| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Biology and
Departments of Pathology and Immunology, Yale University, New Haven, CT 06510
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and IL-4 from both
naive and memory CD4+ T cells, quantified by ELISA or
intracellular cytokine staining. ECs, which lack B7 molecules, use
predominantly leukocyte-function associated Ag 3 (LFA-3) to
provide costimulation. ECs are comparable to or better than PBAMCs,
which use both the LFA-3 and B7 molecules, at costimulating IL-2 and
IL-4 production. ECs are less effective than PBAMCs at costimulating
IFN- production by naive T cells. ECs do not secrete IL-12, and
addition of exogenous IL-12 enables ECs to costimulate IFN- at a
level comparable to that observed with PBAMCs. ECs do not promote
differentiation of naive T cells to Th1-like cells, whereas PBAMCs do.
Again, addition of exogenous IL-12 enables ECs to do so. Transfection
of ECs to express B7-1 or B7-2 is less effective than IL-12
supplementation for restoring these responses. These experiments
suggest that a deficiency in costimulation due to lack of B7 molecule
expression does not fully explain the inability of ECs to activate
resting naive CD4+ T cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The resting T cell population in human blood consists of roughly equal numbers of naive cells, which have not previously encountered Ag, and memory cells, which have undergone a number of changes as a consequence of previous activation. In humans, naive cells generally express the A isoform of CD45 (i.e., CD45RA), whereas memory cells generally express the O isoform (i.e., CD45RO) (7). Typically, naive cells have a more stringent requirement for costimulation than do memory cells, although these differences are probably quantitative rather than qualitative (i.e., the same costimulatory molecules appear to interact with T cells at both stages of differentiation) (8, 9, 10, 11).
Once activated by Ag, naive CD4+ T cells may differentiate
to either Th1-like or Th2-like effector cells. These two T cell subsets
are defined by the cytokines they secrete (12). Th1-like cells
characteristically secrete IL-2, IFN-, and/or lymphotoxin and
serve to activate phagocyte-mediated immune responses. The signature
cytokines of Th2-like cells are IL-4 and/or IL-5. IL-4 is the major
inducer of IgE production by B cells and is therefore a key initiator
of IgE-dependent, mast cell-mediated reactions. IL-5 activates
eosinophils, the effector cells of Th2-mediated inflammatory responses.
Various factors have been found to direct the differentiation of naive
CD4+ T cells to Th1 vs Th2 subsets. The most potent in
vitro factor is the cytokine environment. Differentiation to Th1 is
promoted by the macrophage-derived cytokine IL-12 (13, 14), which also
boosts IFN- production by activated T and NK cells (15, 16). The
production of IL-12 by macrophages can be induced by various bacteria,
intracellular pathogens, viruses, and activated T cells. The role of
IL-12 in Th1 cell differentiation is supported by the finding that
IL-12 p40 knockout mice have severely impaired Th1 responses (17).
Differentiation to Th2 is promoted by IL-4, which may be produced by
mast cells, basophils, and/or a population of T cells (18, 19). IL-4
knockout mice have impaired production of IgE and a paucity of
IL-5-secreting T cells (20, 21). In addition to these cytokines,
differentiation of naive T cells to Th1 vs Th2 may also be affected by
the Ag quantity and affinity or the availability of the specific
costimulatory molecules. In vivo, these factors may be influenced by
the nature of the Ag, its mode of entry, and its tissue distribution
(22).
Vascular endothelial cells (ECs)3 have been thought to act as APCs in vivo, capable of substituting for conventional bone marrow-derived accessory cells (23). Cultured human vascular ECs express class I and (in response to T cell signals) class II MHC molecules (24, 25), displaying peptides to CD8+ and CD4+ T cells, respectively. Previous studies have shown that ECs are able to activate resting allogeneic memory T cells but not resting naive T cells, measured as the production of IL-2 or proliferation (26, 27). In contrast, bone marrow-derived "professional" APCs, such as peripheral blood-adherent mononuclear cells (PBAMCs), are able to activate both naive and memory resting allogeneic T cells. This limited capacity of ECs to activate T cells places them in a separate category of "semiprofessional" APCs, since nonimmunologic tissue cells, such as vascular smooth muscle cells or fibroblasts, are unable to activate any resting T cell populations, although such tissue cells can productively restimulate activated T blasts (28, 29, 30, 31, 32). The different capability of ECs and PBAMCs for activating alloreactive T cells may reside in differences in the Ags displayed and/or in provision of costimulation. Human vascular ECs express LFA-3 but not B7 molecules, whereas human PBAMCs express both LFA-3 and B7-2 and/or B7-1 on their surface (33, 34). It has been proposed that the lack of B7 molecules on ECs may account for the inability of ECs to activate naive CD4+ T cells (27).
In the present study, we wished to determine whether costimulation
provided by ECs was in fact deficient for full activation of naive
CD4+ T cells or whether other factors might contribute to
lack of responsiveness of naive T cells to Ags presented on cultured
ECs. To examine this question, we activate CD4+ T cell
subpopulations with PHA, bypassing signal 1, in the presence of either
ECs or PBAMCs, which act as a source of signal 2. The strength of
signal 2 is measured as augmented cytokine secretion. We find that
costimulation provided by ECs enhances IL-2, IFN-, and IL-4
production from naive as well as memory CD4+ T cells, and
that in general, ECs are comparable or better than PBAMCs as a source
of signal 2. The principal defects observed with ECs compared with
PBAMCs are that ECs are less effective costimulators of IFN-
production by naive CD4+ T cells and that ECs are unable to
promote differentiation of naive cells into Th1 effector cells. These
defects can be remedied by supplementing the EC cocultures with soluble
IL-12, a cytokine produced by activated PBAMCs but not by ECs.
Transfection of ECs to express B7-1 or B7-2 is less effective than
IL-12 supplementation in these assays.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human ECs were isolated from umbilical veins and serially cultured on human plasma fibronectin-coated tissue culture plastic (Falcon, Lincoln Park, NJ) in 20% FCS, medium 199, and 2.5 mM L-glutamine (all from Life Technologies, Grand Island, NY), and EC growth factor (Collaborative Biomedical Products, Bedford, MA) as previously described (35). For the experiments described in this report, ECs were used at passage level 3 to 5, because such cultures are free of contaminating CD45+ leukocytes by immunofluorescence microscopy yet stain uniformly positive for EC markers (von Willebrand factor and CD31).
PBMCs were obtained by leukapheresis from adult volunteer donors and further purified by centrifugation over lymphocyte separation medium (Organon Teknika, Durham, NC) according to the manufacturer’s instructions. Isolated PBMCs were washed three times in HBSS (Mg2+- and Ca2+-free) and either used immediately or suspended in 10% DMSO and 90% heat-inactivated FCS and cryopreserved in liquid nitrogen. No differences were seen in the responses of cells recovered from cryopreservation compared with freshly isolated cells.
CD4+ T cells were isolated from PBMCs by negative selection. In brief, PBMCs were first depleted of monocytes by adherence to human plasma fibronectin-coated petri dishes for 45 min at 37°C. Nonadherent cells were decanted and incubated with a mixture of mAb containing LB3.1 (IgG2b, anti-HLA-DR monomorphic determinant, a gift from Dr. J. Strominger, Harvard University, Cambridge, MA), B-H7 (IgG1, anti-CD8; Biosource, Camarillo, CA), and 3G8 (IgG1, anti-CD16; a gift from Dr. J. Unkeless, Mt. Sinai School of Medicine, New York, NY) at saturating conditions for 30 min at 4°C. The cells were then washed three times with RPMI 1640 (Life Technologies)/5% FCS to remove excess Ab and further enriched by magnetic immunodepletion using goat anti-mouse IgG-bound Dynabeads (Dynal, Lake Success, NY) according to the manufacturer’s instruction. The purity of the isolated CD4+ T cells was determined by direct immunofluorescence labeling with anti-CD4, anti-CD8, anti-CD3, anti-CD16 (for NK cells), anti-CD19 (for B cells), and anti-CD14 (for monocytes) (all Abs from Coulter, Keenesaw, GA), and FACs analysis using a FACSort running LYSIS II software (Becton Dickinson, Mountain View, CA). The CD4+ T cell populations used in these studies typically consisted of >90% CD4+ cells and contained no detectable (<1%) CD8+, CD14+, CD16+ or CD19+ cells. Naive CD4+ T cells (CD45RA+CD45RO-) were negatively selected by adding mAb UCHL.1 (IgG2a, anti-CD45RO, a gift of Dr. P. Beverley, University of London, U.K.) into the mAb mixture, and memory CD4+ T cells (CD45RO+CD45RA-) were enriched by adding mAb B-C15 (IgG1, anti-CD45RA; Biosource, Camarillo, CA) into the mAb mixture. The purity of CD45RA+ or CD45RO+ cells ranged from 90 to 97%.
PBAMCs were purified by incubating PBMCs for 3 h on human plasma
fibronectin-coated plates at 37°C, followed by removal of nonadherent
cells by two washes with HBSS. PBAMCs were incubated in RPMI/10% FCS
at 37°C in a 5% CO2 incubator until use, usually within
1 h of purification. These populations typically consisted of a
mixture of 
80% CD14+ monocytes and a smaller number
(10–20%) of CD19+ B cells; few if any T cells
remained in these preparations.
Reagents and Abs
PHA-L, PMA, ionomycin, monensin, and LPS (Escherichia
coli 055:B5) were all obtained from Sigma, St. Louis, MO.
Recombinant human IFN- was obtained from R&D Systems, Minneapolis,
MN. Trimeric recombinant human CD40 ligand was formed from monomers
tagged with a leucine zipper tail and was a gift from Dr. W. Fanslow,
Immunex, Seatle, WA (36). A human IL-12 Quantikine HS kit was obtained
from R&D Systems.
Inhibitory mAb used in the costimulation studies were anti-TAC
(anti-IL-2 receptor 
-chain/p55 subunit, IgG1, used at 20
µg/ml), a gift from Dr. T. Waldmann, National Institute of Health,
Bethesda, MD; TS2/9 (anti-CD58/LFA-3, IgG1), a gift from Dr. T.
Springer, Center for Blood Research, Boston, MA; IE6
(anti-CD58/LFA-3, IgG1), a gift from Dr. P. Hochman, Biogen,
Cambridge, MA; IT2.2 (anti-CD86, IgG2b) and C8.6
(anti-IL-12, neutralizing mAb, IgG1) from PharMingen, San Diego,
CA; IgG2b negative control from Dako, Glostrup, Denmark; K16/16
(nonbinding IgG1), a gift from Dr. D. Mendrick, Brigham and Women’s
Hospital, Boston, MA; and CTLA4-Ig fusion protein (anti-CD80 and
anti-CD86) and control human Ig, gifts from Dr. G. Gray, Repligen,
Cambridge, MA. All Abs were used at concentrations optimized for
maximal functional inhibition, usually 10 to 50 µg/ml, as determined
in preliminary experiments.
mAb used for intracellular cytokine staining were MQ1-17H12
(anti-IL-2, Rat IgG2a, FITC conjugated), R35-93 (negative control,
Rat IgG2a, FITC conjugated), 4S.B3 (anti-IFN-, mIgG1, FITC or
phycoerythrin (PE) conjugated), MOPC-21 (negative control, mIgG1, FITC
or PE conjugated), MP4-25D2 (anti-IL-4, Rat IgG1, PE conjugated),
R3-34 (negative control, rat IgG1, PE conjugated); all were obtained
from PharMingen.
Human CD80 and CD86 cDNA expression constructs and empty control vectors were kind gifts from Dr. A. Bothwell, Yale University, New Haven, CT (37).
Primary costimulation assay
Tissue culture round-bottom 96-well plates (Falcon) were coated
with human plasma-derived fibronectin and then seeded with 20,000 ECs
or PBAMCs or were mock-seeded with cell-free medium. Purified T cells
(200,000) of defined subpopulations were added to each well in a final
volume of 200 µl of RPMI 1640 medium containing 10% FCS, 2.5 mM
glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The
medium was further supplemented with indicated concentrations of PHA,
usually 3 µg/ml. Inhibitory mAb were also added as indicated in the
text. Medium was collected at 24 h and assayed for cytokines using
human IL-2, IFN-, and IL-4 ELISA kits (Coulter). Effects of mAb on
cytokine secretion were calculated as follows: percentage of
inhibition = (cytokine secreted in control mAb-treated
culture - cytokine secreted in inhibitory mAb-treated
culture)/cytokine secreted in control mAb-treated culture.
Differentiation assay
Tissue culture 24-well plates (Falcon) were coated with human plasma-derived fibronectin and then seeded with 120,000 ECs or PBAMCs or were mock seeded with cell-free medium. Purified naive CD4+ T cells (800,000) were added in 800 µl of RPMI 1640/10% FCS containing 3 µg/ml PHA. This was referred to as the primary culture. In IL-12 supplementation experiments, human rIL-12 (R&D Systems) was added to indicated cultures at a final concentration of 4 ng/ml. A 100-µl sample of the medium was collected at 24 h of culture and measured for cytokine production. After 72 h of culture, T cells were collected and washed twice with RPMI 1640/5% FCS, then rested for another 72 h in 5 ml of RPMI 1640/10% FCS. The cells were recollected, washed, and then recultured with PMA (10 ng/ml) plus ionomycin (1 µM) or with PHA (3 µg/ml) and accessory cells, either in a round-bottom 96-well plate with 200 µl RPMI 1640/10% FCS or in a 24-well plate with 800 µl of RPMI 1640/10% FCS. This was referred to as the secondary culture. Cytokines secreted into the medium of the secondary culture well were measured after 24 h.
Intracellular cytokine staining
For characterization and enumeration of cytokine producers in
the primary cultures, purified naive or memory CD4+ T cells
were activated by PHA alone or by PHA in the presence of either ECs or
PBAMCs for 15 h. Monensin (2 µM) was added for the last 8
h. For analysis of cytokine producers in secondary cultures,
CD4+ T cells were activated by 10 ng/ml PMA plus 1 µM
ionomycin in the presence of 2 µM monensin for 4 h. These
conditions were chosen to optimize detection based on preliminary
experiments. In both cases, the harvested CD4+ T cells were
washed once with staining buffer (Dulbecco’s PBS with 1% FCS) and
then fixed in 200 µl fixation buffer (Dulbecco’s PBS with 4%
paraformaldehyde) for 20 min or overnight at 4°C. The fixed cells
were then washed once with staining buffer, once with permeabilization
buffer (Dulbecco’s PBS with 1% FCS, 0.1% saponin), then incubated
for 30 min at 4°C with a pair of mAb: FITC-conjugated anti-IL-2
mAb and PE-conjugated anti-IFN- mAb, or FITC-conjugated
anti-IFN- mAb and PE-conjugated anti-IL-4 mAb. One
microliter of each mAb was used per test. After two washes with
permeabilization buffer and one wash with staining buffer, the cells
were resuspended in staining buffer and subjected to two-color FACs
analysis using a FACSort running LYSIS II software.
Transient transfection of ECs
Transient transfection of ECs was performed using DEAE-dextran (Promega, Madison, WI) as described previously (37, 38). In brief, ECs were plated at 60% confluence on six-well tissue culture plates (Falcon). Cells were washed twice with 1 mM HEPES/PBS, and 0.5 ml of 1 mM HEPES/PBS containing 3 µg plasmid DNA and 250 µg/ml DEAE-Dextran was added. After 30 min at 37°C, 2 ml of EC growth medium containing 0.08 mM chloroquine (Sigma) was added to each well, and the cells were incubated for an additional 3 h. The medium was then removed, and the cells were incubated with medium 199, containing 20% FCS and 10% DMSO, for 1.5 min. The medium was then removed and replaced with fresh EC growth medium. A sample of transfected cells were analyzed 40 h posttransfection for surface expression of CD80 and CD86 molecules with FITC-conjugated anti-CD80 and PE-conjugated anti-CD86 mAbs (PharMingen). The remainder of the cells was replated on plates coated with human plasma-derived fibronectin for a costimulation assay or a differentiation assay. Transfection efficiencies judged by FACs analysis were typically between 8 and 15%, but occasionally reached a level of 50%.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and
IL-4, as well as IL-2
IFN--treated human ECs can stimulate allogeneic resting
CD4+ T cells to proliferate (39). However, IFN--treated
ECs stimulate fewer CD4+ T cells to secrete IL-2 than do
allogeneic professional APCs, such as PBAMCs (40). It is not known
whether this difference reflects a more limited capacity to present
alloantigens (e.g., fewer or different peptide-MHC complexes) or
provision of less effective costimulation or both. To address this
question, we experimentally separated costimulation from Ag
presentation by using a polyclonal mitogen, PHA, to simulate signals
provided by Ag recognition. The effects of the costimulation provided
by ECs vs PBAMCs were then compared by measuring the extent to which
these cells can augment the production of IFN- and IL-4, as well as
IL-2, as a function of varying PHA concentrations. PHA-treated
CD4+ T cells in the absence of accessory cells produced
little measurable cytokine (Fig. 1
).
Coculture of PHA-treated CD4+ T cells with ECs markedly
increased the production of all three cytokines, whereas coculture
of PHA-treated CD4+ T cells with PBAMCs significantly
increased the production of IL-2 and IFN-, but not IL-4 (Fig. 1).
The concentration of PHA that resulted in optimal costimulation by ECs
or PBAMCs for all three cytokines was 3 µg/ml; this concentration was
used in all subsequent studies.
|
, and IL-4
We and others have found that human ECs are able to activate IL-2
secretion by allogeneic resting memory T cells but are not able to
activate secretion by resting naive T cells (26, 27). In contrast,
PBAMCs can activate both resting naive and memory T cells to secrete
IL-2. To study whether ECs can provide costimulation to naive or memory
T cells, CD4+ T cells were further separated into naive
(CD45RO-) or memory (CD45RA-) subpopulations.
As expected from experiments using total CD4+ T cells,
neither PHA-treated naive nor memory CD4+ T cells produced
much cytokine in the absence of accessory cells (Figs. 2 and 3).
Interestingly, ECs were able to provide effective costimulation for
production of all three cytokines by both naive and memory
CD4+ T cells. In these experiments, memory cells secreted
significantly higher levels of all three cytokines, especially IFN-
and IL-4, than did naive cells, such that memory cell responses (Fig. 3) constituted the majority of the response seen with total
CD4+ T cells (Fig. 1
). Surprisingly, in light of the
allogeneic responses observed previously, ECs were fully comparable to
PBAMCs in providing costimulation for IL-2 secretion by naive
CD4+ T cells (Fig. 2). Indeed, compared with PBAMCs, ECs
consistently provided better costimulation to both naive and memory
CD4+ T cells for the production of IL-4. However, PBAMCs
generally provided better costimulation to naive CD4+ T
cells for the production of IFN- (Fig. 2), although this difference
was not observed in memory T cell responses (Fig. 3).
|
|
and Table I). In these experiments, PHA alone, in
the absence of accessory cells, activated few cytokine producers in
naive or memory CD4+ T cell populations (Table I).
In the presence of ECs or PBAMCs, distinct populations of cytokine
producers were activated. We found that the number of cytokine
producers that were activated was roughly proportional to the net
amount of cytokines detected in the total culture supernatant (compare
Table I with Fig. 2 and Fig. 3). Also consistent with the ELISA
results, ECs costimulated more IL-4 producers and fewer IFN-
producers in the naive CD4+ T cell population and
comparable or greater numbers of all three cytokine producers in
the memory CD4+ T cell population than did PBAMCs (Table I).
|
|
and IL-4 production
is partly mediated by enhanced IL-2 production
The experiments described above indicate that costimulation
provided by ECs increases IFN- and IL-4 as well as IL-2 production
by CD4+ T cells. However, it is possible that IFN- and
IL-4 production may increase indirectly through IL-2-mediated effects
(41, 42). To determine to what extent the increased IL-2 contributes to
the observed costimulation of IFN- and IL-4, mAb (anti-TAC) to
the IL-2R was added to block the response to IL-2 in the cultures. This
mAb reduced IFN- production by 50% and IL-4 production by 75% when
either naive or memory CD4+ T cells were cocultured with
ECs, and it inhibited secretion of these cytokines even more when T
cells were cocultured with PBAMCs. Inhibition of IL-2 signaling did not
appear to affect the production of IL-2 itself, indicating that the
effect was specific and not due to toxicity. However, inhibition in the
presence of ECs was never complete, suggesting that ECs costimulate
IFN- and IL-4 secretion both through IL-2-dependent and -independent
pathways.
ECs use LFA-3 to costimulate IL-2 and IFN- production by naive
and memory CD4+ T cells
The costimulation of cytokine secretion by activated T cells is
thought to result principally from signals provided by cell surface
molecules on the accessory cell. Previous studies from our laboratory
have shown that ECs provide costimulation for IL-2 production through
LFA-3 interaction with CD2, but that human ECs lack of expression of B7
molecules and therefore do not costimulate via T cell CD28 (33, 34). In
contrast, professional APCs such as PBAMCs utilize both signaling
pathways. To explore the role of these molecules in the costimulation
of IFN- and IL-4 as well as IL-2 production by naive and memory
CD4+ T cells, we used a mAb inhibition strategy. As shown
in Figure 5, mAb IE6, reactive with
LFA-3, reduced the secretion of IL-2 and IFN- by 50% or more from
both naive and memory CD4+ T cells that were costimulated
by either ECs or PBAMCs. Interestingly, the same mAb inhibited IL-4
production when PBAMCs were used as accessory cells, yet gave varied
results when ECs were used as accessory cells, such that the maximal
inhibition of IL-4 observed was 30%. Similar results were obtained
with a different inhibitory anti-LFA-3 mAb, TS2/9 (data not shown).
The inhibitory effects of anti-LFA-3 mAb on IFN- secretion were
observed even in the presence of anti-IL-2R mAb (not shown),
suggesting that this ligand contributes to the IL-2-independent effect
on IFN- secretion as well as to the IL-2-dependent response.
|
).
Also as expected, these reagents had no effect on cytokine production
in T cells costimulated by ECs, since ECs lack B7-1 or B7-2 expression.
Surprisingly, we observed nearly complete inhibition of PBAMCs
costimulation by blockade of either LFA-3 or B7 alone.
|
, such B7-expressing ECs showed
increased costimulation for the production of IL-2, IFN-, and IL-4
by naive CD4+ T cells. MAb blocking experiments using
CTLA-4Ig and anti-B7-2 and anti-TAC (not shown) confirmed that
these effects were mediated by the transfected B7 molecules and that
enhanced secretion of IL-2 mediated by B7 molecules did not fully
account for the effects of B7 molecules on IFN- and IL-4 secretion.
These results show that B7 is not necessary for costimulation of naive
or memory T cells by ECs, but can provide additional signals beyond
those provided by LFA-3.
|
production
IL-12 is a potent inducer of IFN- production and Th1
differentiation (13, 14). Activated PBAMCs but not ECs produce IL-12
(Table II). As predicted, neutralizing
mAb to IL-12 did not inhibit IFN- production by naive or memory
CD4+ T cells using ECs as accessory cells, whereas the same
Ab inhibited about 80% of IFN- produced by naive or memory
CD4+ T cells using PBAMCs as accessory cells (Fig. 8). These results suggest that lack of
IL-12 in the EC cultures could contribute to the diminished IFN-
secretion observed with naive CD4+ T cells compared with
cultures with PBAMCs as accessory cells. To further investigate the
effects of IL-12 on IFN- production, we added IL-12 into the
cocultures of naive or memory CD4+ T cells with ECs.
Addition of IL-12 to such cocultures significantly increased IFN-
production by naive CD4+ T cells, but did not strongly
affect the production of IL-2 or IL-4 (Fig. 9). Similar results were obtained when
IL-12 was added to cocultures of memory CD4+ T cells
with ECs (data not shown).
|
|
|
Upon activation, naive CD4+ T cells may differentiate
into either Th1-like or Th2-like effectors depending on their
environment. We next assessed whether ECs could promote differentiation
of activated naive CD4+ T cells to Th1 or Th2 effector
cells. To examine this question, purified naive CD4+ T
cells were initially activated by PHA in the presence of ECs or PBAMCs
or in the absence of accessory cells. After 72 h, the lymphocytes
were recovered, washed, rested for 72 h, and then restimulated in
an accessory cell-independent manner by PMA plus ionomycin. The
cytokines produced in the secondary culture were measured at 24 h.
Naive cells costimulated with ECs or PBAMCs as accessory cells in
primary cultures produced comparable levels of IL-2 in secondary
culture, although this level was not significantly higher than that
produced by naive T cells treated with PHA in the absence of accessory
cells (Fig. 10A). In
contrast, the primary culture with PBAMCs resulted in increased IFN-
production in the secondary culture (Fig. 10, A and
B), while neither accessory cell type appeared to augment
IL-4 production in the secondary culture (data not shown). This
suggests that PBAMCs but not ECs support Th1 differentiation.
|
production
during the secondary stimulation in response to PMA and ionomycin (Fig. 10A). This effect was greater than that observed by adding
IL-12 into the primary cultures with no accessory cells. In contrast to
the effects of IL-12, transfection of ECs to express B7-1 or B7-2 did
not significantly increase Th1 type differentiation (Fig. 10B). These data suggest that lack of IL-12 in the
cocultures with ECs can explain the decreased level of Th1
differentiation compared with that in cocultures with PBAMCs.
Results from intracellular cytokine staining are consistent with the
ELISA results and show that costimulation provided by PBAMCs but not by
ECs in the primary culture supported Th1 differentiation and increased
the IFN- secretors from 0.46% in the absence of accessory cells and
0.33% in the presence of ECs, to 3.39% in the presence of PBAMCs
(Table III). Further, IL-12 addition to
the primary culture with ECs increased IFN- secretors to 3.63% in
the secondary culture (Table III).
|
in the secondary culture with PBAMCs than
with ECs, suggesting that they were still responsive to the IL-12
produced by PBAMCs (Fig. 11A). To confirm that
increased IFN- production in the coculture with PBAMCs is due to the
production of IL-12 by PBAMCs, neutralizing mAb to IL-12 was added to
secondary cultures with PBAMCs. Upon mAb addition, the production of
IFN- in the secondary coculture with PBAMCs was decreased to the
same level as in the coculture with ECs (Fig. 11B). The
simplest interpretation of these experiments is that ECs fail to induce
naive CD4+ T cells to differentiate into Th1 cells because
they are unable to secrete IL-12. However, such T cells remain
responsive to IL-12.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
pretreatment) are able to
stimulate T cell proliferation in response to alloantigens or recall
Ags (39, 44, 45). More recent experiments suggest that only memory
(CD45RO+) CD4+ T cells respond to alloantigens
presented by ECs, measured by production of IL-2 (26, 27). This has led
to the suggestion that ECs are unable to provide adequate costimulation
to activate naive T cells, perhaps because human ECs lack expression of
B7-1 and B7-2 (27).
We and others have previously shown that cultured human ECs can provide
effective costimulation for IL-2 and IFN- production by
CD4+ T cells (34, 40, 46). These results were confirmed in
the present studies and extended to show that ECs can also provide
significant costimulation for IL-4 production by CD4+ T
cells. These effects of ECs are fully comparable to those of PBAMCs. To
study whether ECs can effectively costimulate naive as well as memory
CD4+ T cells, we separated total CD4+ T cells
into naive and memory T cell subsets. Our results show that the total
CD4+ T cell response is dominated by memory cells, since
these cells produced much more cytokines than naive cells. However,
contrary to expectations, our results show that ECs also provide
effective costimulation for naive CD4+ T cells.
In experiments using naive CD4+ T cells, we found that ECs
actually provided better costimulation for IL-4, less effective
costimulation for IFN-, and comparable or better costimulation for
IL-2 production than did PBAMCs. These cytokine measurements were
supported and extended by individual T cell assays using intracellular
cytokine staining technique and FACS analysis, which showed that ECs
appeared to costimulate more IL-2 secretors, more IL-4 secretors, and
fewer IFN- secretors among naive CD4+ T cells than did
PBAMCs. However, we cannot exclude the additional possibility that
increased cytokine production is also due to more cytokine being
produced by each individual activated CD4+ T cell.
Surprisingly, even in the naive T cell population, few T cells made
both IFN- and IL-4. Similar results for human peripheral blood T
cells have been seen by others (47). This lack of detection of double
cytokine producers may imply that the intracellular cytokine staining
technique is too limited in sensitivity to accurately phenotype low
cytokine producers, including most naive T cells.
Costimulation is largely dependent on cell surface ligands, especially
B7 molecules, and in humans, on LFA-3. A key molecular difference
between ECs and PBAMCs is that ECs express LFA-3 but not B7 molecules,
whereas PBAMCs express both LFA-3 and B7 molecules. Both B7-1 and B7-2
costimulate through CD28. Costimulation through CD28 increases IL-2
transcription as well as stabilizing IL-2 mRNA, resulting in a markedly
increased level of IL-2 secretion by newly activated T cells (48, 49). Emerging evidence also shows that a major role of CD28
signaling may be to prevent apoptosis during T cell activation (6).
LFA-3 is expressed widely on hemopoietic and nonhemopoietic tissues and
has been identified in humans as the major ligand for CD2 expressed on
all T cells. Costimulation through CD2 results in enhanced T cell
proliferation and IL-2 transcription (4). However, in some
circumstances, CD2 costimulation appears to deliver more potent signals
than CD28 costimulation, i.e., CD28 costimulation prevents anergy,
whereas CD2 costimulation can actually reverse anergy (50). In the
present study, we find from mAb blocking experiments that LFA-3
may contribute not only to IL-2 transcription but also to
augmentation of IFN- and IL-4. Surprisingly, the effect of LFA-3 on
IL-4 is more clear-cut in experiments using PBAMCs as accessory cells
where B7 molecules are also present. Our transfection experiments show
that B7 signals could augment the results obtained using ECs as
accessory cells. These data are consistent with previous results
showing that B7-transfected fibroblasts can boost anti-EC
allogeneic responses through trans-costimulation (27).
However, our data suggest that lack of costimulation through B7 is not
an adequate explanation for the failure of human ECs to activate
allogeneic naive CD4+ T cells.
Why are ECs less able to provide costimulation than are PBAMCs for
IFN- production by naive CD4+ T cells? IFN-
production can be regulated by cytokine costimulation as well as
surface molecule costimulation. IL-12 and IFN--inducing factor
(IGIF) are two cytokines that augment IFN- production (16, 51, 52),
and there is a synergistic effect on IFN- production between
IGIF and IL-12 (53). Reagents are not yet available for direct
measurement of IGIF. However, IGIF mRNA was detected in total RNA of
ECs by RT-PCR (our unpublished observations). IGIF, synthesized
as a precursor peptide, has to be cleaved by IL-1-converting enzyme
(ICE) to become active (54, 55). Thus the ICE-inhibitory peptide
YVAD can inhibit active IGIF production. In preliminary
experiments, we have observed that addition of YVAD inhibited IFN-
production by naive CD4+ T cells cultured with either ECs
or PBAMCs by 30 to 40%. On the other hand, IL-12 was detected only
in the supernatant of CD4+ T cells cocultured with PBAMCs
but not with ECs. Accordingly, neutralizing anti-IL-12 mAb did not
inhibit IFN- production in cocultures with ECs, whereas the same mAb
inhibited IFN- production by 75% in cocultures with PBAMCs.
Consistent with a recent report of others (46), we found that IL-12 had
little effect on IFN- production by cultured CD4+ T
cells in the absence of ECs, but that addition of IL-12 to cocultures
of T cells with ECs could augment IFN- production by as much as
sevenfold. This suggests that ECs can enhance responsiveness of naive
CD4+ T cells to IL-12.
Why are ECs better able than PBAMCs to provide costimulation for IL-4
production by naive CD4+ T cells? LFA-3 appeared to be a
less important costimulator for IL-4 production than for IL-2 or
IFN-, since anti-LFA-3 mAb had inconsistent effects on IL-4
production in cocultures with ECs. IL-4 production is very dependent on
IL-2 and was reduced by 80% in cocultures with ECs or PBAMCs by
anti-IL-2R mAb. However, increased IL-4 production in cocultures
with ECs is unlikely to be wholly dependent on enhanced IL-2
production, since even in experiments in which ECs costimulated similar
levels of IL-2 production as PBAMCs, the ECs still costimulated
more IL-4 production than PBAMCs. Increased IL-4 production is also
probably not a result of less IFN- production, since a blocking
anti-IFN- receptor mAb did not increase IL-4 production in
cocultures with PBAMCs (our unpublished observation). It has
been shown that mouse IL-6 can induce IL-4 production by naive
CD4+ T cells (56) and that human ECs produce more IL-6 than
PBAMCs in cultures. However, anti-IL-6 mAb failed to reduce IL-4
production in cocultures with ECs to the same level as in cocultures
with PBAMCs (our unpublished observations). Thus, further studies will
be needed to determine which signals presented by ECs constitute the
effective costimulation of IL-4.
The most profound defects in EC costimulation that we have observed is the inability of ECs to promote Th1 differentiation. This was not remedied by transfection of ECs to express B7-1 or B7-2, but could be rescued by addition of exogenous IL-12. Thus, the lack of IL-12 secretion appears to be the major deficiency of ECs as accessory cells compared with bone marrow-derived professional APCs.
Although our current studies have concentrated on the capacity of ECs to provide accessory functions to naive CD4+ T cells, our data also show that ECs appear superior to PBAMCs as accessory cells for the activation of memory CD4+ T cells. This capacity of ECs is consistent with a proposed Ag-presenting function. We would suggest that memory T cells passing luminal capillaries in peripheral tissues would readily sample the Ags present in those tissues as displayed in the form of peptide-MHC complexes on the luminal EC surface. Recognition of Ag by a circulating T cell could rapidly up-regulate adhesion by activation of integrins such as LFA-1 (CD11a/CD18), favoring T cell egress at the site where foreign Ag is displayed. In other words, presentation of Ag by ECs could improve the efficiency of immune surveillance for foreign peptides by selective recruitment of Ag specific T cells. Why are naive cells unaffected by their interaction with ECs? Our data presented here suggest that lack of costimulation due to the absence of B7 molecules is unlikely to be a complete explanation. Perhaps the differences in adhesion molecules expressed by naive cells simply do not allow the T cell-EC interaction to persist long enough for a signal to be delivered. In our in vitro system, the need for adhesion is bypassed by PHA cross-linking of the T cell to the accessory cell surface.
In conclusion, ECs do not express B7 molecules and do not produce
IL-12, yet provide effective costimulation for cytokine production by
naive as well as memory CD4+ T cells. These results suggest
that a deficiency in costimulation, specifically the lack of B7
molecules, does not fully explain the inadequacy of ECs to activate
resting naive CD4+ T cells. However, the deficiency in
IL-12 may impede the capacity of ECs to promote IFN- secretion or
Th1 differentiation. We hypothesize that lack of adhesion between ECs
and naive CD4+ T cells may be the major factor that
prevents interactions between these two cell types. This may account
for why naive T cell activation and differentiation occurs primarily in
secondary lymphoid organs, outside of the bloodstream, where
professional APCs are available to provide more effective interaction.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jordan S. Pober, Boyer Center for Molecular Medicine, Yale University, School of Medicine, 295 Congress Avenue, New Haven, CT 06536-0812. E-mail address:
3 Abbreviations used in this paper: ECs, endothelial cells; PBAMCs, peripheral blood-adherent mononuclear cells; LFA-3, leukocyte function-associated Ag 3; PE, phycoerythrin; ICE, IL-1-converting enzyme.
Received for publication February 27, 1998. Accepted for publication April 30, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-treated fibroblasts. J. Immunol. 138:385.[Abstract]
B site and p65 homodimers. J. Biol. Chem. 270:933. production. J. Immunol. 159:3247.[Abstract]
-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J. Immunol. 156:4274.[Abstract]
-inducing factor in enhanced production of IFN-. J. Immunol. 159:2125.-inducing factor and regulates LPS-induced IFN-gamma production. Nature 386:619.[Medline]
inducing factor mediated by interleukin-1beta converting enzyme. Science 275:206.This article has been cited by other articles:
|
|
 |
|
  Y. Suarez, B. R. Shepherd, D. A. Rao, and J. S. Pober Alloimmunity to Human Endothelial Cells Derived from Cord Blood Progenitors J. Immunol., December 1, 2007; 179(11): 7488 - 7496. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Shiao, N. C. Kirkiles-Smith, B. R. Shepherd, J. M. McNiff, E. J. Carr, and J. S. Pober Human Effector Memory CD4+ T Cells Directly Recognize Allogeneic Endothelial Cells In Vitro and In Vivo J. Immunol., October 1, 2007; 179(7): 4397 - 4404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Tellides and J. S. Pober Interferon-{gamma} Axis in Graft Arteriosclerosis Circ. Res., March 16, 2007; 100(5): 622 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Shiao, J. M. McNiff, and J. S. Pober Memory T Cells and Their Costimulators in Human Allograft Injury J. Immunol., October 15, 2005; 175(8): 4886 - 4896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Choi, J. Walker, K. Talbert-Slagle, P. Wright, J. S. Pober, and L. Alexander Endothelial Cells Promote Human Immunodeficiency Virus Replication in Nondividing Memory T Cells via Nef-, Vpr-, and T-Cell Receptor-Dependent Activation of NFAT J. Virol., September 1, 2005; 79(17): 11194 - 11204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kummer, A. Lev, Y. Reiter, and B. C. Biedermann Vascular Endothelial Cells Have Impaired Capacity to Present Immunodominant, Antigenic Peptides: A Mechanism of Cell Type-Specific Immune Escape J. Immunol., February 15, 2005; 174(4): 1947 - 1953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Choi, J. Walker, S. Boichuk, N. Kirkiles-Smith, N. Torpey, J. S. Pober, and L. Alexander Human Endothelial Cells Enhance Human Immunodeficiency Virus Type 1 Replication in CD4+ T Cells in a Nef-Dependent Manner In Vitro and In Vivo J. Virol., January 1, 2005; 79(1): 264 - 276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Kreisel, A. M. Krasinskas, A. S. Krupnick, A. E. Gelman, K. R. Balsara, S. H. Popma, M. Riha, A. M. Rosengard, L. A. Turka, and B. R. Rosengard Vascular Endothelium Does Not Activate CD4+ Direct Allorecognition in Graft Rejection J. Immunol., September 1, 2004; 173(5): 3027 - 3034. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tomescu, W. K. Law, and D. H. Kedes Surface Downregulation of Major Histocompatibility Complex Class I, PE-CAM, and ICAM-1 following De Novo Infection of Endothelial Cells with Kaposi's Sarcoma-Associated Herpesvirus J. Virol., September 1, 2003; 77(17): 9669 - 9684. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Schmidt, H. Krook, A. Maeda, O. Korsgren, and B. Benda A New Murine Model of Islet Xenograft Rejection: Graft Destruction Is Dependent on a Major Histocompatibility-Specific Interaction Between T-Cells and Macrophages Diabetes, May 1, 2003; 52(5): 1111 - 1118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Kreisel, A. S. Krupnick, K. R. Balsara, M. Riha, A. E. Gelman, S. H. Popma, W. Y. Szeto, L. A. Turka, and B. R. Rosengard Mouse Vascular Endothelium Activates CD8+ T Lymphocytes in a B7-Dependent Fashion J. Immunol., December 1, 2002; 169(11): 6154 - 6161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. L. W. Yun, A. A. DeCarlo, C. Collyer, and N. Hunter Modulation of an Interleukin-12 and Gamma Interferon Synergistic Feedback Regulatory Cycle of T-Cell and Monocyte Cocultures by Porphyromonas gingivalis Lipopolysaccharide in the Absence or Presence of Cysteine Proteinases Infect. Immun., October 1, 2002; 70(10): 5695 - 5705. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. M. Mazanet and C. C. W. Hughes B7-H1 Is Expressed by Human Endothelial Cells and Suppresses T Cell Cytokine Synthesis J. Immunol., October 1, 2002; 169(7): 3581 - 3588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Segal, A. Bihorac, and M. Koc Circulating endothelial cells: tea leaves for renal disease Am J Physiol Renal Physiol, July 1, 2002; 283(1): F11 - F19. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Mercier, H. Gahery-Segard, M. Monteil, R. Lengagne, J.-G. Guillet, M. Eloit, and C. Denesvre Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product J. Virol., February 22, 2002; 76(6): 2899 - 2911. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. L. W. Yun, A. A. Decarlo, C. Collyer, and N. Hunter Hydrolysis of Interleukin-12 by Porphyromonas gingivalis Major Cysteine Proteinases May Affect Local Gamma Interferon Accumulation and the Th1 or Th2 T-Cell Phenotype in Periodontitis Infect. Immun., September 1, 2001; 69(9): 5650 - 5660. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Batten, A. M. McCormack, M. L. Rose, and M. H. Yacoub Valve interstitial cells induce donor-specific T-cell anergy J. Thorac. Cardiovasc. Surg., July 1, 2001; 122(1): 129 - 135. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M. Marelli-Berg, D. Scott, I. Bartok, E. Peek, J. Dyson, and R. I. Lechler Activated Murine Endothelial Cells Have Reduced Immunogenicity for CD8+ T Cells: A Mechanism of Immunoregulation? J. Immunol., October 15, 2000; 165(8): 4182 - 4189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Van Rhijn, L. H. Van den Berg, W. M. J. Bosboom, H. G. Otten, and T. Logtenberg Expression of accessory molecules for T-cell activation in peripheral nerve of patients with CIDP and vasculitic neuropathy Brain, October 1, 2000; 123(10): 2020 - 2029. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Lawson, A. M. McCormack, D. Moyes, S. Yun, J. W. Fabre, M. Yacoub, and M. L. Rose An Epithelial Cell Line That Can Stimulate Alloproliferation of Resting CD4+ T Cells, But Not After IFN-{gamma} Stimulation J. Immunol., July 15, 2000; 165(2): 734 - 742. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kawai, M. Seki, H. Watanabe, J. W. Eastcott, D. J. Smith, and M. A. Taubman Th1 transmigration anergy: a new concept of endothelial cell-T cell regulatory interaction Int. Immunol., June 1, 2000; 12(6): 937 - 948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Dengler and J. S. Pober Human Vascular Endothelial Cells Stimulate Memory But Not Naive CD8+ T Cells to Differentiate into CTL Retaining an Early Activation Phenotype J. Immunol., May 15, 2000; 164(10): 5146 - 5155. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Gatti, M. A. Velleca, B. C. Biedermann, W. Ma, J. Unternaehrer, M. W. Ebersold, R. Medzhitov, J. S. Pober, and I. Mellman Large-Scale Culture and Selective Maturation of Human Langerhans Cells from Granulocyte Colony-Stimulating Factor-Mobilized CD34+ Progenitors J. Immunol., April 1, 2000; 164(7): 3600 - 3607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Murakami, W. Ma, R. Fuleihan, and J. S. Pober Human Endothelial Cells Augment Early CD40 Ligand Expression in Activated CD4+ T Cells Through LFA-3-Mediated Stabilization of mRNA J. Immunol., September 1, 1999; 163(5): 2667 - 2673. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
