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Program in Molecular Cardiobiology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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after
target cell contact. In the present study, we have analyzed these
polyclonal populations at a single cell level by cloning at limiting
dilution and propagating the resulting CTL clones in the absence of EC.
Phenotypically stable, alloreactive EC-selective CTL preferentially
emerge from cocultures in which EC or EC + BLC are the initial
stimulating cell types compared with cocultures stimulated by BLC alone
(p = 0.005). Compared with BLC-stimulated CTL,
EC-stimulated CTL clones often fail to secrete IFN- after target
cell contact (p = 0.0006) and constitutively
express CD40 ligand (CD40L) at rest (p = 0.0006).
The absence of IFN- secretion does not result from a switch to IL-4
secretion. The expression of CD40L inversely correlates with the
secretion of IFN- after target cell contact (p =
0.0001), but correlations of CD40L expression and failure to secrete
IFN- with EC-selective killing did not reach statistical
significance. We conclude that in a microenvironment in which
allogeneic EC are in close contact with infiltrating CD8+ T
cells, such as within a graft arterial intima, CTL subsets may emerge
that display EC selectivity or express CD40L and secrete little IFN-
after Ag contact. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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upon target cell
recognition. Since these lines were propagated continuously in the
presence of EC, it was unclear whether the unusual properties of these
lines, namely EC selectivity and low IFN- secretion, resulted from
stable, characteristic features of unusual CTL that had been
preferentially expanded in the presence of EC, or whether they were
consequences of transient EC-mediated phenotypic modulations of
conventional (i.e., cell type-unrestricted, IFN--secreting) CTL. The
present study was designed to answer this question by cloning T cells
from the polyclonal CTL lines at limiting dilution and then propagating
these T cell clones in the absence of EC. In this study, we report that
the presence of EC in the initial polyclonal T cell culture favors the
expansion of phenotypically stable CTL clones that display both EC
selectivity and poor IFN- secretion. We also observed that a
significant number of EC-stimulated CTL express CD40 ligand (CD40L,
officially designated CD154) at rest. This marker correlated strongly
with poor IFN- secretion. BLC also stimulated EC-selective CTL and
CD40L+ CTL, but this was much less common. We conclude that
EC promote the expansion of unusual CTL at the expense of conventional
CTL and that these unusual CTL clones do not require subsequent EC
contact to maintain their unusual phenotypes. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were obtained from healthy volunteers by density-gradient centrifugation of leukapheresis products and stored in liquid nitrogen, as described previously (3). These populations were used to isolate responder cells for the CTL differentiation cultures as well as feeder cells for cloning at limiting dilution. CD8+ T lymphocytes were isolated by positive selection (2). In brief, Dynabeads (Dynal, Lake Success, NY) coated with an anti-CD8 mAb were incubated with the PBMC suspension to bind CD8+ T cells, and nonbinding cells were removed by extensive washing. The magnetic beads were detached from the responder cells by applying Detachabead according to the manufacturer’s instructions. The responder population obtained by this procedure was routinely >98% CD8/CD3 positive and >99% viable, as shown by trypan blue exclusion.
HUVEC were obtained from umbilical cords by enzymatic digestion and maintained in culture, as described (3). Such EC cultures are free from detectable CD45+ contaminating leukocytes and uniformly express von Willebrand factor and CD31. B lymphoblastoid cells (BLC) from the same donor as the EC were grown from EBV-immortalized cord blood mononuclear cells (4). After 6–8 wk in culture, BLC lines were uniformly CD19 positive. HUVEC and BLC were used as stimulator cells in cocultures and as target cells in effector assays. When BLC were used as stimulator or feeder cells, they were pretreated with mitomycin C (Sigma, St. Louis, MO) to prevent proliferation (2). The erythroleukemia cell line K562 was obtained from American Type Culture Collection (Manassas, VA) and grown in RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Life Technologies, Grand Island, NY).
CTL differentiation and cloning
The procedure for CTL differentiation has been described previously (2). In brief, purified CD8+ T cells were incubated with stimulator cells (EC, BLC, or both) in 96-well microculture plates (Falcon; Becton Dickinson, Bedford, MA) at a responder to stimulator cell ratio of 2.5–7.5:1. All cultures were maintained at 37°C in 5% CO2 room air. In each experiment, 18–30 microculture replicates per stimulator-responder combination were initiated. The medium for coculture consisted of RPMI 1640 supplemented with 10% human AB serum (Irvine Scientific, Santa Ana, CA), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. The medium was supplemented with exogenous IL-2 (R&D Systems, Minneapolis, MN) on day 3 (final concentration: 5–10 ng/ml). On day 7, the medium was changed and the CTL were transferred to fresh stimulator cells. Cultures were again fed with fresh medium plus IL-2 on day 10. On day 14, the CTL microcultures were tested for cytotoxicity against the stimulator cell type (EC or BLC, respectively) using a calcein fluorescence release assay (5), as described below.
From each experiment, the three microcultures that displayed the highest level of cytotoxicity were chosen for cloning by limiting dilution following published protocols (6), (7), with minor modifications as follows. The lymphocytes were counted and suspended in complete cloning medium (RPMI 1640, 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 ng/ml IL-2, and 1 µg/ml PHA-L (Sigma) at 1000, 100, or 10 cells/ml). These cell suspensions were distributed to round-bottom 96-well plates (100 µl/well), resulting in input cell numbers of 100 (24 replicates), 10 (24 replicates), and 1 (48 replicates) per well. Each well was additionally supplemented with feeder cells consisting of 50,000 irradiated (30 Gy) PBMC autologous to the responder CD8+ T cells and 2,000 mitomycin C-treated BLC autologous to the stimulator cell. In most experiments, EC-stimulated CTL were distributed into 96-well plates that also contained stimulator EC at subconfluent density (5–10,000 cells/well). On day 7, the clones were fed with the same medium, except that PHA was not included. Beginning on day 14, cultures were inspected daily for the presence of expanding clones. These were collected into 5 ml complete cloning medium plus fresh feeder cells and expanded in tissue culture flasks. The CTL clones were maintained in culture by repetitive weekly restimulations (0.5 x 106 CTL/ml complete cloning medium plus feeder cells). On day 21, the cloning plates were analyzed for the final number of expanded clones per dilution to assess cloning efficiency and conformance to "single hit" responses.
Cytotoxicity assay
Cytotoxicity by CTL was measured at least 7 days after the last restimulation with feeder cells and PHA by a calcein fluorescence release assay (5), as described (2). In brief, target cells were loaded with calcein-AM (Molecular Probes, Eugene, OH), washed, and incubated with effector CTL at titered E:T ratios (30:1, 10:1, 3:1) for 4 h at 37°C. The supernatant was then harvested and calcein release was measured using a fluorescence plate reader (Cytofluor 2; Perseptive Biosystems, Framingham, MA; excitation wavelength 485 nm, emission wavelength 530 nm). Percent specific killing was calculated as (release sample - spontaneous release)/(maximal release - spontaneous release) x 100%. Spontaneous release was obtained by adding medium alone; maximal release was obtained by adding lysis buffer (50 mM sodium borate, 0.1% Triton X-100, pH 9).
Cytokine measurements
IFN-, TNF, and IL-4 were measured in the CTL assay
supernatant that was collected 18–24 h after the cytotoxicity assay
was started. After harvesting the supernatant to measure calcein
release, medium was replaced (RPMI 1640, 10% human serum AB, without
IL-2) and the cultures were further incubated at 37°C. The
supernatant was collected from all E:T ratios tested per each
individual clone, pooled, and stored frozen at -70°C. Cytokine
concentration was determined by an ELISA using commercially available
Ab pairs (monoclonal mouse anti-human IFN- (MAB285), monoclonal
mouse anti-human TNF (MAB610), monoclonal mouse anti-human IL-4
(MAB604), biotinylated polyclonal goat anti-human IFN- (BAF285),
biotinylated polyclonal goat anti-human TNF (BAF210), and
biotinylated mouse anti-human IL-4 (BAF204), all from R&D Systems),
according to the manufacturer’s instructions.
Immunophenotyping of the CTL clones
CTL were collected for immunophenotyping at least 7 days after restimulation with feeder cells and PHA. CTL were either fixed with paraformaldehyde, spun onto gelatin-coated slides, permeabilized, and double stained for CD8 and perforin as described previously (2), or processed unfixed for FACS analysis. In the latter case, 50,000 CTL/sample were washed once with ice-cold PBS/1% BSA and incubated with saturating concentrations of directly FITC- or PE-conjugated mouse anti-human CD8, CD3, CD25, CD2, CD28 (all from Coulter Immunotech, Miami, FL), or CD40L (TRAP1; PharMingen, San Diego, CA)), or nonconjugated anti-human FasL, Fas (both PharMingen), or Mac-1 (LM2/1, gift from Dr. D. Altieri, Yale Medical School, New Haven, CT) mAbs for 30 min at 4°C. Cells were washed three times with ice-cold PBS/BSA and either fixed with 1% paraformaldehyde in PBS (conjugated first Ab) or incubated with an FITC-conjugated, goat anti-mouse IgG (H + L) secondary Ab (F(ab')2) (50 µl/sample, 1/50 dilution; Boehringer Mannheim, Indianapolis, IN) for another 30 min at 4°C. Cells were washed three times before fixation with 1% paraformaldehyde in PBS. Samples were analyzed using FACSort (Becton Dickinson, San Jose, CA) by gating on viable cells and collecting 3000 events per sample.
Quantitative competitive RT-PCR
Total RNA was isolated from 5 x 106 resting
CTL using a guanidinium isothiocyanate-based RNA isolation kit (RNeasy
mini kit; Qiagen, Santa Clarita, CA), according to the manufacturer’s
instructions. A total of 2 µg of total RNA (final volume: 20 µl)
was suspended in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM
MgCl2, 20 mM DTT, 16.5 µg/ml oligo(dT)15 (Program for
Critical Technology in Molecular Medicine, Yale University Department
of Pathology, New Haven, CT), 0.5 mM dNTP (New England Biolabs,
Beverly, MA), and 40 U RNAsin (Promega, Madison, WI), and reverse
transcribed with 200 U Superscript (Life Technologies) for 60 min at
45°C. After 15 min of heat inactivation at 70°C, the reaction tubes
were incubated for 5 min on ice. A total of 3 U RNase H (Life
Technologies) was added, and the reaction was incubated for 20 min at
37°C. A total of 80 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 8) was
added, and the samples were stored at 4°C and analyzed within 1 mo.
The sequences for the genes of interest (CD3
, perforin, FasL,
IFN-, and CD40L) were analyzed for primer annealing sites using the
primer analysis software Oligo version 4.0 (National Biosciences,
Plymouth, MN). To easily rule out amplification of genomic DNA, the 5'
and 3' primer annealing sites were placed on two different exons of the
gene. Table I
shows the primer pairs (all
5' to 3' direction) that were used for PCR. All full-size templates
could be amplified from cDNA obtained from PHA-treated PBMC, and
competitor cDNA was shortened by 70–100 nucleotides applying rPCR. The
competitor cDNA was amplified and the weight concentration was
determined by comparison with the known amount of nucleotide size
standard (Lambda DNA BstEII digest; New England Biolabs) on
the same gel. For each competitor, a 100 pg/ml and a 100 fg/ml stock
solution was prepared in TE buffer and stored at 4°C. The cDNA
obtained from the CTL clones (1 µl) was mixed with the same volume of
competitor cDNA, added at four different concentrations (10-fold
dilutions). This template mix was amplified in a PCR reaction (final
volume: 10 µl) containing 0.2 mM dNTP, 250 nM of each primer, and 5
U/ml Taq (Boehringer Mannheim, Indianapolis, IN) through the
following protocol: 5-min initial denaturation at 95°C, then 35
cycles of denaturing 30 s at 95°C, annealing 30 s at the
gene-specific annealing temperature, and elongating 1 min at 72°C,
with incubating for 10 min at 72°C for final extension. The PCR
products were resolved on a 1.5% agarose gel, stained with ethidium
bromide, and photographed under UV transillumination (Eagle Eye;
Stratagene, La Jolla, CA). The resulting pictures were scanned (Scan
Jet IIcx; Hewlett Packard, Palo Alto, CA), band intensities were
quantified (National Institute of Health Image 1.61), and the
competitor concentration of equivalent band intensity to the test
samples was determined. This concentration was taken as the full-size
cDNA concentration present in the sample. For each sample, the
concentration of perforin, FasL, IFN-, and CD40L was normalized to
the concentration of CD3 (arbitrarily set to be 100,000 U (8)).
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The TCRVß profile of the clones was determined by RT-PCR, according to published methods applying a set of 22 Vß family-specific primers (9). A total of 1 µl of cDNA was amplified in a PCR reaction (final volume: 10 µl) containing 0.2 mM dNTP, 250 nM of each primer, and 5 U/ml Taq (Boehringer Mannheim, Indianapolis, IN) through the following protocol: 5-min initial denaturation at 95°C, then 30 cycles of denaturing 30 s at 95°C, annealing 30 s at 55°C, and elongating 1 min at 72°C with final extension for 10 min at 72°C.
Statistical analysis
Data analysis of limiting dilution cloning was performed
according to likelihood maximization using a computer program kindly
provided by Dr. C. Orosz (Ohio State University, Columbus, OH). The
outcome of various treatments between paired groups was tested for
significant differences using 
2 analysis. Results from
different groups in multivariable experiments were compared by ANOVA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To generate clonal lines, primary 2-wk cultures of
CD8+ T cells stimulated by EC, BLC, or EC + BLC (see
Materials and Methods) were cloned by limiting dilution. The
cloning conditions included irradiated PBMC as feeder cells (autologous
to the responder T cells), mitomycin C-treated BLC (autologous to the
stimulator cells), IL-2 (20 ng/ml), and PHA-L (1 µg/ml). Pilot
experiments in the absence of PHA led to
CD3+CD8-CD4- clones that failed
to display cytotoxicity. Stimulator EC were usually present during the
limiting dilution cloning if EC were present in the initial stimulator
cultures, but their absence did not seem to influence the cloning
efficiency (p = 0.71) nor change the phenotype
(p = 0.30) of the emerging CTL clones.
Therefore, the data from cloning in the absence and presence of EC have
been pooled for purposes of statistical analysis. Overall, 66 CTL lines
were cloned using cells from 10 different donors and resulting in 7
different allocombinations. With only one exception, all 66 limiting
dilution clonings conformed to single hit kinetics for clonal growth
(Fig. 1A). The observed
frequencies were much higher than in primary cultures, reported
previously (2), indicating that clonal expansion of CTL precursors had
occurred during the initial 2-wk coculture. The fraction of
alloreactive T cells present in the 2-wk microcultures that were
capable of expansion varied between 0.2% and 25%. Surprisingly, this
frequency was significantly higher for cultures stimulated with EC,
which expanded least during the 2-wk primary culture than for cultures
stimulated with both cell types or BLC alone (Fig. 1B).
Eighteen of these CTL clones were studied by identifying the
TCRVß-chain expressed by these cells (Fig. 1C). A total of
4 of 18 CTL lines expressed two, 8 of 18 expressed one TCRVß-chain,
and 4 of 18 CTL lines were negative for all of the 20 TCRVß families
tested, consistent with expected frequencies for human CD8+
T cell clones (10). Only 2 of 18 CTL lines expressed more than two
TCRVß-chains, suggesting that they were in fact oligoclonal. For the
cytolytic functional studies reported in this work, all of the cloned
CTL lines (including the confirmed oligoclonal ones) were included in
the analysis, but only true clones, confirmed by TCRVß analysis, are
presented in the analysis of CTL phenotype.
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, in which the data are separated
according to the initial allogeneic stimulator cell type (EC, BLC, or
EC + BLC). Only 11 T cell clones of the 94 were not cytolytic for EC or
BLC. These clones arose from EC (n = 6), BLC
(n = 3), and EC + BLC (n = 2) lines and
were not further characterized in this study. We operationally defined
EC-selective clones as those that display percent specific lysis of EC
2x percent specific lysis of BLC. By this definition, 17 of 94 CTL
clones were EC selective. Nine of these EC-selective CTL clones were
derived from lines stimulated with EC alone, four from lines stimulated
with EC + BLC, and, surprisingly, four arose from lines stimulated with
BLC alone that had never been in contact with cultured EC before the
cytotoxicity assay. As shown in Table II,
the
presence of EC in the initial cocultures increased the frequency of
outgrowth of EC-selective CTL clones (p <
0.005). Mixed stimulator cell populations (i.e., EC + BLC) seem to be
even more efficient inducers of EC-selective CTL than EC alone (not
shown), although the number of clones analyzed is too small to allow
separate statistical analysis. This trend is consistent with our
previous observation that mixed stimulator cells accentuate EC
selectivity of polyclonal CTL lines (2).
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About 50% of the clones analyzed continued to expand for at least 8
wk. We defined such clones as being long-term CTL. We tested some of
the long-term EC-selective CTL clones for NK-like, allospecific, and
class I MHC-dependent killing (Fig. 3).
EC-selective CTL clones did not lyse the NK cell target K562. Pooled EC
were not lysed by EC-selective CTL clones, consistent with
allospecificity (Fig. 3A). EC-selective CTL clones were
inhibited by mAb against class I MHC and CD8 to the same extent as
conventional, i.e., cell type-unrestricted CTL clones tested in
parallel (i.e., by about 35–50% at E:T ratios of 30:1; Fig. 3B). Unfortunately, HLA-typed EC lines were not available to
directly test class I MHC restriction of these clones. However, these
data are consistent with alloreactive, class I MHC-restricted CTL that
lack NK activity. The pattern of EC-selective, alloreactive, class I
MHC-restricted CTL clones is also consistent with the characteristics
of the 2-wk CTL lines described previously (2), some of which were
tested on HLA-typed EC cultures, supporting the interpretation that
alloreactive EC-selective killing exhibited by EC-stimulated CTL lines
results from characteristics of individual CTL clones that have been
expanded within the original cultures.
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We applied FACS analysis, immunostaining, and competitive RT-PCR
to analyze the phenotypes of long-term (8-wk) CTL clones. All long-term
CTL clones analyzed were CD3/CD8 double positive, but the level of CD8
was variable (Fig. 4B).
Specifically, some EC-selective CTL clones seemed to contain a
CD8dim subpopulation, whereas conventional CTL clones were
uniformly CD8bright+. Interestingly,
CD3+CD8dim cells were present in populations
established as monoclonal by TCRVß analysis and probably do not
represent a differential T cell population.
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). FasL was detectable on only 4 of 39
resting CTL clones: 2 EC-selective CTL clones contained a subpopulation
of FasL+ cells, and 2 BLC-stimulated conventional CTL
clones were weakly FasL+ (not shown). However, mRNA for
FasL was detectable even in surface FasL- cells and varied
over a broad range (Fig. 5). It is possible that such
mRNA+, surface- clones contain intracellular
pools of FasL protein (11), but this was not explored in the present
study.
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C).
mRNA levels for CD40L were 10–100-fold higher in CTL clones that
expressed CD40L on their surface than on CD40L-negative cells (Fig. 5B). CD40L was also found on some EC-stimulated CTL clones
that did not display EC selectivity, but not on BLC-stimulated CTL
clones. To determine whether EC stimulators favored the emergence of
CD40L+ CTL, we prospectively analyzed the next 44 CTL
clones, including 23 that were stimulated by EC and 21 that were
stimulated by BLC. As shown in Table IV, CTL that have been initially
stimulated by EC are much more likely to express CD40L at rest than CTL
that were not (p = 0.0006).
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-chain
(4 conventional and 2 EC selective) and CD45RA (4 conventional and 2 EC
selective). A total of 12 of 16 cytolytic CTL clones tested (10
conventional and 2 EC selective) were positive for Mac-1 (CD11b/CD18).
A total of 3 of 7 tested CTL clones (1 conventional and 2 EC selective)
expressed CD28. Only 2 of 7 (both EC selective) were CD25+
7 days after the last restimulation, but all CTL clones (13
conventional, 1 EC selective) tested expressed Fas (CD95) on their
surface. Cytokine secretion by the CTL clones
Forty-one long-term CTL clones were analyzed for their capacity to
secrete IFN-. All clones tested secreted IFN- in response to PHA
(not shown). However, differences emerged when CTL clones were
activated by target cells (Fig. 6). None
of the 10 EC-selective clones, but 8 of 14 EC-stimulated and 16 of 17
BLC-stimulated conventional CTL clones secreted significant amounts of
IFN- (>25 pg/ml), irrespective of the target cell in the assay
(Fig. 6B). The level of secretion of IFN- did not
correlate with cytotoxicity at the single clone level in any of the
groups analyzed. To test whether EC stimulators favored the emergence
of CTL, which were poor secretors of IFN-, we prospectively analyzed
the next 43 CTL clones, including 22 that were stimulated by EC and 21
that were stimulated by BLC. As shown in Table III CTL that have been
initially stimulated by EC are much more likely to be poor IFN-
secretors than CTL that were not (p = 0.0006).
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mRNA levels at rest were not significantly lower in
EC-selective CTL clones (p = 0.22), and they
did not correlate at all with the capacity to secrete IFN- after
target cell contact (Fig. 5B). Interestingly, we observed
several clones that had IFN- mRNA levels >2000 CD3 units at
rest, but did not secrete measurable amounts of IFN- even after
target cell lysis (e.g., clone 11.3.2.; Fig. 5B). This
suggests that additional translational or posttranslational control
mechanisms of IFN- production must be occurring.
The pattern of TNF secretion by CTL after lysis of EC was similar to
the pattern observed for IFN-. However, the EC-selective CTL clones
produced TNF after BLC contact, indicating a partial dissociation of
cytokine production and cytotoxicity in these CTL clones (Fig. 6C). These data also suggest that EC-selective clones can
recognize BLC, but it was not tested whether such recognition is
allorestricted. None of the CTL clones analyzed (EC selective and
conventional) secreted measurable amounts of IL-4 after target cell
contact (not shown). Thus, lack of IFN- secretion does not reflect
immune deviation to a Tc2-like phenotype (14).
Correlations among EC-stimulated characteristics
In the preceding analyses, we prospectively analyzed the effects
of EC upon CTL differentiation. The conclusions of our initial
experiments, which held up through completion of the analyses,
indicated that EC stimulation favors EC selectivity, poor IFN-
secretion, and CD40L expression. A retrospective analysis of the
initial clones suggested that these three traits were linked. In a
final series of experiments, we prospectively analyzed more than 40
additional clones to determine the statistical significance of the
relationships among EC selectivity, poor IFN- production, and CD40L
expression (Table V). We found that CD40L
expression correlated strongly with poor secretion of IFN- after
target cell lysis (p = 0.0001). However,
neither CD40L expression nor poor secretion of IFN- after target
cell lysis reached statistical significance as indicators of EC
selectivity (p = 0.12 and p =
0.06, respectively). Apparent dissociation of IFN- secretion and EC
selectivity would be surprising since polyclonal CTL lines appeared to
display both characteristics (2). It is more likely that these traits
are linked, albeit less tightly than poor IFN- secretion and CD40L
expression, and that if we had further increased the numbers of clones
prospectively analyzed, the correlation between poor IFN- secretion
and EC selectivity would have reached statistical significance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although EC-selective CTL preferentially emerged from EC-stimulated cultures, a majority of long-term CTL clones from these same cultures actually displayed a cell type-unrestricted, i.e., conventional pattern of killing. We had noted previously that EC suppress growth of conventional CTL stimulated by BLC (2). The simplest explanation of our results is that EC also suppress growth of conventional CTL stimulated by EC, but that such clones can be expanded under the conditions of limiting dilution culture in the presence of feeder cells. In other words, the cloning conditions allow emergence of conventional CTL that were silent (or poorly expanded) in the polyclonal lines. However, we cannot rule out that the cloning conditions that we optimized for growth at limiting dilution allow some EC-selective CTL to convert to a more conventional specificity.
A major surprise of these studies was the production of four EC-selective clones from cultures that had never seen EC in vitro. In theory, EC-selective killing could arise from a target structure formed by a peptide derived from an EC-specific protein (e.g., von Willebrand factor) not synthesized by BLC (15). If so, it is hard to imagine how such clones could be activated by BLC. Alternatively, cell-selective CTL may arise from a requirement for unusual accessory or adhesive interactions that would favor killing of EC over BLC (e.g., binding to E-selectin or ICAM-2, adhesion molecules expressed on EC, but not BLC (19)). Such an accessory molecule-based explanation has recently been offered to account for cell-selective killing of renal epithelial cells by CTL (20). It is possible that the BLC-stimulated clones that display EC selectivity also fit into this category. If this explanation is true, the generation of EC-selective CTL arising from stimulation by BLC raises the possibility that some or all EC-stimulated CTL that display EC selectivity are also selective because of accessory interactions rather than cell-specific peptides. This possibility will also be explored in our future studies.
Cytolytically active CTL clones fulfill the definition of an effector T cell. Expression of Mac-1 and perforin is a recognized marker of effector CTL (21), and these molecules are also found in a majority of our EC-selective and conventional CTL clones. CD40L is also an effector cell marker, but more typically on CD4+ Th cells (12, 13). We identified CD40L as the most consistent surface marker for EC-stimulated CTL. However, the correlation of CD40L expression with EC-selective killing pattern did not reach statistical significance. Although we think these traits are probably linked, and that statistical significance would become clearer in a larger analysis, the points remain that some CD40L-expressing CTL may exhibit a conventional target cell profile, and that not all EC-selective CTL are CD40L positive. CD40L-CD40 signals have been shown to induce B cell activation and Ab isotype switching (22, 23), dendritic cell maturation (24, 25), as well as macrophage (26) and EC activation (27). Transiently expressed CD40L on CD8+ T cell clones has been shown to be functionally active (28). If EC-stimulated CTL express this important costimulatory molecule so persistently in vivo, it may well substitute for CD4+ T cells and amplify immune responses in the absence of class II MHC-restricted signals.
The second major difference between EC- and BLC-stimulated CTL was the
capacity to secrete IFN- after target cell lysis. Long-term
EC-selective CTL did not secrete IFN- in response to EC nor BLC. The
threshold of integrated TCR activation events required for IFN-
secretion has been reported to be orders of magnitude higher than that
for cytolysis (29). Our data would conform with this model if
EC-selective CTL were activated by a very rare EC-specific Ag signal,
sufficient to trigger killing, but insufficient to induce cytokine
secretion. However, EC-selective CTL clones can secrete TNF in response
to BLC, but not EC. This observation indicates EC-selective CTL are
responsive to BLC, and supports the notion that peptide recognition is
not the basis of the cell type-selective killing by EC-selective CTL
clones, although we did not show that TNF synthesis was actually
alloantigen dependent.
The effects of EC upon CTL differentiation reported in our previous
study and extended here to the clonal level have implications for both
transplantation and vascular biology. For example, our data suggest
that the differentiation and/or expansion of conventional CTL
precursors are likely to be suppressed in a microenvironment in which
EC are in close apposition to infiltrating T cells, e.g., in the intima
of the arterial wall. Those CTL that do emerge may be EC selective,
poorly secrete IFN- after activation, and constitutively express
CD40L. In allografts, EC-selective CTL may mediate endothelialitis, the
harbinger of therapy-resistant acute vascular rejection (30). Acute
endothelialitis may evolve into chronic graft arteriosclerosis, the
principal cause of cardiac and renal graft failure (31, 32). On the
other hand, IFN- has been shown in mouse heart transplant models to
be essential for intimal expansion in subacute/chronic allograft
rejection, despite the fact that it is not required for acute
parenchymal rejection (33). In contrast, elevated CD40L and perforin
mRNA levels have been shown to be independent risk factors for acute
kidney transplant rejection (34, 35). CD40L is also relevant in chronic
pathobiology of the arterial intima, contributing to the formation of
atheromata in hyperlipidemic mice (36), possibly triggering acute
coronary syndromes in humans by promoting macrophage production of
tissue factor and matrix metalloproteinases (26) and mediating arterial
intimal expansion in a heterotopic heart transplantation model in mice
(37). Finally, our new data suggest that coexpression of CD40L and
perforin may be a useful marker to identify unusual, EC-stimulated CTL
in situ in the setting of intimal disease. We conclude by noting that
endothelial cells may not only be activators of circulating memory T
cells, but may influence the outcome of immune reactions by mediating
novel forms of immune deviation.
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Acknowledgments |
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Footnotes |
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2 Current address: Medizinische Universitaetsklinik, Bruderholzspital, 4101 Bruderholz, Switzerland.
3 Address correspondence and reprint requests to Dr. Jordan S. Pober, Boyer Center for Molecular Medicine, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06510. E-mail address:
4 Abbreviations used in this paper: EC, endothelial cells; BLC, B lymphoblastoid cells; CD40L, CD40 ligand; FasL, Fas ligand.
Received for publication January 14, 1999. Accepted for publication March 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), and BLC (
). Between 5 and 8 wk after the coculture was
started, the clones were tested at least once for lysis of EC or BLC,
both derived from the original donor of stimulator cells. For each
individual clone, percent specific lysis of EC is displayed on the
y-axis, and percent specific lysis of BLC on the
x-axis. Ten percent specific lysis was taken as
threshold ofsignificant killing for both target cell types (dotted
lines). CTL that lysed both target cells <10% were defined as
nonkillers. For those clones that were tested in more than one CTL
assay, the mean values of cytolysis are displayed.
