MM chloroquine after
ovemight pulsing with the relevant endothelial cell line (o).
EIT ratio, effector to target cell ratio. After 1 week, the
lymphocytes were recovered and used as effectors in a 4-hour
cytotoxicity assay with the relevant endothelial cell target.
vascularized allograft rejection, since it forms a contiguous barrier, bearing surface donor alloantigens,
to circulating recipient lymphocytes. Immunohistological data in human vascularized grafts undergoing
rejection support this notion, because they frequently
demonstrate widespread microvascular damage,
which is initially seen in those venules, arterioles, and
small veins enveloped by lymphocytes.26 However,
analysis of mononuclear cell infiltrates in rejected
allografts is not sufficient to determine which lymphocyte subset is primarily responsible for damaging
the donor tissue, nor does it clarify the sequence of
events underlying the rejection process. For this
reason, we analyzed the signals and restriction elements used by unprimed lymphocyte subsets to generate a functional response in vitro to allogeneic ECs
of microvascular origin. The results demonstrated
that resting microvascular ECs are unable to trigger a
functional response by allogeneic monocyte-depleted
T cells. These observations confirm the data obtained
using ECs derived from large veins as stimulators27
and suggest that T cells, unlike their functional
response to genetically unrelated lymphoid cells,
require additional signals to proliferate and differentiate into allospecific effectors. Because ECs express
surface class I MHC determinants with a density
comparable to lymphoid cells,' it is likely that their
inability to stimulate allogeneic T cells reflects either
defective "accessory" cell function (i.e., soluble factor production or antigen processing capabilities) or
insufficient expression of MHC class II allodeterminants to be recognized by allospecific T cells. The
induction of alloantigen-specific T-cell proliferation
and cytotoxicity to allogeneic ECs in the presence of
low concentrations of IL-1 or IL-2 (Figure 1 and
Table 1) would suggest that surface alloantigens
displayed by ECs are normally recognized by precursor T cells, whereas the IL-1 mediated activation
pathway involved in the afferent limb of the T-celldependent immune response28 is defective.
This conclusion is supported by the experiments
performed with CD8' cells (Figure 1 and Table 1),
which were induced to lyse the sensitizing EC line if
as little as 2 units/ml IL-2 were present in the 7-day
cultures. This result suggests that CD8+ cells express
TABLE 2. Proliferative and Cytotoxic Activity of Allogeneic Endothelial Cell-Specific CD4+ T-Cell Clone (AT.30.1)
Proliferationt
Inhibition (%)
cpm
...
2,812
...
66,332
...
5,835
6,002
0
25,331
2.5
24,698
38.2
15,639
44.0
14,208
0
26,112
33.6
16,838
45.2
13,901
Cytolysist
Inhibition (%)
Lysis (%)
Stimulators
Cofactors
Blocking MAb*
+
...
...
...
None
+
...
...
...
IL-2 (2 units/ml)
...
Auto ACs
+
...
Allo ACs
0
...
+
78.0
Auto ACs
71.8
8.0
+
Irrelevant
Auto ACs
53.8
39.0
+
Anti-CD3
Auto ACs
28.4
63.6
+
Anti-CD4
Auto ACs
+
64.0
18.0
Auto ACs
W6/32
+
CA-141
74.9
4.0
Auto ACs
+
L243
71.0
9.0
Auto ACs
ND
...
74.0
+
Anti-LFA-1
20.3
Auto ACs
71.4
+
Anti-LFA-3
ND
...
8.5
Auto ACs
MAb, monoclonal antibody; IL-2, interleukin-2; Auto ACs, autologous accessory cells; Allo ACs, allogeneic
accessory cells; LFA, lymphocyte function associated; ND, not done.
*The indicated antibodies (10 ,ug/ml) were added at the beginning of the culture or during the 4-hour cytotoxic assay.
tAT.30.1 cloned cells were recovered, rested for 18 hours in the absence of stimulating antigen or IL-2, and cultured
(5 x 104) for 72 hours in the presence of the indicated cofactors, with or without irradiated endothelial cells (lx 104)
from the line originally used as stimulator. Irradiated autologous (auto AC) or allogeneic (allo AC) monocytes were
added at a monocyte to T-cell ratio of 0.2: 1. Twelve hours before harvesting the cultures, 1 gCi [3H]thymidine was
added.
*Cloned AT.30.1 cells were washed and added to 5`Cr-labeled endothelial cells from the original stimulator line at
a T-cell to endothelial cell ratio of 50: 1, with or without 10 ,ug/ml of the indicated antibodies. Four hours later, `tCr
release was evaluated in the supernatants, and specific lysis was calculated as described in "Materials and Methods."
Pardi and Bender T-Cell Alloreactivity to Microvascular Endothelium
1277
TABLE 3. Cytotoxic Activity of CD4+ T-Cell Clones Induced by Major Histocompatibility Complex Class II Allogeneic
Endothelial Cells
Ia+ EC45t
Ia- EC45
Clone
Blocking MAb*
Lysis (%)
Inhibition (%)
Lysis (%)
Inhibition (%)
AT.45.3
...
65.0
0
8.1
0
CA-141
28.1
56.8
7.5
9.7
W632
63.8
1.9
8.3
0
...
AT.45.8
58.3
0
2.1
0
CA-141
19.3
66.9
3.0
0
W632
60.0
0
2.6
0
MAb, monoclonal antibody; EC, endothelial cell. EC45-specific cloned cells were recovered, rested for 24 hours in
the absence of stimulating cells, and assayed for cytotoxic activity against 51Cr-labeled EC45 cells at a T-cell to EC ratio
of 25: 1.
*The indicated MAbs (10 ,tg/ml) were added during the 4-hour chromium release assay.
tTo induce major histocompatibility complex class II expression, EC45 cells were pretreated for 72 hours with 60
units/ml y-interferon before the cytotoxicity assay.
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
functional IL-2 receptors on recognition of ECspecific alloantigens but that they do not progress
into the differentiation pathway leading to allospecific effector CTLs. The presence of IL-1 or autologous ACs could, therefore, provide the additional
signals required for the production of IL-2 by CD8+
cells,29 thus driving an autocrine allospecific response
by this lymphocyte subset. The demonstration of an
autocrine loop, driven by allogeneic class I MHC
antigens and entirely mediated by CD8+ lymphocytes, has precedent, as "helper-independent" cytotoxic lymphocytes have been described both in murine and human experimental systems.23,30 Blocking
experiments (Figure 3a) carried out in the final lytic
assay using allogeneic EC-primed CD8+ cells as
effectors demonstrated that, as expected, membrane
class I MHC allodeterminants on ECs represent the
major target and presumed stimulus for this lymphocyte subset. The CD3/TcR and CD8 molecular complexes on the effector cells appear to be involved in
the recognition and subsequent lysis of the stimulating EC line. These findings confirm observations
made by other investigators'4 and indicate that class
I MHC antigens displayed by ECs are fully capable of
triggering allospecific CD8+ CTL precursors. This
functional correlation between CD8+ cells and EC
class I MHC alloantigens is consistent with the
molecular constraints originally described in the classical mixed lymphocyte reaction model3' and subsequently confirmed by functional studies performed
on lymphocytes infiltrating class I MHC incompatible
grafts.32-35
The analysis of the CD4+ cell-mediated anti-EC
response provided interesting and somewhat unexpected results. The existence of CD4+ cells capable
of differentiating into cytotoxic effectors has been
clearly demonstrated when allogeneic MHC class II
antigen positive cells have been used as stimulators,36
and it was confirmed in the present study using
y-interferon-treated Ia-positive ECs as stimulators
(Table 3). Nonetheless, the likelihood of detecting
such activity in CD4+ lymphocytes cultured for 1
week with allogeneic ECs seemed low, since HLA-
DP, -DQ, and -DR antigens are not detectable on
microvascular ECs under these conditions (Figure 4).
Clonal analysis of the CD4+ cell-mediated anti-EC
cytotoxic response demonstrates that, at least in
some instances, the same cell can be induced to
proliferate in response to the relevant EC alloantigens and to differentiate toward the final effector
CTLs (Table 2). The presence in the sensitizing
culture of autologous ACs appears to be a condition
necessary and sufficient for generating a detectable
response by CD4+ cells in our experimental model.
The activity of autologous ACs in the culture system
is not replaceable by IL-1 or IL-2 (Figure 1 and Table
1), but it is completely abrogated by the treatment of
ACs with 500 ,uM chloroquine before alloantigen
pulsing (Figure 5). This strongly suggests that intracellular processing of allogeneic EC-derived antigens
is required to trigger the recognition of allogeneic EC
determinants by CD4+ lymphocytes. Results obtained with an allogeneic EC-specific CD4+ clone
(Table 2) suggest that self-MHC class Il-expressing
autologous ACs, in addition to processing EC-derived antigens, provide the necessary restriction elements for CD4+ cells, as shown by the inhibition
observed with anti-Ia antibodies and by the inability
of ACs bearing MHC antigens unrelated to those of
the responding T cells to stimulate a proliferative
response to the sensitizing EC line. The most likely
explanation for these findings is that alloantigen
processing and presentation in the context of selfMHC membrane molecules by ACs is a requirement
for the stimulation of allospecific responses by CD4+
lymphocytes. This interpretation is further supported
by other studies using cell-free minor histocompatibility antigens as stimulators of a T-cell-mediated
cytotoxic response in the murine system37-39 and,
more recently, by the elegant demonstration that
peptide fragments derived from the hypervariable
domain of MHC molecules physically associate with
membrane Ia antigens on antigen-presenting cells
and effectively stimulate an allospecific T-cell response.40'41 The EC-specific alloantigens responsible
for the induction of CD4+ cell-mediated anti-EC
1278
Circulation Research Vol 69, No 5 November 1991
responses remain elusive, as resting ECs do not
express detectable surface HLA-DP, -DQ, or -DR
antigens, which are typically involved in the trigger-
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
ing of CD4+ lymphocyte-mediated activities (Figure
4 and Reference 33). Particularly difficult to understand is how antigen presentation by autologous ACs
leads to recognition and lysis of allogeneic ECs. Since
no monocytes were added to the cytotoxicity assays
and since the resting EC targets lack detectable class
II MHC determinants, the possibility exists that
cytolysis by the CD4+ CTLs is not specific for classical MHC class II (or class I) molecules. This is
surprising in light of the generally accepted view that
all or nearly all CD4+ T-cell functions are MHC class
LI restricted.42 On the other hand, since we have not
yet tested the effects on cytotoxicity of a large panel
of MHC class ll-specific antibodies, the possibility
remains that ECs may express quantities of MHC
class II antigens that are sufficient to trigger cytotoxicity but insufficient to generate an immunofluorescent signal or to stimulate clonal expansion. Alternatively, class II MHC determinants shed either from
the activated CD4+ effectors or autologous monocytes may have passively adsorbed onto allogeneic
EC targets during the 4-hour chromium release assay
and combined with EC alloantigens in a manner
recognized by CD4' CTL. There is precedent for
passive adsorption of Ia molecules onto T cells that
have been incubated with allogeneic non-T mononuclear cells.43 Finally, it is possible that non-MHCencoded EC antigens might play a major role in the
sensitization of an allospecific CD4+ CTL precursor.
This concept is supported by the existence of ECspecific alloantigens apparently responsible for allograft rejection in HLA-identical individuals44 and
by recent reports demonstrating a role for CD4+
cytolytic4546 and noncytolytic47 cells specific for nonMHC-encoded determinants. All of these possibilities remain to be explored.
In conclusion, our results indicate that the generation of a functional immune response to allogeneic
vascular endothelial cells, which are defective in AC
activity, requires a complex interaction between different cell types having specialized functions. In
addition to the role of CD4' and CD8' precursor
lymphocytes capable of clonal expansion, cytokine
production, and allo-EC-specific cytotoxicity, our
data underscore the importance of ACs of host origin
in the inductive phase of the response to allogeneic
tissues. ACs autologous to the responder lymphocytes appear to be critical not only as a source of
soluble factors, such as IL-1, involved in the clonal
expansion and subsequent differentiation of allo-EC
specific precursors but also as elements capable of
uptaking and processing the alloantigens required to
sensitize CD4+ precursors in association with the
appropriate restriction elements.48 These data lend
support to the observations made in vivo in experimental allograft rejection models, showing that adoptive transfer of whole mononuclear cell populations
to a sublethally irradiated host is consistently more
effective in promoting allograft rejection than selected lymphocyte subsets of the helper or cytotoxic
lineage (for review, see Reference 26). Moreover,
they provide evidence for the existence of a sequential activation process, involving ACs and CD4+ and
CD8' T cells, in the response to nonlymphoid allogeneic tissues, as suggested by histological data that
shows the serial appearance of activated cells belonging to these lineages in cellular infiltrates associated
with graft rejection.49,50
Acknowledgments
We gratefully acknowledge Edgar G. Engleman
(Stanford University), in whose laboratory some of
the experiments were performed, for his advice and
critical review of the manuscript. We also thank
Jennie Annatone, Georgi Danley, and Kristie Vollono for preparation of the manuscript, Leslie Tackett for assistance with endothelial cell culture, and
Silvia Heltai for her help with cytofluorography.
References
1. Stastny P, Nunez G: The alloantigens of endothelial cells, in
Jaffe EA (ed): Biology of Endothelial Cells. Boston, Martinus
Nijhoff Publishers, 1984, pp 377-384
2. Marlene LR, Coles MI, Griffin RJ, Pmerance A, Yacoub MH:
Expression of class I and II major histocompatibility antigens
in normal and transplanted human heart. Transplantation
1986;41:776-781
3. Hall BM, Bishop GA, Duggin GG, Horvath JS, Philips J, Tiller
DJ: Increased expression of HLA-DR antigens on renal
tubular cells in renal transplants: Relevance to the rejection
response. Lancet 1984;2:247-249
4. DeWaal RMW, Bogman JJ, Maars CN, Cornelissen LMH,
Tax WJM, Koene RAP: Variable expression of Ia antigens on
the vascular endothelium of mouse skin allografts. Nature
1983;303:426-429
5. Dvorak HF, Mihm MC, Dvorak AM, Barnes BA, Manseau EJ,
Galli SJ: Rejection of first set skin allograft in man: The
microvasculature is the critical target of the immune response.
J Exp Med 1979;105:322-335
6. Pardi R, Bender JR, Engleman EG: Lymphocyte subsets
differentially induce class II human leukocyte antigens on
allogeneic microvascular endothelial cells. J Immunol 1987;
139:2585-2595
7. Pober JS, Gimbrone MA Jr, Cotran RS, Reiss CS, Burakoff
SJ, Fiers W, Ault KA: Ia expression by vascular endothelium
is inducible by activated T cells and by human interferon. JExp
Med 1983;157:1339-1351
8. Brasile L, Zerbe T, Rabin B, Clarke J, Abrams A, Cerilli J:
Identification of the antibody to vascular endothelial cells in
patients undergoing cardiac transplantation. Transplantation
1985;40:672-678
9. Blankert JJ, Van ES, Kunz HW, Cramer DV, Paul LC:
Genetics of the antibody response to an endothelial transplantation antigen in the rat. Hum Immunol 1986;15:125-129
10. Blankert JJ, Muizert Y, Van ES, Paul LC: Immunogenicity of
the non-MHC endothelial antigen EAG-1 in various tissues of
the rat. Transplantation 1987;43:736-742
11. Schook LB, Wood N, Mohanakumar T: Identification of
human vascular endothelial cell/monocyte antigenic system
using monoclonal antibodies. Transplantation 1987;44:412-416
12. Clayberger C, Uyehara T, Hardy B, Eaton J, Karasek M,
Krensky AM: Target specificity and cell surface structures
involved in the human cytolytic T lymphocyte response to
endothelial cells. J Immunol 1985;135:12-20
13. Geppert TD, Lipsky PE: Dissection of defective antigen
presentation by interferon-y treated fibroblasts. J Immunol
1987;138:385-392
Pardi and Bender T-Cell Alloreactivity to Microvascular Endothelium
Downloaded from http://circres.ahajournals.org/ by guest on August 3, 2017
14. Kayroska A, Lipsky PE: Dissection of the functions of antigenpresenting cells in the induction of T cell activation. JImmunol
1985;135:2953-2958
15. Moissec P, Cavender D, Ziff M: Production of IL-1 by human
endothelial cells. J Immunol 1986;136:2486-2494
16. Leeuwenberg JFM, Von Asmuth EJU, Jeunhomme TMAA,
Buurman WA: IFN-y regulates the expression of the adhesion
molecule ELAM-1 and IL-6 production by human endothelial
cells in vitro. J Immunol 1990;145:2110-2114
17. Sieff CA, Tsai S, Fawer DV: Interleukin-1 induces cultured
human endothelial cell production of granulocyte-macrophage
CSF. J Clin Invest 1987;79:48-53
18. Bender JR, Pardi R, Karasek MA, Engleman EG: Phenotypic
and functional characterization of lymphocytes that bind
human microvascular endothelial cells in vitro. J Clin Invest
1987;79:1679-1688
19. Lifson J, Raubitschek A, Benike C, Koths K, Ammann A,
Sondel P, Engleman EG: Purified interleukin 2 induces proliferation of fresh human lymphocytes in the absence of
exogenous stimuli. J Biol Response Mod 1986;5:61-66
20. Weiner MS, Bianco C, Nussenzweig V: Enhanced binding of
neuraminidase-treated sheep erythrocytes to human T lymphocytes. Blood 1973;42:939-944
21. Wysocki LJ, Sato VL: Panning for lymphocytes: A method of
cell separation. Proc Natl Acad Sci U S A 1978;75:2844-2849
22. Watson J: Continuous proliferation of murine antigen specific
helper T lymphocytes in culture. J Exp Med 1979; 150:
1510-1519
23. Fishwild DM, Benike CJ, Engleman EG: Activation of HLArestricted EBV-specific cytotoxic T cells does not require
CD4+(helper) T cells or exogenous cytokines. J Immunol
1988;140:199-204
24. Ziegler K, Unanue E: Identification of a macrophage antigenprocessing event required for I-region-restricted antigen presentation to T lymphocytes. J Immunol 1981;127:1869-1880
25. Ziegler HK, Unanue E: Decrease in macrophage antigen
catabolism caused by ammonia and chloroquine is associated
with inhibition of antigen presentation to T cells. Proc Natl
Acad Sci USA 1981;79:175-179
26. Mason DW, Moris PJ: Effector mechanisms in allograft rejection. Annu Rev Immunol 1986;4:119-153
27. Geppert TD, Lipsky PE: Accessory cell-T interactions
involved in anti-CD3 induced T4 and T8 cell proliferation:
Analysis with monoclonal antibodies. J Immunol 1986;137:
3065-3073
28. Kaye J, Gillis S, Mizel SB, Shevach EM, Malek TR, Dinarello
CA, Lachman LB, Janeway CA: Growth of a cloned helper T
cell line induced by a monoclonal antibody specific for the
antigen receptor: Interleukin 1 is required for the expression
of receptors for interleukin 2. J Immunol 1984;133:1339-1350
29. Mizuochi T, McKean DJ, Singer A: IL-1 as a cofactor for
lymphokine-secreting CD8+ murine T cells. J Immunol 1988;
141:1571-1577
30. Isakov N, Biel LW, Bach FH: Induction of class I antigen:
Disparate pancreatic islet cell graft rejection by in vivo administration of cloned helper-independent cytolytic T lymphocytes. Transplant Proc 1985;17:727-731
31. Engleman EG, Benike CJ, Glickman E, Evans RL: Antibodies
to membrane structures that distinguish suppressor/cytotoxic
and helper T lymphocyte subpopulations block the mixed
lymphocyte reaction in man. J Exp Med 1981;153:193-202
32. Moreau JF, Vie H, Peyrat MA, Soulillou JP: Function and cell
surface markers of cloned T lymphocytes isolated from human
renal allograft biopsies. Transplant Proc 1985;17:810-814
33. Flomenberg N, Duffy E, Dupont B: HLA class II specific T
lymphocyte clones with dual alloreactive functions. Scand J
Immunol 1984;19:237-245
1279
34. Stepkowski SM, Duncan WR: The role of TDTH and T,
populations in organ graft rejection: Functional analysis of
graft infiltrating T cells. Transplantation 1986;42:406-410
35. Zeei A, Pung J, Zerbe TR, Kaufman C, Rabin B, Griffith BP,
Hardesty RC, Duquesnoy RJ: Allospecificity of activated T
cells grown from endomyocardial biopsies from heart transplant patients. Transplantation 1986;41:620-625
36. Mayer TG, Lazarovits AI, Boyle LA, Sullivan ER, Leary CP,
Fuller AA, Fuller TC, Bhan AK, Colvin RB, Collins AB,
Kurnick JT: Functional allospecific T lymphocytes isolated
from human renal allograft biopsies. Transplant Proc 1985;17:
816-819
37. Pilarski LM: In vitro cytotoxic T cell responses to cell-free
minor histocompatibility antigens: Requirement for antigen
presenting cells. Transplantation 1987;43:286-290
38. Golding H, Singer A: Role of accessory cell processing and
presentation of shed H-2 alloantigens in allospecific cytotoxic
T lymphocyte responses. J Immunol 1984;133:597-660
39. Weinberger 0, Germain RN, Springer TA, Burakoff SJ: Role
of syngeneic Ia+ AC in the generation of allospecific CTL
responses. J Immunol 1982;129:694-703
40. Parham P, Clayberger C, Zorn SL, Ludwig DS, Schoolnik GK,
Krensky AM: Inhibition of alloreactive cytotoxic T lymphocytes by peptides from the 2 domain of HLA-A2. Nature
1987;325:625-627
41. Clayberger C, Parham P, Rothbard J, Ludwig DS, Schoolnik
GK, Krensky AM: HLA-A2 peptides can regulate cytolysis by
human allogeneic T lymphocytes. Nature 1987;330:763-760
42. Littman DR: The structure of the CD4 and CD8 genes. Annu
Rev Immunol 1987;5:561-592
43. Yu DTJ, McCune JM, Fu SM, Winchester RJ, Kunkel HG:
Two types of Ia-positive T cells: Synthesis and exchange of Ia
antigens. J Exp Med 1980;152:895-908
44. Brasile L, Clarke J, Galouzis T, Cerilli J: The clinical significance of the vascular endothelial cell antigen system: Evidence
for genetic linkage between the endothelial cell antigen system
and the major histocompatibility complex. Transplant Proc
1985;17:741-743
45. Erb P, Grogg D, Troxler M, Kennedy M, Fluri M: CD4+ T
cell-mediated killing of MHC class II positive antigen presenting cells: Characterization of target cell recognition by in vivo
and in vitro activated CD4+ killer T cells. J Immunol 1990;
144:790-795
46. Korngold R, Sprent J: Variable capacity of L3T4+ T cells to
cause lethal graft vs. host disease across minor histocompatibility barriers in mice. J Eip Med 1987;165:155-166
47. Miconnet I, Huchet R, Bonardelle D, Motta R, Canon C,
Garay-Rojas E, Kress M, Reynes M, Halle-Pannenko 0,
Bruley-Rosset M: Graft vs. host mortality induced by noncytolytic CD4+ T cell clones specific for non H2 antigens.
J Immunol 1990;145:2123-2131
48. Sherwood RA, Brent L, Rayfield LA: Presentation of alloantigens by host cells. Eur J Immunol 1986;16:569-577
49. McWhinnie DL, Thompson JF, Taylor HM, Chapaman JR,
Bolton EM, Carter NP, Wood RFM, Morris PJ: Leukocyte
infiltration patterns in renal allografts assessed by immunoperoxidase staining of 245 sequential biopsies. Transplant Proc
1985;17:560-564
50. Hoshinaga K, Mohanakumar T, Goldman MH, Wolfgang TC,
Szenpetery S, Lee HM, Lower RR: Clinical significance of in
situ detection of T lymphocyte subsets and monocyte/
macrophage lineages in heart allografts. Transplantation 1984;
38:634-641
KEY WORDS * cytotoxic T lymphocytes * microvascular
endothelium * alloreactivity * allograft rejection
Signal requirements for the generation of CD4+ and CD8+ T-cell responses to human
allogeneic microvascular endothelium.
R Pardi and J R Bender
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Circ Res. 1991;69:1269-1279
doi: 10.1161/01.RES.69.5.1269
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