| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06510
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
secretion (4). Further studies of EC-stimulated
CTL clones confirmed these unusual features, especially EC selectivity
and low production of IFN-, which correlated closely with
constitutive surface expression of CD40 ligand (CD154)
(5), a molecule that is not usually seen on
CD8+ T cells. Although our initial studies highlighted the capacity of EC to stimulate an unusual CTL response, these analyses were limited to a relatively late time point, i.e., after CD8+ T cell clonal expansion had progressed sufficiently to permit measurement of cytolysis or after cloning by limiting dilution with further propagation in culture. In the present study, we have used 5-(and -6)-carboxyfluorecein diacetate succinimidyl ester (CFSE) labeling to identify and selectively analyze proliferating lymphocytes (6), which allowed us to look at earlier events in the primary coculture system. We have used this approach to compare EC with professional APC, e.g., B lymphoblastoid cells (BLC), as stimulators of CTL by analyzing the kinetics of clonal expansion, the identity of the resting T cells that are able to respond to alloantigen by proliferation, and the phenotype of the proliferating T cell populations during their clonal expansion. We have also been able to analyze the effects of costimulation blockade, costimulatory augmentation, and cyclosporine (CsA) on both clonal expansion and CTL maturation. Our experiments show that EC stimulate a smaller number of alloreactive CD8+ T cells drawn entirely from the memory T cell subset compared with BLC, which stimulate expansion of both naive and memory alloreactive T cells. Interestingly, EC-stimulated CTL appear to retain an early activation phenotype characterized by persistent expression of CD69, CD25, and L-selectin (CD62L). In the presence of exogenous IL-2, which is required for CTL generation in these cocultures with EC or BLC stimulators, we have not detected a role for costimulation of CD8+ T cells through B7, LFA-3, ICAM-1, or CD40. CsA inhibits EC- and BLC-stimulated cocultures primarily by reducing clonal expansion despite the presence of exogenous IL-2 and does not appear to significantly affect other CTL differentiation events. We conclude that EC cause a form of immune deviation resulting in activation of unusual CTL effector populations that could be important for allogeneic responses that develop within the graft vessel wall.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
PBMC were obtained from healthy volunteers by density-gradient centrifugation of leukapheresis products and were stored in liquid nitrogen as described previously (7). CD8+ T cells were isolated from PBMC by positive selection (4) using Dynabeads (Dynal, Lake Success, NY). The selected population obtained by this procedure was routinely >98% CD8+/CD3+ by flow cytometry and >99% viable, as shown by trypan blue exclusion. Use of positively selected CD8+ T cells in this assay had previously been shown to yield identical results compared with negative selection (4). Naive and memory subsets of T cells were isolated from the CD8-selected population by further negative selection. CD8+ T cells were incubated with anti-CD45RA (B-C15; 1 µl/107 cells) or anti-CD45RO mAb (5 µl/107 cells; UCHL-1, both mAb from Biosource, Camarillo, CA), respectively, for 30 min at 4°C. After three washes, cells were incubated with magnetic beads coated with goat anti-mouse IgG antiserum (Dynal) for 30 min at 4°C. Cells not attached to beads were recovered during application of a magnet; negatively selected populations obtained in this manner were routinely >95% double-positive for CD8 and either CD45RA or CD45RO, respectively, and were >99% viable.
HUVEC were isolated from umbilical cords by enzymatic digestion and maintained in culture, as described (8). EC cultures were free from detectable CD45+ contaminating leukocytes and uniformly expressed von Willebrand factor and CD31. BLC from the same donors as the HUVEC were generated by EBV immortalization of cord blood mononuclear cells (4). After 6–8 wk in culture, BLC lines were uniformly CD19-positive. HUVEC and BLC of the same donor were used as stimulator cells in cocultures and as target cells in cytotoxicity assays. When BLC were used as stimulator cells in cocultures, they were pretreated with mitomycin C (50 µg/ml in PBS, 30 min; Sigma-Aldrich, St. Louis, MO) to prevent proliferation. This treatment did not affect the outcome of cultures stimulated with HUVEC and was routinely omitted.
CTL generation
The procedure for CTL differentiation has been described in detail elsewhere (4). In brief, purified CD8+ T cells were incubated with EC or BLC stimulator cells in 96-well microculture plates (Falcon; Becton Dickinson, Bedford, MA) at responder-to-stimulator cell numbers of 200,000/20,000 for EC cocultures and 100,000/20,000 for BLC cocultures. A total of 24–48 microculture replicates were initiated for each experimental group within a given responder-stimulator combination. All cultures were maintained in 5% CO2 room air at 37°C. 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 further supplemented with exogenous IL-2 (R&D Systems, Minneapolis, MN; or National Cancer Institute, Frederick, MD) on day 3 (final concentration, 5 ng/ml (12.5 U/ml, R&D conversion table)). On day 7, T cells were transferred to fresh stimulator cells in fresh medium containing IL-2, and cultures were fed with fresh medium containing IL-2 on day 10. On day 14, CTL were harvested from microculture wells, counted manually in a hemocytometer, and subjected to further analyses described below either from individual wells or after pooling cells from 3–12 microwells. CD8+ T cells in the absence of any stimulator cells but receiving the same IL-2 treatment were cultured in replicate wells as unstimulated controls.
In experiments involving the addition of blocking mAb, mAb were added at the initiation of cocultures at final concentrations indicated below; treatment with mAb was repeated at times of medium change on days 3, 7, and 10. The mAb used were TS2/9 (anti-CD58; 1:200 dilution of ascites; gift of T. Springer, Center for Blood Research, Boston, MA), M3 (anti-CD40; 10 µg/ml; gift of W. C. Fanslow, Immunex, Seattle, WA), and D5 (anti-ICAM-1; 10 µg/ml; gift of Dr. D. Altieri, Yale Medical School, New Haven, CT). The inhibitory potential of mAbs used in these studies had been extensively demonstrated previously (9, 10); saturating concentrations of all mAb were used. To exclude interference of blocking Abs with cytotoxicity assays, recovered cells were washed before use.
For costimulation experiments, anti-CD28 mAb (clone 37407.111; R&D Systems) was added at the initiation of cocultures at 10 µg/ml and again at times of medium change on days 3, 7, and 10. For preactivation of EC with CD40 ligand, CD40 ligand trimer (gift from W. C. Fanslow) was added to EC at 100 ng/ml for 24 h before initiation of cocultures.
For experiments involving CsA, Sandimmune Neoral (liquid formulation, 100 mg/ml; Novartis, East Hanover, NJ) was added at initiation of cocultures or 4 h before cytotoxicity assays at 500 ng/ml. Cells recovered from cocultures were washed before use in cytotoxicity assays.
Cytotoxicity assay
Cytotoxicity by CTL was measured by a calcein fluorescent dye release assay as described previously (4, 11). In brief, target cells were loaded with calcein-AM (Molecular Probes, Eugene, OR) and incubated with effector CTL from individual microculture wells for 4 h at 37°C. In experiments involving CsA or mAb, CTL were washed three times before addition to cytotoxicity assays. The supernatant was harvested and calcein release was quantitated with a fluorescence plate reader (Cytofluor 2; Perseptive Biosystems, Framingham, MA; excitation wavelength, 485 nm; emission wavelength, 530 nm). Killing was considered positive for individual microculture wells if released fluorescence in a sample well exceeded the mean + 3 SD of the spontaneous release. Percent specific killing was calculated as follows: (release sample - spontaneous release)/(maximal release - spontaneous release) x 100. Spontaneous release was obtained by medium alone, and maximal release was obtained by adding lysis buffer (50 mM sodium borate and 0.1% Triton X-100 (pH 9)). Corresponding BLC or EC were used as targets for BLC-stimulated or EC-stimulated cultures, respectively. In most experiments, the cytotoxicity of EC-stimulated cultures against BLC autologous to the EC was also measured to confirm EC selectivity.
Immunophenotyping of CTL from microcultures
For flow cytometric analysis, T cells were harvested from microculture wells, and cells were further processed either from individual wells or after pooling of cells from 6–18 wells, as indicated. Cells were washed twice in ice-cold PBS/1% BSA and incubated with saturating concentrations of directly FITC- or PE-conjugated mouse anti-human CD3, CD8, CD25, CD69, CD45RA/RO, CD62L, HLA-DR (all from Coulter Immunotech, Miami, FL), or CD154 (TRAP-1; PharMingen, San Diego, CA) for 1 h at 4°C. Cells were washed twice in ice-cold PBS/1% BSA and fixed with 1% paraformaldehyde in PBS. Samples were analyzed using FACSort (Becton Dickinson, San Jose, CA) and CellQuest analysis software by gating on viable cells and collecting 5000 gated events per sample. For intracellular staining for flow cytometric analysis, cells were washed twice in ice-cold PBS and fixed in 4% paraformaldehyde in PBS for 15 min at 4°C. After washing twice, cells were incubated for 15 min in permeabilization solution consisting of 1% FCS, 0.1% saponin (Sigma-Aldrich), and 0.1% sodium azide. Cells were then incubated with primary unconjugated mouse anti-human perforin mAb (T-Cell Sciences, Woburn, MA) in permeabilization solution for 1 h at 4°C. After washing twice in permeabilization solution, cells were incubated with PE-conjugated goat anti-mouse IgG antiserum for 1 h at 4°C. Cells were finally washed twice in permeabilization solution and once in PBS/1% BSA and stored in PBS/1% BSA until analysis by flow cytometry as above. For immunofluorescence microscopy, CTL were fixed, spun onto glass slides, permeabilized, and stained as described previously (4). Cells were examined with a Microphot FXA microscope (Nikon, Natick, MA).
Labeling studies of CD8+ T cells with CFSE
Labeling and FACS analysis of CD8+ T cells were performed as described elsewhere (6, 12). In brief, total CD8+ T cells or separated CD45RA+/RO+ subsets were washed twice in cold PBS and then incubated in 0.25 µM CFSE (Molecular Probes) in PBS at 37°C for 15 min. Cells were again washed twice before addition to cocultures. At indicated times, CFSE-labeled cells were collected, washed in PBS/1% BSA twice, and fixed in PBS/1% paraformaldehyde. For two-color fluorescence studies, CFSE-labeled cells were incubated with saturating concentrations of directly PE-conjugated mouse anti-human mAbs reactive with CD3, CD8, CD69, CD25, CD62L, CD45RA, CD45RO, HLA-DR, and CD154 for 30 min at 4°C, washed twice, and subjected to flow cytometric analysis. Unlabeled CD8+ T cells and cells fixed immediately after CFSE labeling were used as negative and positive internal controls for staining, respectively.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To analyze the extent of lymphocyte expansion after stimulation
with allogeneic EC or BLC, CD8+ T cells were
labeled with CFSE before addition to microwell cocultures. Fig. 1
is a representative example of
two-color FACS analysis of surface CD8 labeled with PE-conjugated mAb
and CFSE content after 14 days of coculture with EC or BLC.
Unstimulated CFSE-labeled CD8+ T cells and
unlabeled CD8+ T cells are included as positive
and negative staining controls. In both types of cocultures,
CD8+ T cells fall into discrete
CFSEhigh and CFSElow
populations. CFSEhigh cells, recovered from
cocultures with either type of stimulator cell, express the same
intensity of CFSE fluorescence as unstimulated
CD8+ T cells or CD8+ T
cells immediately after labeling (not shown), indicating that no cell
divisions have occurred in these populations.
CFSElow/CD8+ T cells show a
fluorescence intensity comparable to that of unlabeled
CD8+ T cells. These populations represent cells
that have been activated by alloantigen and have undergone multiple
cell divisions such that there has been a dilution of the CFSE signal
below the limit of detection. Although the number of cell divisions
that these alloactivated CD8+ T cells have
undergone cannot be exactly defined, a minimum of seven to eight rounds
of cell divisions is required to dilute CFSE to background levels of
fluorescence. Further enhancement of the CFSE signal to detect more
rounds of division was not possible because higher concentrations of
CFSE inhibited CD8+ T cell proliferation (not
shown). The absence of cells with intermediate CFSE labeling, which are
readily detectable in PHA-activated cultures (our unpublished
observation), is consistent with the interpretation that the
alloreactive precursor populations of CD8+ T
cells (as low as 1:20,000 for EC stimulators) are too small to be
detectable until multiple divisions have occurred (7).
|
). On day 3 of the
coculture period, no significant population of
CFSElow cells was detectable either after EC or
BLC stimulation (not shown). With BLC stimulators, significant numbers
of alloactivated (CFSElow) T cells became
detectable on day 7, expanding until day 10 and then either leveling
off in absolute number (Fig. 2, Exp. A) or expanding further
(Fig. 2, Exp. B) until day 14. Total numbers of
expanded CD8+ T cells always reached at least
100% of input cell numbers with BLC stimulation. On day 14 of
coculture, essentially all microculture wells stimulated with BLC
consistently tested positive for cytotoxicity (Fig. 2, insets) with mean specific killing exceeding 30%. With EC
stimulation, low but significant numbers of alloactivated, expanded
CD8+ T cells only became detectable on day 10.
Such cells usually expanded moderately further until day 14. However,
total cell numbers of alloactivated (CFSElow)
cells after EC stimulation never reached more than 50–60% of input
cell numbers. After 14 days, 
15–25% of microculture wells
stimulated with EC were positive for cytotoxicity (Fig. 2, insets), and mean specific killing remained below 15%.
Control experiments, demonstrating the absence of cytotoxicity of
EC-stimulated CTL vs corresponding BLC, confirmed the EC specificity of
killing in these cocultures as reported previously (Ref. 4
and not shown). Extension of EC-stimulated cocultures beyond day 14 by
restimulation and refeeding failed to further enhance the absolute
number of alloactivated cells or the extent of cytotoxicity. Indeed, in
most EC-stimulated cultures the absolute and relative sizes of the
expanded (CFSElow) cell pool actually contracted
between day 14 and day 21 (not shown). The number of surviving,
nondividing (CFSEhigh) CD8+
T cells (relative to input cell number) was not significantly different
between BLC or EC stimulation (Fig. 1
). These data show that BLC
stimulation of CTL consistently displayed significantly greater
expansion of relative and absolute numbers of alloactivated cells than
EC stimulation. This greater expansion of the alloactivated population
correlates with and can explain the higher degree of measured cytotoxic
activity of cells recovered from BLC-stimulated cocultures.
|
After 14 days of microwell coculture, recovered
CD8+ T cells were analyzed by two-color flow
cytometry for CFSE fluorescence levels and for expression of several
surface activation markers or intracellular perforin expression. CFSE
labeling of CD8+ T cells in these experiments
allowed the separate analysis of surface Ags and perforin expression
for unactivated (CFSEhigh) and for
alloactivated, expanded (CFSElow) cells. At
14 days after stimulation with BLC, the alloactivated
CD8+ T cells were almost exclusively
CD45RO+ (memory phenotype) and expressed high
levels of HLA-DR and very low levels of CD25 and CD69. These last two
markers, indicative of early activation, had fallen to levels
comparable to those of unstimulated, IL-2-treated control cells (Fig. 3A). Expression of L-selectin
(CD62L) was significantly reduced compared with that in control cells
but was not completely absent. In day 14 cultures stimulated with EC,
the alloactivated, expanded CD8+ T cells
(CFSElow) were also exclusively of
CD45RO+ phenotype and expressed high levels of
HLA-DR. In striking contrast to BLC-stimulated
CD8+ T cells, alloactivated
(CFSElow) T cells in EC-stimulated cultures
retained high expression levels of the early activation markers CD69
and CD25. In addition, almost all activated cells were positive for
CD62L, an even higher percentage than that observed in unstimulated
control cells (Fig. 3A). In cocultures extended beyond 14
days, these phenotypic differences were essentially maintained (not
shown); in most EC-stimulated cocultures, the number of alloactivated T
cells began to decline after more than 14 days (see above). Comparisons
of 10-day cultures, i.e., 3 days after restimulation, showed some
expression of CD25 and CD69 in BLC-stimulated cultures, confirmimg that
the low levels seen in day 14 cultures did represent a decline from
peak levels. However, even at day 10 the level and percentage of
positive cells were still markedly lower than those in EC-stimulated
cultures (not shown). In BLC- or EC-stimulated cocultures, CD154 was
not detectable on alloactivated (CFSElow) cells
by flow cytometry (Fig. 3A).
|
B). After stimulation with either cell type, 90% of the
alloactivated CD8+ T cell population expressed
detectable levels of perforin; however, the expression level per cell
was markedly higher after stimulation with BLC. Nondividing
CD8+ T cells in coculture and unstimulated,
IL-2-treated control cells did not show detectable perforin expression
(not shown). Activation of CD45RA+ (naive) and/or CD45RO+ (memory) subsets
Although all of the alloactivated CD8+ T
cells recovered at day 14 expressed CD45RO, these cells could have
arisen from resting memory (CD45RO+) or resting
naive (CD45RA+) subsets or both. To directly
identify the subset(s) of CD8+ T cells that were
activated by stimulation with BLC or EC, cocultures were set up using
CFSE-labeled, purified CD45RA+ or
CD45RO+ CD8+ resting T
cells. As in the case of unseparated CD8+ T
cells, populations were analyzed after 14 days by two-color flow
cytometry and cytotoxicity assays. After stimulation with BLC, the
proportion and absolute numbers of alloactivated
(CFSElow) CD8+ T cells were
similar in both subsets, indicating that both naive and memory
CD8+ T cells were activated. The expression of
surface activation markers and perforin was very similar for both
subsets after BLC stimulation and was comparable to the findings from
studies of unseparated CD8+ T cells (Fig. 4A; perforin not shown),
namely showing low expression of CD25 and CD69. A high proportion
(90%) of cells in the CD45RA+ (naive) subset
had become CD45RA/RO double-positive after activation. These results
differ from those obtained after stimulation with EC, when
alloactivated CD8+ T cells
(CFSElow) were only detectable in the
CD45RO+ (memory) subset (Fig. 4A).
Expression of surface activation markers was similar to findings with
unseparated CD8+ T cells (Fig. 4A)
showing high levels of CD25 and CD69 in alloactivated cells. However,
all alloactivated cells expressed perforin again at a markedly lower
level than after stimulation with BLC (not shown).
|
B). Similarly to experiments with unseparated
CD8+ T cells, only about 20% of EC-stimulated
microwell cocultures showed detectable cytotoxicity (Fig. 4B), and the percentage of specific killing was markedly
lower than that observed in BLC-stimulated cocultures (not shown). Effect of inhibition or augmentation of costimulation upon EC-stimulated CTL differentiation
Previous studies have shown that neither differences in production
of IL-12 nor MHC class I expression levels could account for the
differences in the capacities of EC vs BLC to stimulate and expand CTL
(4). BLC also differ from EC regarding the costimulator
molecules expressed by these cell types (13), and these
differences are reflected in the pathways used to stimulate IL-2
production. The influence of CD8+ T cell
costimulation by EC upon CTL generation was examined in cocultures
using blocking mAbs. Because these cocultures were also fed routinely
with exogenous IL-2, these blocking experiments examine features of T
cell activation and differentiation independent of IL-2 synthesis.
Inhibition of costimulation with mAbs vs LFA-3, ICAM-1, or CD40 did not
influence any of the parameters analyzed after 14 days of coculture,
including T cell numbers, extent of cytotoxicity, perforin positivity
and expression level, and expression of surface activation markers
(Fig. 5).
|
A).
|
B). Effect of CsA on CTL differentiation
To address the influence of the immunosuppressant CsA on the
differentiation of CTL after stimulation with BLC or EC, cocultures
were performed in the presence of 500 ng/ml of CsA added at various
time points after coculture initiation. In the presence of CsA, the
absolute number of alloactivated cells (i.e.,
CFSElow) was markedly reduced with both types of
stimulator cells (Fig. 7), suggesting
general inhibition of the expansion/proliferation of alloactivated
CD8+ T cells even in the presence of exogenous
IL-2. In case of stimulation with BLC, the absolute size of the
alloactivated (CFSElow) cell population was
reduced by 75% after 7, 10, and 14 days compared with cultures
without CsA (Fig. 7, left half of panel). In EC-stimulated cultures,
alloactivated CD8+ T cells in the presence of CsA
could only be detected on day 14, and the absolute number of expanded
(activated) cells was reduced by 80% compared with control cultures
without CsA (Fig. 7, right side of panel). The influence of CsA on
cytotoxic activity of CTL from cocultures was examined, using T cells
recovered at day 14. CsA suppressed cytotoxicity displayed by
cocultures after stimulation with either BLC or EC (Fig. 8). Addition of CsA at the initiation of
BLC-stimulated cocultures resulted in a reduction in microwells testing
positive for cytotoxicity from 100% to 20%, with a corresponding
decrease in mean percentage of specific killing. Similarly, in
EC-stimulated cocultures, detectable cytotoxicity was completely lost
after early addition of CsA. Treatment of cocultures for 4 h
before the cytotoxicity assay still resulted in a moderate decrease in
cytotoxicity (Fig. 8) without affecting cell numbers (not shown) in
both types of cocultures, but the magnitude of this effect was marginal
compared with that produced by persistent CsA exposure. However,
persistent presence of CsA in cocultures did not influence the
expression of surface activation markers and perforin expression after
stimulation with either BLC or EC on those CD8+ T
cells that did proliferate (Fig. 9).
These observations suggest that the primary action of CsA is to reduce
clonal expansion, despite the presence of IL-2, to a level that falls
below the sensitivity of the cytotoxicity assays. Those cells that do
expand appear resistant to the effects of CsA, and a smaller number of
CsA-resistant clones emerge in the EC-stimulated cultures than in the
BLC-stimulated cultures, which is consistent with the lower overall
frequency of EC-responsive CTL.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and
CD40L (CD154) expression (5).
In the present study, EC-selective CTL were analyzed and compared with
conventional CTL directly from primary cocultures with stimulating
cells, thus eliminating potential selection bias and long-term culture
changes that may occur during cloning procedures. Two principal
findings of our study are that CD8+ T cells
expanded less well after stimulation with EC than with BLC and that
stimulation of CD8+ T cells by EC exclusively
activated memory (CD45RO+) cells to differentiate
into EC-selective CTL, whereas BLC stimulators were able to recruit
both naive and memory CD8+ T cells to
differentiate into CTL. Specifically, there was a 5- to 20-fold
reduction in the final absolute number of expanded (i.e.,
alloactivated) CD8+ T cells after EC stimulation
compared with that after BLC stimulation. Therefore, on the basis of
these data, the reduced cytotoxicity seen with EC-selective CTL appears
to be largely due to much lower E:T ratios in these cytotoxicity
assays. However, EC-stimulated CTL also expressed significantly lower
amounts of perforin in individual cells (Fig. 2, Exp.
B), which could also contribute to the difference in
cytotoxicity. The smaller number of expanded, alloactivated
CD8+ T cells in EC-stimulated cocultures can in
principle be explained either by a lower frequency of precursor cells
or by fewer generations of cell division during cocultures. To account
for the observed difference in CTL at the end of cocultures, precursor
frequencies for BLC-stimulated CTL would have to be 5–20 times
higher than for EC-responsive CD8+ T cells.
Alternatively, the difference in absolute numbers of CTL at the end of
cocultures could be caused by two to four fewer rounds of cell division
in EC-stimulated cultures due to generally slower kinetics of
CD8+ T cell divisions or to a delayed start of T
cell expansion in the presence of EC stimulation. As technical
limitations preclude the exact determination of generational cycles in
our cocultures, neither hypothesis could be tested experimentally.
Considering that EC stimulation is only capable of activating memory
CD8+ T cells, the precursor frequency in
EC-stimulated cultures at most can represent half of that in
BLC-stimulated cultures. Moreover, previous studies determined that the
precursor frequency in CD8+ T cells comparing EC
to monocyte stimulators was 5 times lower when measured by limiting
dilution analysis of IL-2 production (7). Moreover, if
EC-selective CTL arise from precursors that only recognize EC-derived
peptides, this precursor pool of pre-CTL may be even smaller than that
determined by limiting dilution analysis. On the other hand,
proliferation after stimulation with EC does appear to be delayed
compared with BLC stimulation (7), and EC reduce T cell
proliferation stimulated by BLC in mixed cocultures (4).
Thus, both factors may contribute to the lesser capacity of EC to
stimulate alloreactive T cell populations. The inability of EC to
activate naive T cells has been proposed to be due to limitations in
Ag-independent adhesion compared with other, professional APC
(9) or to reduced capacity of cytokine secretion,
especially IL-12 (9, 20). Previous observations that
supplementation of EC-stimulated cocultures with IL-12 did not improve
the generation of CTL (4) render the latter alternative
less likely.
CTL from EC-stimulated cultures continued to express surface markers CD69, CD25, and L-selectin (CD62L) at the end of the coculture period, representing an early, immature activation phenotype. CD69 and CD25 are generally considered early activation markers (21, 22, 23), and L-selectin is usually lost rapidly during the course of T cell activation (24, 25, 26). Also, the expression level of perforin per cell in EC-selective CTL was markedly lower than in corresponding conventional CTL. In contrast, on BLC-stimulated CTL, the expression of CD69 and CD25 had subsided, and CD62L expression, highly expressed in CD8+ T cells at the beginning of coculture, had largely been lost. The findings in BLC-stimulated CD8+ T cells are in accordance with numerous in vitro and in vivo reports showing the loss of early activation markers 2–3 days after activation of T cells by BLC or other professional APC (21, 22, 23). A direct correlation between the number of cell divisions and loss of CD25 and CD69 also has recently been reported (27). Time course of the activation phenotype on T cells after (allo)activation by EC has not been reported previously, and the basis for the retention of this early activation phenotype of EC-specific CTL is currently unknown. Several (not mutually exclusive) explanations appear possible. First, the phenotype seen on EC-stimulated CTL might represent an immature state of activation caused by slower or delayed activation of CD8+ T cells by EC, such that the EC-selective CTL lag behind their conventional counterparts in the down-regulation of early activation markers and in the synthesis of perforin. Such defective activation in EC-stimulated cocultures may be caused by differences in EC-mediated adhesion, costimulation, or cytokine production. However, the persistence of the phenotypic differences between EC- and BLC-stimulated CTL beyond 14 days of coculture seems to argue against a simple temporal lag in the EC cocultures. It also appears unlikely that the differential phenotype of CTL after stimulation with EC or BLC should be exclusively due to fewer cell divisions after EC stimulation, especially because similar differences in the expression of early activation markers could already be detected after 10 days of coculture with EC. If the differences in expansion and phenotype between the cocultures were entirely due to the number of cell divisions, the number of cell divisions at day 14 after stimulation with EC would be expected to be more equivalent to the cell divisions at day 10 after stimulation with BLC, and cell phenotypes would likely correspond. Also, the up-regulation of CD62L was only seen in EC-stimulated cultures and represents a unique feature of the EC-selective CTL phenotype. EC-derived effects could also account for the increased expression of CD69, even on nondividing (CFSEhigh) CD8+ T cells, in EC-stimulated cocultures compared with IL-2-treated control cultures (not shown). EC have been shown to secrete IL-7 and IL-15 and could thus up-regulate the expression of CD69 and CD25 (28, 29, 30, 31). Finally, continuing activation of CD8+ T cells by alloantigen in cocultures with EC but not with BLC could account for the described phenotype. Persistent expression of early activation markers has been demonstrated on T cells in disease states associated with continuous long-term immune activation, especially HIV and CMV infection or rheumatoid arthritis (32, 33, 34). In the current study, persistent T cell activation might theoretically result from a greater persistence of EC than BLC in the cocultures such that EC would continue to activate CD8+ T cells. EC stimulators (in contrast to BLC stimulators) were not treated with mitomycin for growth arrest, but pilot experiments failed to show any difference in CTL generation irrespective of mitomycin treatment (not shown). In addition, no viable EC were detectable by microscopy in coculture wells after 5 days.
Previous studies from our group and others have emphasized differences in the costimulators used by human EC and professional APC. In IL-2-supplemented EC-stimulated cultures, clonal expansion, surface phenotype, perforin positivity, and cytotoxicity toward EC were unchanged in the presence of blocking Abs to the three most relevant accessory receptor/ligand pairs involved in the interaction of EC and T cells: LFA-1/ICAM-1, CD2/CD58, and CD40/CD154. HUVEC do not express the ligands for either CD28 (B7.1 and B7.2; Ref. 10) or CD137, the human equivalent for mouse 4-1BB (ILA; T. J. Dengler, unpublished observation), and the T cell costimulatory potential of VCAM-1 and E-selectin are thought to be of minor importance (20, 35). However, exogenous costimulatory enhancement with a comitogenic anti-CD28 mAb was also without effect on CTL generation. The central role of the CD40/CD154 interaction for the priming of professional APC as stimulators of CTL has been demonstrated extensively (14, 15, 36), but we did not observe a similar effect on EC in our experiments using recombinant CD40 ligand trimer. These results of our costimulation studies are in accordance with the classic concept that T cell costimulation by accessory cells primarily induces increased IL-2 production (37), an effect that was bypassed in our experiments by exogenous IL-2.
Finally, as EC-selective CTL might play an important part in the pathogenesis of forms of rejection (e.g., endothelialitis) that are less amenable to treatment, we investigated the sensitivity of EC-selective CTL to CsA. Our results demonstrate a similar inhibition of alloactivated CD8+ T cell expansion by CsA in EC- or BLC-stimulated cocultures. In BLC-stimulated cultures, the proportion of CD8+ T cells expanding despite the presence of CsA increased over time and was markedly higher than in EC-stimulated cocultures, which is again compatible with a delayed activation process in the presence of EC stimulators. Interestingly, the suppression of proliferation occurs in the presence of significant amounts of exogenous IL-2. Similar suppression of T cell growth by CsA has been reported previously (38, 39) and may be explained by more recent data indicating that CsA can inhibit T cell activation by additional mechanisms such as TGF-ß production, up-regulation of the cdk inhibitor p21, and inhibition of IL-2 receptor expression (40, 41, 42). Unspecific toxic or apoptotic cell death induced by CsA can be excluded as a cause of the reduced cell numbers in our experiments on the basis of the low concentration of CsA (500 ng/ml) used and the absence of apoptotic cells. CsA also reduced cytotoxicity, primarily via the reduction in CTL numbers, and consequently reduced E:T ratios. Differentiation or maturation of CTL at the level of the individual effector cell was not affected by CsA, as indicated by normal expression of activation markers and perforin. Late treatment of cocultures with CsA seemed to have a marginal inhibitory effect on cytotoxicity, which is in line with previous reports suggesting inhibition of perforin degranulation by CsA (43, 44). Taken together, EC stimulation of CD8+ T cells for CTL generation does not appear to impart any CsA resistance.
The effects of EC upon CTL activation and differentiation described here and in our previous studies have significant implications for transplantation and vascular biology. As shown earlier (4), the generation of highly cytotoxic, conventional CTL is suppressed by EC, a finding with particular pertinence for microenvironments in which EC are closely apposed to infiltrating immune cells, such as the vascular intima. On the other hand, stimulation of CD8+ T cells by EC appears to favor the differentiation to a unique population of CTL displaying a variety of characteristic features, including an immature early activation phenotype, low perforin content, reduced capacity for clonal expansion, and cell type selectivity. These results strengthen a function of vascular EC as semiprofessional APC of intermediate stimulatory capacity as proposed earlier (7, 9) and help to define an evolving concept of the vascular endothelium as an immunomodulatory cell type. In the intimal space, where EC are likely to represent the predominant APC, that may create a distinct immunologic compartment endowed with specific regulatory characteristics, similar to other organ-specific immune functions. For example, the immaturity and comparatively ineffective clonal expansion of EC-stimulated CTL and the suppression of conventional CTL suggest that the predominant modulatory role of the endothelium might be inhibitory for immune-mediated injury. Based on our in vitro findings and recent clinical data (3), such EC-mediated immunoregulation can be overcome and can result in activation and differentiation of CTL selectively directed against EC. By analogy to other organ-specific CTL (17, 18), the generation of EC-selective CTL could be an independent, tissue-specific alloimmune process occurring in the absence of generalized alloactivation. Vascular rejection and intimal arteritis could be interpreted as forms of "organ"-specific endothelial rejection mediated by such EC-selective CTL, developing independently of wide-spread parenchymal rejection. Further characterization of EC-selective CTL and their activation requirements could lead to a better understanding of the pathogenesis and to novel therapeutic approaches for immune-mediated vascular pathology.
|
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 06510.
3 Abbreviations used in this paper: EC, endothelial cell; BLC, B lymphoblastoid cell; CFSE, 5-(and -6)-carboxyfluorecein diacetate succinimidyl ester; CsA, cyclosporine; CD62L, CD62 ligand; CD40L, CD40 ligand.
Received for publication December 20, 1999. Accepted for publication March 3, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production. J. Immunol. 159:3247.[Abstract]
chain/CD25 promoter is a target for nuclear factor of activated T cells. J. Exp. Med. 188:1369.This article has been cited by other articles:
|
|
 |
|
  S. A. Gerber and J. S. Pober IFN-{alpha} Induces Transcription of Hypoxia-Inducible Factor-1{alpha} to Inhibit Proliferation of Human Endothelial Cells J. Immunol., July 15, 2008; 181(2): 1052 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. D. Manes and J. S. Pober Antigen Presentation by Human Microvascular Endothelial Cells Triggers ICAM-1-Dependent Transendothelial Protrusion by, and Fractalkine-Dependent Transendothelial Migration of, Effector Memory CD4+ T Cells J. Immunol., June 15, 2008; 180(12): 8386 - 8392. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Coupel, A. Moreau, M. Hamidou, V. Horejsi, J.-P. Soulillou, and B. Charreau Expression and release of soluble HLA-E is an immunoregulatory feature of endothelial cell activation Blood, April 1, 2007; 109(7): 2806 - 2814. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Choy, Y. Wang, G. Tellides, and J. S. Pober Induction of inducible NO synthase in bystander human T cells increases allogeneic responses in the vasculature PNAS, January 23, 2007; 104(4): 1313 - 1318. [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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
L. Zheng, T. F. Gibson, J. S. Schechner, J. S. Pober, and A. L. M. Bothwell Bcl-2 Transduction Protects Human Endothelial Cell Synthetic Microvessel Grafts from Allogeneic T Cells In Vivo J. Immunol., September 1, 2004; 173(5): 3020 - 3026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Manavalan, S. Kim-Schulze, L. Scotto, A. J. Naiyer, G. Vlad, P. C. Colombo, C. Marboe, D. Mancini, R. Cortesini, and N. Suciu-Foca Alloantigen specific CD8+CD28- FOXP3+ T suppressor cells induce ILT3+ ILT4+ tolerogenic endothelial cells, inhibiting alloreactivity Int. Immunol., August 1, 2004; 16(8): 1055 - 1068. [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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
T. J. Dengler, D. R. Johnson, and J. S. Pober Human Vascular Endothelial Cells Stimulate a Lower Frequency of Alloreactive CD8+ Pre-CTL and Induce Less Clonal Expansion than Matching B Lymphoblastoid Cells: Development of a Novel Limiting Dilution Analysis Method Based on CFSE Labeling of Lymphocytes J. Immunol., March 15, 2001; 166(6): 3846 - 3854. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
