| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Cellular and Developmental Biology, "La Sapienza" University, Rome, Italy; and
Department of Immunology, Imperial College of Medicine, Hammersmith Hospital, London, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dendritic cells (DC)3 have been shown to play essential roles in the induction of T cell-mediated immune reactions. Nevertheless, these APC need to be stimulated to perform their task efficiently. The activation of DC reflects the changes in their function from cells specialized for Ag uptaking and processing into APC specialized for delivering T cell-stimulatory signals (9, 10). This means that DC can change both phenotype and function depending on their maturational states. However, this may also mean that depending on their maturational states, they can be differentially regulated by suppressive stimuli. The recent evidence in mice that fully mature DC are refractory to both inhibitory and activating stimuli (8, 11) supports this hypothesis. Moreover, the phenotypical characterization of DC strongly suggests that these represent a heterogeneous cell population with different functions depending on their preparations from different anatomical compartments, with additional differences between DC from mice and humans (12). This suggests that not only the degree of DC activation/maturation, but also the murine or human origin could be crucial for the characterization of their role in the activation and suppression of immune response.
In the mouse system, anergic T cells have been shown to exert suppression by regulating the APC function of immature DC through a cell contact mechanism (8, 13). Therefore, in the present work, we studied the effect of human anergic T cells on DC in two stages of maturation defined by us, on the basis of their MHC, costimulatory, and proapoptotic molecule expression, as immature and mature.
We have focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and on the fate of DC and responder T cells. We found that both immature and mature DC were susceptible to suppressive signals. However, while the suppressive effect on immature DC was due to down-regulation of their Ag-presenting capacity, in the presence of mature DC, suppression was mediated through apoptosis of these APC, which, in turn, favored responder T cell deletion.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The synthetic peptides corresponding to residues 307–319 and 100–115 of influenza hemagglutinin (HA) and the synthetic peptide corresponding to the hypervariable region of HLA-A2 (residues 92–120) were synthesized using F-moc chemistry (7). The peptides were HPLC purified, and amino acid analysis was conducted.
Monoclonal Abs
The OKT3 mAb (anti-human CD3; American Type Culture Collection (ATCC), Manassas, VA) was purified from culture supernatant on protein A-Sepharose beads by standard methods. Eluted Ab was dialyzed against three changes of PBS. Anti-CD40 (G28-5) Ab was obtained from ATCC. Antagonistic anti-CD40 Ab (mouse IgG1, azide free) was purchased from Serotec (Oxford, U.K.). Dr. D. H. Lynch (Immunex, Seattle, WA) kindly provided antagonistic anti-CD95 (M3) Ab. Anti-mouse IgG (used for cross-linking of CD40 mAb) was purchased from Sigma-Aldrich (St. Louis, MO). L243 (IgG2a, anti-DR) was purchased from ATCC. R-PE-conjugated anti-human CD154 Ab TRAP-1 (mouse IgG1) was purchased from BD PharMingen (San Diego, CA). Hamster anti-human CD95L Ab (IgG) clone 4H9 was obtained from Immunotech (Coulter Company, Marseille, France). FITC-conjugated hamster anti-human Bcl-2 Ab was purchased from BD PharMingen. Anti-CD1a (OKT6) was obtained from ATCC, and PAM-1 (anti-human mannose receptor) was kindly provided by G. Peri (Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy). CD86-specific Ab (Bu63) was kindly provided by P. Beverly (London, U.K.). Anti-CD14 mAb (LeuM3) and nonconjugated and R-PE-conjugated anti-CD4 (Leu3a) mAb were purchased from BD PharMingen. Mouse IgM anti-human CD95 Ab (CH11) was purchased from Upstate Biotechnology (Lake Placid, NY).
T cell clones and L cells transfected with human MHC molecules
The T cell clone F17 (14), specific for the HA 307–319 and restricted by DRB1*1101, and T cell clone M3, specific for HA 100–115 and restricted by DRB1*0101, were derived from PBMC isolated from a DRB1*0101/DRB1*1101 individual. The T cell clone EL26 specific for HLA-A2 92–120 and restricted by DRB1*1502 was used as an irrelevant clone in suppression experiments (15). The G12-alloreactive T cell clone, specific for DRB1*0101, was derived from a DRB1*0401/DRB1*1301 individual (5). T cell clones were used for functional assays at least 1 wk after their last stimulation. Accessory cell-free preparations of T cells were obtained as described previously (5, 7). Briefly, T cells were purified on Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) 10 days after restimulation and washed five times by slow-speed centrifugation (210 g x 5 min) before use in the experiments. The L cell transfectants expressing HLA-DRB1*0101 (5-3.1) and cotransfected with human CD80 (5-3.1/B7) were previously described (16).
Renal cell preparation and culture
The renal tubular epithelial cells (RTEC) were purified from
human renal tissue biopsy from a DR15-expressing individual and
cultured, as previously described (17). RTEC, when
required for anergy induction, were cultured in medium 199 (20% FCS, 2
mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml
streptomycin), insulin transferrin selenite (5 µg/ml;
Sigma-Aldrich), triiodiotironine (3 x 10-8
M; Sigma-Aldrich), and hydrocortisone (5 x
10-8 M; Sigma-Aldrich) with IFN-
(500
U/ml).
Two-stage culture system for anergy induction in T cell clones
T cell anergy was obtained as previously described (5, 7). Briefly, T cells were incubated overnight (o.n.) with
immobilized mAb anti-CD3 (OKT3), harvested, and washed twice. To
assess lack of proliferation and IL-2 production, the cells and the
supernatants were tested in proliferation and CTLL assay, respectively
(7). The cells that did not proliferate and did not
synthesize IL-2 were considered anergic. They were used after either 24
or 72 h of culture in RPMI 1640 medium containing 10%
human serum (HS) either in suppression experiments (after irradiation)
or in cytotoxic assays. To maintain T cells in a resting state
for 72 h, they were cultured in 10% HS medium in the presence of
a suboptimal dose of rIL-2 (7). T cell clones were also
anergized by coculture with cognate RTEC treated with IFN- to allow
them to express MHC class II molecules, as described elsewhere
(17).
Generation and culture of DC
Blood-derived DC were prepared from PBMC isolated from whole blood of healthy donors. PBMC fractions were incubated with a mixture of purified mAbs (OKT3 and mouse anti-human Ig, Fab specific, at saturating concentrations for 30 min at 4°C). The cells were washed twice to remove excess Abs, and the Ab-bound cells were removed by magnetic immunodepletion. Briefly, mAb-treated cells were incubated with magnetic microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) coated with sheep anti-mouse Ig for 15 min at 4°C, and bead/mAb-coated cells were removed by passage through a magnetic column (MiniMACsystem; Miltenyi Biotec), as specified by the manufacturer’s instructions. The remaining cells were allowed to adhere to six-well plates for 40 min at 37°C in a humidified CO2 incubator. After removal of the nonadherent cells, the adherent cells were cultured in RPMI 1640–2% HS supplemented with 50 ng/ml GM-CSF and 100 U/ml rIL-4 (Boehringer Mannheim, Indianapolis, IN). This protocol allowed us to obtain 90% pure human DC. When necessary, adherent cells were detached by incubation with Mg2+- and Ca2+-free PBS 1x containing 0.5 mM EDTA at 37°C. After 6 days of culture, DC that were CD14-, mannose receptor+, HLA-class II+, and CD4+ were defined immature DC. Immature DC tended to reach spontaneous maturation by up-regulating CD40, HLA-DR, CD86, and CD95 during a further 3 days of culture in the presence of GM-CSF and IL-4. Spontaneous maturation was helped by stimulating these immature DC with LPS (Sigma-Aldrich) for 40 h (1 µg/ml). These DC, cultured for at least another 9 days, were defined mature DC. Both immature and mature DC were used as APC. In the experiments in which T cell clones specific for HA peptides were used, the DC were prepulsed o.n. with the specific peptide at concentration usually between 5 and 20 µg/ml, depending on the responder T cell clone.
Phenotypical analysis of DC alone in culture or cocultured with T cell clones
Phenotypical characterization of immature and mature DC was performed by staining the cells with the following mAbs at 4°C for 30 min: L243, CH11, PAM-1, Bu63, Leu3a, and LeuM3. After washing, the cells were incubated with FITC-conjugated IgG and IgM-specific F(ab')2 goat anti-mouse Ab (BioSource International, Nivelles, Belgium) and analyzed by using a BD Biosciences FACSCalibur flow cytometer (FACS). CD86 and HLA-DR expression on DC cocultured with anergic, resting, and activated T cells was assessed by incubating the cells with Bu63 and L243. Briefly, 104 immature DC were cultured for 48 h either alone or in the presence of 105 either anergic or responder or anergic and responder T cells in flat-bottom 96-well plates in RPMI 1640–5% HS without any added growth factor. Isotype-matched mAb were always included. Bcl-2 expression in DC treated o.n. with cross-linked anti-CD40 mAb G28-5 was assessed by intracellular staining with the FITC-conjugated hamster anti-human Bcl-2. The isotype control utilized was FITC-conjugated Armenia hamster IgG mAb (BD PharMingen). Briefly, fixation of 1x PBS washed cells was obtained by incubating them with 1x PBS-2% paraformaldehyde for 15 min at room temperature. Cells were washed in staining buffer (1x PBS-0.5% BSA, 0.02% sodium azide). Permeabilization of cells was obtained by incubating them with 1x PBS-0.5% BSA, 0.02% sodium azide, 0.5% saponine, for 15 min. After washing in staining buffer, the cells were incubated for 15 min with 20 µl anti-Bcl-2 or isotype control. After two washes in staining buffer, stained cells were analyzed by FACS.
CD154 and CD95L expression analysis on anergic and responder T cell clones
CD154 and CD95L expression was measured at different times of culture with either mAb OKT3 in the absence of accessory cells, or DRB1*0101/CD80-transfected L cells, or with RTEC, or with PMA and ionomycin (PMA plus I) (see below). The cells were incubated with R-PE-conjugated anti-human CD40L Ab TRAP-1 (BD PharMingen) and with the hamster anti-CD95L Ab, followed by incubation with FITC-conjugated mouse anti-hamster Ab (BioSource International). Stained cells were analyzed by FACS.
T cell suppression assay
Responder T cells (5 x 103) were cultured with 5 x 103 irradiated either immature or mature DC in flat-bottom microtiter plates in a total volume of 200 µl in RPMI 1640 medium (Life Technologies, Paisley, Scotland) supplemented with 10% HS, 2 mM L-glutamine, 50 IU/ml penicillin, and 10 µg/ml streptomycin in the absence or in the presence of 1.5 x 104 irradiated anergic T cells. As control, 5 x 103 responder T cells were cultured with 5 x 103 mature DC in the presence of T cells activated either with 0.05 µM PMA (Sigma-Aldrich) plus 0.5 µM I (PMA plus I; Calbiochem, La Jolla, CA) for 4 or 14 h or with OKT3 in the presence of accessory cells o.n. Wells of cells cocultured for 48 h were pulsed with 1 µCi of [3H]TdR (Amersham International, Amersham, U.K.) and harvested onto glass fiber filters 18 h later. Proliferation was measured as [3H]TdR incorporation by liquid scintillation spectroscopy. The results are expressed as mean of the triplicate cultures. SEs were routinely <10%.
Cytotoxic assay
The capacity of anergic T cells (effector cells) to kill DC or responder T cells (target cells) was assessed by standard 51Cr release assay in which 3 x 103 target cells, previously labeled with 51Cr for 1 h, were cultured in a 96-well round-bottom plate with 9 x 103 effector cells (E:T ratio, 3:1). T cells either untreated or treated with PMA plus I were also used as control effectors, and their killing activity was always <10%. After 4 h, 51Cr release in the supernatants was determined on a ME Plus gamma scintillation counter (Micromedic Systems, Huntsville, TN). The percentage of specific lysis was calculated as follows: 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The recent evidence that immature, but not fully mature murine DC
were susceptible to the inhibitory effects of anergic T cells
(8) supports the idea that suppression can be exerted by
anergic CD4+ T cells only in the presence of DC
that have not reached a complete maturational state. This evidence
prompted us to verify whether human anergic
CD4+ T cells could exert a similar differential
effect on DC taken at different stages of their maturation. First, we
studied the proliferative response of alloreactive T cell clones to DC
in the presence of anergic T cells. At this aim, the alloreactive T
cell clone G12, specific for DRB1*0101, was anergized by plastic-bound
OKT3 mAb, as previously described (7). An irrelevant T
cell clone, EL26, was also anergized and used as control. Anergy
induction was always assessed by demonstrating lack of proliferation
and IL-2 production in rechallenge experiments in response to cognate
APC, as previously reported (7). The phenotypical
characterization of DC used at different stages of maturity was also
performed, and Fig. 1
shows the
expression of HLA-DR, CD86, CD95, CD1a, and mannose receptor molecules
expressed by DC defined as immature and mature APC (see Materials
and Methods). Immature DC, stained after 6 days of culture
in the presence of IL-4 and GM-CSF, showed already a certain degree of
MHC class II, CD1a, and mannose receptor molecule expression, but were
negative for CD86 and CD95 molecules (Fig. 1, a,
c, and e). Induction of maturation significantly
up-regulated MHC class II, CD86, and CD95 molecule expression, while
CD1a presented a slight increase (Fig. 1, b, d,
f, and h). On the contrary, a decrease of mannose
receptor expression was observed (Fig. 1, i and
l). The effect of anergic T cells on DNA synthesis of T cell
clones activated in the presence of immature and mature DC is reported
in Fig. 2. The results show that human
anergic T cells act as suppressor cells in the presence of DC taken at
the two different maturational states. The irrelevant anergic T cell
clone (EL26; see Materials and Methods) did not exert
any suppressive activity. In the murine system, the suppressive effect
of anergic T cells was explained by their ability to induce
down-regulation of MHC and costimulatory molecules on immature, and not
on fully mature DC (8). In this study, we verified whether
anergic T cells could also affect the expression of these molecules on
human DC. The results are reported in Fig. 3. The anergic clone M3, specific for the
HA 100–115 and restricted by DRB1*0101, did not affect the HLA-DR and
CD86 molecule expression on immature DC during cognate interaction. On
the contrary, activated T cells were able to induce efficiently
up-regulation of these markers after 40 h of coculture. Resting
nonanergic T cells could also induce a significant increase of CD86
expression and a slight increase of HLA-DR. Neither anergic nor
activated T cells, used as control, could induce up-regulation of CD86
and HLA class II molecules on immature DC that did not present the Ag
recognized by their TCR. The phenotypical analysis of DC, cultured in
the presence of both responder and anergic T cells (responder:anergic
cell ratio 1:3), was also performed using the alloreactive T cell
clone, G12. The results in Fig. 3c demonstrate that the
presence of anergic cells could almost completely abrogate the
up-regulation of HLA-DR and CD86 molecules induced by responder cells.
Indeed, although after coculture with responder T cells, CD86
expression on DC was almost double than that observed in control DC,
CD86 expression in coculture with anergic T cells or with anergic and
responder T cells was not modified. Although less evident, the same
effect was observed for HLA-DR molecule expression. Moreover, no
modification of expression of MHC class II and CD86 molecules was
observed on cognate fully mature DC in the presence of either anergic
or responder or activated T cells (data not shown).
|
|
|
The evidence that anergic T cells could suppress APC functions of
fully mature DC, without modifying MHC and costimulatory molecule
expression, prompted us to clarify this suppressive phenomenon. The
induction of apoptosis, which is a matter of debate in the infectious
tolerance (3), is one of the mechanisms that we
investigated. We decided to address this issue because we had at least
two evidences that the CD95-mediated apoptotic pathway could be
involved. First, in our hands, fully mature human DC expressed a high
amount of CD95, differently from the immature ones (Fig. 1
, e and f). Second, we and others have demonstrated
that human anergic CD4+ T cell clones do express
functional CD95L following anergy induction either via T-T presentation
or culture with OKT3 Ab (18, 19). In this study, we
confirm the expression of CD95L on human Ag-specific and alloreactive T
cell clones anergized with OKT3 (Fig. 4a). To exclude
activation-induced cell death phenomena (20), we verified
first whether activated T cells could inhibit responder T cell
proliferation. At this aim, we performed proliferation assays by mixing
responding T cells either with anergic T cells or with T cells
activated either with PMA plus I or OKT3 in the presence of accessory
cells. We found that only anergic, and not fully activated T cells,
could mediate suppressive effects (Fig. 4b). We further
verified the possibility that the observed suppression of T cell
proliferation was dependent on CD95-mediated apoptosis. The
alloreactive T cell clone G12 and the Ag-specific T cell clones M3 (HA
100–115 specific and DRB1*0101 restricted) and F17 (HA 307–319
specific and DRB1*1101 restricted) were anergized by using OKT3 mAb.
After either 24 or 72 h, anergic T cells were mixed with responder
T cells at 3:1 ratio, and mature CD95-positive DC were added. The
experiments in Fig. 4c show that the addition of an
antagonistic anti-CD95 Ab to the culture (M3 mAb) completely
abrogated T cell suppression. The addition of an irrelevant anergic T
cell clone in the assay did not induce any inhibition of proliferation
of responding T cells; the proliferation was unchanged when the
antagonistic anti-CD95 Ab M3 was added to the culture (data not
shown). Having established that the CD95-CD95L system could account for
the suppressive activity of CD4+ anergic T cells,
we verified which cells (APC and/or responder T cells) were killed by
anergic T cell clones. First, we analyzed whether anergic T cells were
able to directly mediate killing by CD95-CD95L interaction. At this
aim, we performed a cytotoxic assay in which anergic T cells were used
as effectors and either CD95+ responder T cell
clones (19) or CD95+ mature DC were
used as targets (Fig. 5a).
This experiment showed that DC were the principal targets of the
anergic killer cells, although a poor cytotoxic effect was also exerted
toward the responder T cells. Moreover, a proliferation assay was
performed to study the killing activity of anergic T cells in the
conditions in which suppression is exerted. At this aim, either
responding T cells or DC were separately incubated with the
antagonistic anti-CD95 Ab M3 prior to mixing the three cell types
(responder T cells, anergic T cells, and mature DC). We found that the
suppressive effect of anergic T cells was reduced in a significant
degree not only when DC, but also responder T cells, were preincubated
with the antagonistic anti-CD95 Ab M3 (Fig. 5b). Only
when both cell types were preincubated with M3 was the protective
effect total. To understand this unexpected result and further support
the idea that responder T cells, in addition to DC, were killed by
anergic cells, we performed a cytotoxic assay in which all three cell
types (responder T cells, anergic T cells, and cognate mature DC) were
present in the same culture. The ratio of DC, anergic, and responding T
cells used was the same as that utilized in proliferation experiments.
This time we marked with 51Cr either the
responder T cells or the DC that were present in the same well. Again,
anergic T cells were the effectors in the assay. The results in Fig. 5c show that in these culture conditions, both DC and T
cells were induced to die in a significant extent and that cell death
was mainly mediated via the CD95-CD95L pathway. As a control, we also
showed that an irrelevant anergic T cell clone (EL26) failed to induce
cell death of either DC or responder T cells. This latter experiment
strongly suggests that the activation of apoptotic pathways by anergic
T cells is an Ag-specific phenomenon in which cognate recognition is a
substantial requirement. Indeed, anergic T cells kill both APC and
responder T cells only when the three cell types come into contact by
the interaction of molecules involved in Ag recognition.
|
|
To investigate further the different effect of anergic and
activated T cells on DC functions, we decided to study the contribution
of other molecular interactions in this system. First, we analyzed the
expression of CD154 by anergic T cells to understand whether their
capacity to kill (at least DC) via CD95-CD95L pathway could be due to a
defect in CD154 expression (21, 22). In fact, it is well
known that engagement of CD40 by CD154 prevents both spontaneous and
CD95-induced cell death of DC (23, 24, 25). In addition, the
inability of anergic T cells to induce up-regulation of MHC and
costimulatory molecules on immature DC, as observed by us above,
strongly suggested the possibility of an impairment of CD154 expression
on these T cells (26, 27). In Fig. 6a, we report the expression
of CD154 at different points of time following activation with PMA plus
I or antigenic stimulation (DRB1*0101/CD80-expressing transfectants
pulsed with the relevant peptide were used as APC) or during anergy
induction (OKT3 treatment). It was clear that T cells, activated with
either PMA plus I or antigenic stimulation, showed a significant
up-regulation of CD154 that was not observed following anergy
induction. T cells treated with OKT3 in the presence of accessory cells
also presented CD154 up-regulation (data not shown). Moreover, we found
that T cells anergized following Ag presentation by RTEC
(17) failed to induce a significant up-regulation of CD154
(Fig. 6b). Reexpression of this molecule after 3 days of
resting and rechallenge with Ag was also impaired in anergized T cells
(Fig. 6c). Altogether, these data confirm that failure of
CD154 expression is a feature of T cell anergy. Therefore, we
hypothesized that a defective interaction of CD40 molecules with CD154
could account for CD95-mediated apoptosis of DC during cognate
interaction. To test this hypothesis, we cross-linked CD40 molecules on
DC with an anti-CD40 Ab and performed killing experiments. Fig. 7a shows the protective effect
of CD40 activation on CD95-mediated apoptosis of DC. As a control, the
same cross-linked anti-CD40 mAb was able to protect DC from
apoptosis induced by 1 µg/ml of the agonistic anti-CD95 CH11 Ab
(percentage of apoptosis induced by CH11 = 34, percentage of
apoptosis after cross-linking of CD40 = 15), determined by FACS,
as previously demonstrated (19). In the same experiment,
both untreated and PMA plus I-activated T cells induced only modest
apoptosis (10% of 51Cr release), which was
completely abrogated following treatment with antagonistic
anti-CD95 mAb. A further evidence of the protective role of
CD40-CD154 interaction derived from the experiments in which an
antagonistic anti-CD40 Ab engaged CD40 molecules on DC. Indeed
(Fig. 7b), when CD40 molecules were hidden on DC, either
anergic T cells or activated T cells, added into the assay, induced
significant T cell suppression. These results reinforce the role of
CD40 as regulatory molecule of APC function and elucidate a new
mechanism exerted by anergic T cells for mediating suppressive
phenomena.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
DC are unique in their ability to present Ag due to the relatively high levels of MHC class II and important costimulatory molecules, including CD80, CD86, and CD40 (9, 10). The expression of these potent activatory molecules develops sequentially during a process termed maturation, and equips DC to deliver activation signals (9, 10, 28, 29). On the other hand, Th cells are instrumental in DC maturation (a phenomenon called conditioning or licensing of DC) (26), but can also provide signals regulating DC death (30, 31). Recent data support the importance of CD40-CD154 interaction in DC/T cell cross-talk. Cognate interaction between DC and T cells results in CD154 up-regulation on T cells. CD40 triggering on DC enhances the expression of MHC and accessory molecules, favors their survival, prolongs DC/T cell interaction, and increases T cell activation (9, 10). In addition, costimulatory signals (32, 33) such as those mediated by B7/CD28 molecules can stabilize CD154 up-regulation on T cells and concur to a strong T cell activation. Indeed, lack of interaction among these important molecules can induce anergy (17, 34, 35). Consequently, a defective activation of CD40 expressed on DC might result in a defective activation of both DC and T cells. Recently, it has been described that triggering of CD40 on DC also regulates both spontaneous and CD95-mediated apoptotic pathways (23, 24, 25). In this work, we present data confirming the regulatory role of CD40 on the susceptibility or resistance to CD95-induced apoptosis in DC. The first derives from the observation that low expression of CD154 on anergic T cells favors DC apoptosis, while high levels of CD154 on properly activated T cells result in their survival. To corroborate this finding, we show that the protective effect of CD40 triggering is abolished (in the presence of activated T cells) when DC are treated with an antagonistic anti-CD40 Ab, whereas it is restored when DC are treated with a cross-linked anti-CD40 Ab in the presence of anergic cells. Altogether, these observations strongly support the conclusion that a defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cell clustering around DC during Ag recognition could be the primary event of suppressive phenomena. On the contrary, properly activated T cells expressing high levels of CD154, despite their capacity to up-regulate CD95L expression, favor activation and/or survival of both DC and responder T cells.
The susceptibility or resistance to CD95-mediated apoptosis is strictly dependent on antigenic stimulation. This issue is very well documented in B and T lymphocytes and macrophages (40, 41, 42, 43). The terminal fate of DC after Ag presentation, however, is not completely clarified. Concerning the T cell compartment, it is well known that human naive T cells are resistant to apoptosis during their first encounter with Ag, but once they have been activated, become susceptible to Ag-driven cell death, unless they are again properly activated (42). T cell activation that led to protection from cell death is particularly dependent on the quality and quantity of TCR triggering and costimulatory molecules (42, 44). This means that memory T cells can be deleted once they have accomplished their task and Ag concentration is decreased (45). The recent evidence that murine mature DC can undergo rapid apoptosis in the presence of Ag-specific CD4+ T cells (30) suggests that also DC can be eliminated once they have accomplished their function of APC. However, it is not clear how mature DC regulate their survival when their task has not been concluded. Our data suggest that, as observed in T cells, mature DC can decide to live or to die according to the quality and quantity of signals that they receive from the T cells to which they present Ag. It has been recently demonstrated that both DC and T cell persistence in vivo is dependent on CD40-CD154 interactions, and that CD40 triggering greatly increases the period for which DC function as APC (46). We found that CD95+ DC survive when they encounter properly activated specific CD95L+, CD154+ Th cells, whereas they die in the presence of not properly activated CD95L+, CD154-defective T cells (namely anergic T cells). Altogether, these data suggest that cognate recognition between T cells and DC regulates the CD95/CD95L pathway of apoptosis not only in T cells, but also in DC, and that CD40 triggering of DC by Th cells regulates both their Ag-presenting capacity and survival.
Worthy of note, the susceptibility to CD95/CD95L-mediated apoptosis is a character acquired by DC following maturation in response to either a second stimulus, such as bacterial products, or prolonged culture, both favoring their full maturation. Indeed, in our experiments, we found that DC, activated for 6 days with GM-CSF and IL-4, did not express detectable levels of CD95, and consequently were not susceptible to anergic cell-mediated apoptosis. On the contrary, after 3 more days of culture in vitro, these cells up-regulate CD95 and become responsive to CD95-mediated pathway of apoptosis. Previous studies have shown, in a similar experimental system, a correlation between CD95 expression on human DC and vulnerability to CD95-mediated apoptosis during 7 days of culture in vitro, although in that case only susceptibility to agonistic anti-CD95 Abs and not to CD95L+ anergic cells has been addressed (24).
However, what is really new and interesting in our study is the evidence that anergic T cells can always suppress the APC function of DC. In a first step, during their maturation process in the presence of responder Th cells, CD154-defective anergic T cells can antagonize responder T cell-activating signals delivered to the APC. This results in the inhibition of the up-regulation of MHC and costimulatory molecules on DC. Indeed, we have shown an impaired capacity of anergic T cells to induce up-regulation of HLA-DR and CD86 molecules on immature DC. In a second step, when DC reach maturation and become refractory to down-regulation of MHC and costimulatory molecules, anergic T cells can exert suppression by triggering apoptotic pathways in these APC. We provide evidence that CD95-mediated death signals of human anergic T cells are strongly dependent upon cognate interaction, in which recognition of DC by both responder and anergic T cells is a critical parameter. In fact, irrelevant anergic T cells can never exert suppression because in the absence of cognate interaction, they do not cause the death of any cell type in coculture experiments.
The fact that induction of cell death via CD95-CD95L interaction may be one of the mechanisms involved in peripheral tolerance is not surprising (38, 39). It is well known that impairment of this death pathway is responsible for disregulation of the immune response (36, 37). Several groups have reported that T cells, phenotypically resembling anergic T cells, are involved in maintaining T cell tolerance in vivo, by acting as regulatory/suppressor cells (47, 48, 49, 50, 51). All these studies highlight the complexity of the in vivo interactions of multiple regulatory-suppressive mechanisms that might be involved. In this context, the induction of anergic T cells defective in CD154 expression can be another way of maintaining peripheral tolerance. Such tolerant lymphocytes may play a regulatory role by interfering with activation/maturation or inducing deletion of the most potent APC, the DC. In the context of transplantation, new tolerant CD4+ T cells can be induced by recognition of transplantation Ags on B7-negative organ parenchymal cells (17) exposed to inflammatory factors. Such regulatory CD4+ T cells may also exert linked suppression (7) by affecting activation and survival of host DC and/or passenger DC imported with the graft.
In conclusion, our data allow us to try and depict a possible scenario of T cell suppression that could occur in vivo in the sites of active immune responses, i.e., lymphoid tissue, when anergic T cells come into contact with DC and/or with DC and responder T cells that recognize Ag on the same DC. T cell activation could be suppressed at least in three different ways: 1) via a block of DC full maturation (i.e., inhibition of MHC class II and CD86 molecule up-regulation); 2) via a shortage of DC availability through CD95-CD95L-mediated apoptosis; and 3) via CD95-CD95L-mediated apoptosis of responder T cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Loredana Frasca, Departimento di Biologia Cellulare e dello Sviluppo, Università di Roma "La Sapienza," Via dei Sardi, 70-00185, Rome, Italy. E-mail address: enza.piccolella{at}uniroma1.it
3 Abbreviations used in this paper: DC, dendritic cell; HA, hemagglutinin; HS, human serum; I, ionomycin; MFI, mean fluorescence intensity; o.n., overnight; RTEC, renal tubular epithelial cell.
Received for publication June 22, 2001. Accepted for publication November 2, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells. J. Immunol. 162:3231.-treated renal tubular epithelial cells induce allospecific tolerance. Kidney Int. 53:679.[Medline]
, but strongly enhanced by interleukin-10. Eur. J. Immunol. 25:1943.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Carvalho-Gaspar, N. D. Jones, S. Luo, L. Martin, M. O. Brook, and K. J. Wood Location and Time-Dependent Control of Rejection by Regulatory T Cells Culminates in a Failure to Generate Memory T Cells J. Immunol., May 15, 2008; 180(10): 6640 - 6648. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Frasca, M. Nasso, F. Spensieri, G. Fedele, R. Palazzo, F. Malavasi, and C. M. Ausiello IFN-{gamma} Arms Human Dendritic Cells to Perform Multiple Effector Functions J. Immunol., February 1, 2008; 180(3): 1471 - 1481. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. P. Lewkowich, N. S. Herman, K. W. Schleifer, M. P. Dance, B. L. Chen, K. M. Dienger, A. A. Sproles, J. S. Shah, J. Kohl, Y. Belkaid, et al. CD4+CD25+ T cells protect against experimentally induced asthma and alter pulmonary dendritic cell phenotype and function J. Exp. Med., December 5, 2005; 202(11): 1549 - 1561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Mallone, S. A. Kochik, H. Reijonen, B. Carson, S. F. Ziegler, W. W. Kwok, and G. T. Nepom Functional avidity directs T-cell fate in autoreactive CD4+ T cells Blood, October 15, 2005; 106(8): 2798 - 2805. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Scotta, L. Tuosto, A. M. Masci, L. Racioppi, E. Piccolella, and L. Frasca Hypervariable region 1 variant acting as TCR antagonist affects hepatitis C virus-specific CD4+ T cell repertoire by favoring CD95-mediated apoptosis J. Leukoc. Biol., August 1, 2005; 78(2): 372 - 382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kriehuber, W. Bauer, A.-S. Charbonnier, D. Winter, S. Amatschek, D. Tamandl, N. Schweifer, G. Stingl, and D. Maurer Balance between NF-{kappa}B and JNK/AP-1 activity controls dendritic cell life and death Blood, July 1, 2005; 106(1): 175 - 183. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Smith, T. Patterson, and M. E. Pauza Transgenic Ly-49A Inhibits Antigen-Driven T Cell Activation and Delays Diabetes J. Immunol., April 1, 2005; 174(7): 3897 - 3905. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Weiss, V. Donkova-Petrini, L. Caccavelli, M. Balbo, C. Carbonneil, and Y. Levy Human immunodeficiency virus-driven expansion of CD4+CD25+ regulatory T cells, which suppress HIV-specific CD4 T-cell responses in HIV-infected patients Blood, November 15, 2004; 104(10): 3249 - 3256. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. H. Munn, M. D. Sharma, and A. L. Mellor Ligation of B7-1/B7-2 by Human CD4+ T Cells Triggers Indoleamine 2,3-Dioxygenase Activity in Dendritic Cells J. Immunol., April 1, 2004; 172(7): 4100 - 4110. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Guillot, S. Menoret, C. Guillonneau, C. Braudeau, M. G. Castro, P. Lowenstein, and I. Anegon Active suppression of allogeneic proliferative responses by dendritic cells after induction of long-term allograft survival by CTLA4Ig Blood, April 15, 2003; 101(8): 3325 - 3333. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-P. Min, D. Zhou, T. E. Ichim, G. H. Strejan, X. Xia, J. Yang, X. Huang, B. Garcia, D. White, P. Dutartre, et al. Inhibitory Feedback Loop Between Tolerogenic Dendritic Cells and Regulatory T Cells in Transplant Tolerance J. Immunol., February 1, 2003; 170(3): 1304 - 1312. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. James, D. Scott, J.-G. Chai, M. Millrain, P. Chandler, and E. Simpson HY peptides modulate transplantation responses to skin allografts Int. Immunol., November 1, 2002; 14(11): 1333 - 1342. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or in
the presence (
) of G12 anergic cells (G12A) at 3:1
ratio between anergic and responder cells. The M3 mAb (5 µg/ml) was
used to protect from apoptosis only DC (DC) or responder T cells (G12R)
or both (DC + G12R), as indicated. Proliferation was assessed as
reported in Fig. 2
