T cell suppression exerted by regulatory T cells represents a well-established phenomenon, but the mechanisms involved are still a matter of debate. Recent data suggest that anergic T cells can suppress responder T cell activation by inhibiting Ag presentation by dendritic cells (DC). In this study, we focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and analyzed the fate of DC and responder T cells. To address this issue, we have cocultured human alloreactive or Ag-specific CD4+ T cell clones, rendered anergic by incubation with immobilized anti-CD3 Ab, with autologous DC and responder T cells. We show that anergic T cells affect either Ag-presenting functions or survival of DC, depending whether immature or mature DC are used as APC. Indeed, MHC and costimulatory molecule expression on immature DC activated by responder T cells is inhibited, while apoptotic programs are induced in mature DC and in turn in responder T cells. Ligation of CD95 by CD95L expressed on anergic T cells in the absence of CD40-CD40L (CD154) interaction are critical parameters in eliciting apoptosis in both DC and responder T cells. In conclusion, these findings indicate that the defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cells could be the primary event in determining T cell suppression and support the role of CD40 signaling in regulating both conditioning and survival of DC.

Animal models of transplantation and autoimmune diseases provide clear evidence that tolerance can be transferred by T cells from a tolerant to a naive host. This phenomenon has been referred to as infectious tolerance (1, 2). Many models have been proposed to explain the mechanisms of T cell suppression and infectious tolerance (3). Several years ago, Waldmann et al. (4) proposed that anergic T cells could compete for stimulation at the site of Ag presentation, preventing activation of reactive T cells. A similar phenomenon has been described in the human system (5), in which T cells, rendered tolerant, were shown to act as suppressor cells in vitro. The mechanism proposed was that anergic/suppressor T cells acted by disrupting the collaborative clusters of responding cells by competing for APC (3) surface and locally produced activating factors, and not by secreting inhibitory cytokines as shown in other models (6). A further demonstration that the regulatory effect of anergic T cells was dependent upon cell contact has been provided by our subsequent experiments, in which we explored the ability of human anergic T cells to affect linked suppression in Ag-specific and allospecific responses (7). More recently, the analysis of the mechanism, whereby murine allospecific anergic T cells, cocultured with bone marrow-derived dendritic cells, inhibited maturation of these APC and suppressed responder T cell activation, strongly suggested to us that suppression observed in our human system could be mediated also through the APC (8).

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.

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.

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).

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 × 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).

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 × 10−8 M; Sigma-Aldrich), and hydrocortisone (5 × 10−8 M; Sigma-Aldrich) with IFN-γ (500 U/ml).

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-γ to allow them to express MHC class II molecules, as described elsewhere (17).

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 1× 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 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 1× PBS washed cells was obtained by incubating them with 1× PBS-2% paraformaldehyde for 15 min at room temperature. Cells were washed in staining buffer (1× PBS-0.5% BSA, 0.02% sodium azide). Permeabilization of cells was obtained by incubating them with 1× 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 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.

Responder T cells (5 × 103) were cultured with 5 × 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 × 104 irradiated anergic T cells. As control, 5 × 103 responder T cells were cultured with 5 × 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%.

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 × 103 target cells, previously labeled with 51Cr for 1 h, were cultured in a 96-well round-bottom plate with 9 × 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 × (experimental release − spontaneous release)/(maximum release − spontaneous release).

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. 3 c 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).

FIGURE 1.

Analysis of surface molecules expressed by immature and mature DC. Cells were stained with anti-HLA-DR (a and b), anti-CD86 (c and d), anti-CD95 (e and f), anti-CD1a (g and h), and anti-mannose receptor (i and l) mAbs, and analyzed by FACS. Isotype-matched controls (filled histograms) are also indicated. Mature DC obtained with or without LPS treatment presented similar phenotypes. This analysis was performed on each DC preparation.

FIGURE 1.

Analysis of surface molecules expressed by immature and mature DC. Cells were stained with anti-HLA-DR (a and b), anti-CD86 (c and d), anti-CD95 (e and f), anti-CD1a (g and h), and anti-mannose receptor (i and l) mAbs, and analyzed by FACS. Isotype-matched controls (filled histograms) are also indicated. Mature DC obtained with or without LPS treatment presented similar phenotypes. This analysis was performed on each DC preparation.

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FIGURE 2.

Anergic T cells inhibit T cell proliferation induced by mature and immature DC. G12 (5 × 103) was cultured for 72 h with 5 × 103 DRB1*0101-expressing mature (a) or immature (b) DC in the absence or in the presence of anergic T cells obtained after treatment with OKT3 (G12A). EL26-alloreactive T cell clone (HLA-A2 specific) treated with OKT3 was used as irrelevant anergic clone (EL26A). The results, obtained in a proliferation assay, are expressed as the mean of triplicated cultures + SD and derived by one representative experiment. Similar results were obtained with the Ag-specific T cell clone F17.

FIGURE 2.

Anergic T cells inhibit T cell proliferation induced by mature and immature DC. G12 (5 × 103) was cultured for 72 h with 5 × 103 DRB1*0101-expressing mature (a) or immature (b) DC in the absence or in the presence of anergic T cells obtained after treatment with OKT3 (G12A). EL26-alloreactive T cell clone (HLA-A2 specific) treated with OKT3 was used as irrelevant anergic clone (EL26A). The results, obtained in a proliferation assay, are expressed as the mean of triplicated cultures + SD and derived by one representative experiment. Similar results were obtained with the Ag-specific T cell clone F17.

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FIGURE 3.

Anergic T cells fail to up-regulate MHC class II and CD86 molecule expression on immature DC. a and b, 2 × 104 immature DRB1*0101-expressing DC, either unpulsed or pulsed with HA 100–115, were cultured in RPMI 1640–2% HS in 48-well plates, in the absence of any growth factors, either alone (DC) or with resting M3 T cell clone (med) or M3 T cell clone rendered anergic with immobilized OKT3 Ab in the absence of accessory cells (OKT3) or stimulated with PMA plus I (PI). The cells were cocultured at 37°C for 40 h. After incubation, HLA-DR and CD86 expression on DC was assessed by FACS. Data shown are from one representative of five separate experiments performed with different T cell clones. The percentage of inhibition of DR and CD86 up-regulation, calculated as 100 − (mean fluorescence intensity (MFI) in OKT3 cultures/MFI in PMA plus I cultures × 100), was always >50%. c, Immature DRB1*0101-expressing DC (4 × 104) were cultured in RPMI 1640–2% HS, in the absence of any growth factors, either alone (DC) or with resting alloreactive G12 T cell clone (med) or both anergic and responder G12 (med + OKT3) (responder:anergic T cell ratio 1:3). HLA-DR and CD86 expression was estimated as described above. Results are representative of one of two independent experiments.

FIGURE 3.

Anergic T cells fail to up-regulate MHC class II and CD86 molecule expression on immature DC. a and b, 2 × 104 immature DRB1*0101-expressing DC, either unpulsed or pulsed with HA 100–115, were cultured in RPMI 1640–2% HS in 48-well plates, in the absence of any growth factors, either alone (DC) or with resting M3 T cell clone (med) or M3 T cell clone rendered anergic with immobilized OKT3 Ab in the absence of accessory cells (OKT3) or stimulated with PMA plus I (PI). The cells were cocultured at 37°C for 40 h. After incubation, HLA-DR and CD86 expression on DC was assessed by FACS. Data shown are from one representative of five separate experiments performed with different T cell clones. The percentage of inhibition of DR and CD86 up-regulation, calculated as 100 − (mean fluorescence intensity (MFI) in OKT3 cultures/MFI in PMA plus I cultures × 100), was always >50%. c, Immature DRB1*0101-expressing DC (4 × 104) were cultured in RPMI 1640–2% HS, in the absence of any growth factors, either alone (DC) or with resting alloreactive G12 T cell clone (med) or both anergic and responder G12 (med + OKT3) (responder:anergic T cell ratio 1:3). HLA-DR and CD86 expression was estimated as described above. Results are representative of one of two independent experiments.

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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. 4,a). 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. 4,b). 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. 4,c 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. 5,a). 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. 5,b). 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. 5 c 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.

FIGURE 4.

a and b, Only CD95L+ anergic and not CD95L+-activated T cells suppress T cell proliferation induced by mature DC. CD95L expression on T cell clone M3 either untreated (M3) or treated with OKT3 (M3A) or with PMA plus I (M3PI) was analyzed after staining with anti-CD95L mAb by FACS. MFI are also reported. Similar results were obtained with F17 clone. Responder T cell clone F17 (5 × 103 cells/well) was cultured with 5 × 103 HA 307–319-pulsed DC in the absence or in the presence of 1.5 × 104 F17 anergic cells (F17A) or in the presence of 1.5 × 104 F17 stimulated with OKT3 in the presence of accessory cells (F17S) or in the presence of 1.5 × 104 F17 stimulated with PMA plus I (F17PI). The reported data are representative of similar results obtained with M3 T cell clone. c, Anti-CD95 antagonistic mAb blocks suppressive phenomena mediated by anergic T cells. A total of 5 × 103 responder T cell clones G12, M3, and F17 was cultured with the specific DC in the absence or in the presence of anergic T cells (G12A, M3A, and F17A) at 3:1 ratio anergic:responder cells. The T cell cultures were either untreated or treated with 5 μg/ml antagonistic anti-CD95 Ab (M3 mAb). Results are expressed as reported in Fig. 2 legend.

FIGURE 4.

a and b, Only CD95L+ anergic and not CD95L+-activated T cells suppress T cell proliferation induced by mature DC. CD95L expression on T cell clone M3 either untreated (M3) or treated with OKT3 (M3A) or with PMA plus I (M3PI) was analyzed after staining with anti-CD95L mAb by FACS. MFI are also reported. Similar results were obtained with F17 clone. Responder T cell clone F17 (5 × 103 cells/well) was cultured with 5 × 103 HA 307–319-pulsed DC in the absence or in the presence of 1.5 × 104 F17 anergic cells (F17A) or in the presence of 1.5 × 104 F17 stimulated with OKT3 in the presence of accessory cells (F17S) or in the presence of 1.5 × 104 F17 stimulated with PMA plus I (F17PI). The reported data are representative of similar results obtained with M3 T cell clone. c, Anti-CD95 antagonistic mAb blocks suppressive phenomena mediated by anergic T cells. A total of 5 × 103 responder T cell clones G12, M3, and F17 was cultured with the specific DC in the absence or in the presence of anergic T cells (G12A, M3A, and F17A) at 3:1 ratio anergic:responder cells. The T cell cultures were either untreated or treated with 5 μg/ml antagonistic anti-CD95 Ab (M3 mAb). Results are expressed as reported in Fig. 2 legend.

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FIGURE 5.

Anergic T cells exert suppressive effects by killing both DC and responder T cells, mainly through the CD95-CD95L system. a, Nine × 103 G12 anergic T cells used as effectors were cultured with 3 × 103 51Cr-labeled DRB1*0101+ mature DC (DC) or with 3 × 103 51Cr-labeled G12 T cells (G12R) pretreated or not with antagonistic anti-CD95 Ab (M3 mAb). Mature DC (DC) and untreated G12 (G12R) cells were used as targets. The results are expressed as percentage of specific lysis and are representative of two experiments. b, Responder T cells of clone G12 (5 × 103 cells/well) were cultured with 5 × 103 specific mature DC in the absence (□) 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 legend. c, Nine × 103 G12 anergic T cells (□) or 9 × 103 EL26 irrelevant anergic T cells (▨) were cultured separately with 3 × 103 DRB1*0101+ unlabeled DC plus 3 × 103 51Cr-labeled responder G12 (DC plus 51Cr-G12R) or with 3 × 103 51Cr-labeled DC plus 3 × 103 unlabeled G12 (51Cr-DC plus G12R). Both 51Cr-labeled target cells (51Cr-DC or 51Cr-G12R) were pretreated or not with M3 mAb. The results expressed as percentage of specific lysis are representative of two experiments.

FIGURE 5.

Anergic T cells exert suppressive effects by killing both DC and responder T cells, mainly through the CD95-CD95L system. a, Nine × 103 G12 anergic T cells used as effectors were cultured with 3 × 103 51Cr-labeled DRB1*0101+ mature DC (DC) or with 3 × 103 51Cr-labeled G12 T cells (G12R) pretreated or not with antagonistic anti-CD95 Ab (M3 mAb). Mature DC (DC) and untreated G12 (G12R) cells were used as targets. The results are expressed as percentage of specific lysis and are representative of two experiments. b, Responder T cells of clone G12 (5 × 103 cells/well) were cultured with 5 × 103 specific mature DC in the absence (□) 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 legend. c, Nine × 103 G12 anergic T cells (□) or 9 × 103 EL26 irrelevant anergic T cells (▨) were cultured separately with 3 × 103 DRB1*0101+ unlabeled DC plus 3 × 103 51Cr-labeled responder G12 (DC plus 51Cr-G12R) or with 3 × 103 51Cr-labeled DC plus 3 × 103 unlabeled G12 (51Cr-DC plus G12R). Both 51Cr-labeled target cells (51Cr-DC or 51Cr-G12R) were pretreated or not with M3 mAb. The results expressed as percentage of specific lysis are representative of two experiments.

Close modal

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. 6,a, 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. 6,b). Reexpression of this molecule after 3 days of resting and rechallenge with Ag was also impaired in anergized T cells (Fig. 6,c). 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. 7,a 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. 7 b), 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.

FIGURE 6.

Anergic CD4+ T cells show impaired expression of CD154 during anergy induction and upon rechallenge with Ag. a, Two × 105 cells of M3 T cell clone were either untreated (M3) or anergized with immobilized OKT3 Ab in the absence of accessory cells (M3A) or stimulated with 2 × 105 L cells, expressing the human CD80 and HLA-DRB1*0101 molecules, prepulsed o.n. with 10 μg/ml HA 100–115 (M3S) or with PMA plus I (M3PI) in 48-well plates. The cells were cultured at 37°C for either 6 (filled histogram) or 24 h (open histogram), as indicated, and CD154 expression was assessed by staining with PE-conjugated anti-human CD154 mAb. Isotype-matched controls (dotted lines) and MFI are also indicated. Similar results were obtained with F17 T cell clone. b, EL26 T cells were either untreated (EL26) or cultured with PMA plus I (EL26PI) or OKT3 in the absence of accessory cells (EL26A) or with IFN-γ-pretreated RTEC expressing the restriction molecule DR15 and pulsed with peptide HLA-A2 92–120 (EL26RTEC). CD154 expression was assessed after 24 h of culture. Isotype-matched controls (filled histograms) and MFI are also indicated. c, T cell clone M3 was anergized (M3A) or activated with mitomycin C-treated cognate transfectants (M3S). After 3 days of resting in the presence of suboptimal dose of IL-2, the cells were rechallenged with cognate transfectants, and CD154 expression was assessed after 6, 18, and 24 h, as indicated. MFI and isotype-matched controls (filled histograms) are also indicated. Data shown are representative of three separate experiments performed with different T cell clones (F17 and EL26).

FIGURE 6.

Anergic CD4+ T cells show impaired expression of CD154 during anergy induction and upon rechallenge with Ag. a, Two × 105 cells of M3 T cell clone were either untreated (M3) or anergized with immobilized OKT3 Ab in the absence of accessory cells (M3A) or stimulated with 2 × 105 L cells, expressing the human CD80 and HLA-DRB1*0101 molecules, prepulsed o.n. with 10 μg/ml HA 100–115 (M3S) or with PMA plus I (M3PI) in 48-well plates. The cells were cultured at 37°C for either 6 (filled histogram) or 24 h (open histogram), as indicated, and CD154 expression was assessed by staining with PE-conjugated anti-human CD154 mAb. Isotype-matched controls (dotted lines) and MFI are also indicated. Similar results were obtained with F17 T cell clone. b, EL26 T cells were either untreated (EL26) or cultured with PMA plus I (EL26PI) or OKT3 in the absence of accessory cells (EL26A) or with IFN-γ-pretreated RTEC expressing the restriction molecule DR15 and pulsed with peptide HLA-A2 92–120 (EL26RTEC). CD154 expression was assessed after 24 h of culture. Isotype-matched controls (filled histograms) and MFI are also indicated. c, T cell clone M3 was anergized (M3A) or activated with mitomycin C-treated cognate transfectants (M3S). After 3 days of resting in the presence of suboptimal dose of IL-2, the cells were rechallenged with cognate transfectants, and CD154 expression was assessed after 6, 18, and 24 h, as indicated. MFI and isotype-matched controls (filled histograms) are also indicated. Data shown are representative of three separate experiments performed with different T cell clones (F17 and EL26).

Close modal
FIGURE 7.

CD40-CD154 interaction protects DC from cell death. a, Stimulation via CD40 protects DC from death induced by anergic T cells. A total of 9 × 103 anergic G12 T cells was cultured in the presence of 3 × 103 51Cr-labeled DRB1*0101+ mature DC either untreated or treated with M3 mAb or cross-linked with anti-CD40 mAb (G28-5 mAb). The results are expressed as the percentage of specific 51Cr release and are representative of two experiments. These experiments were repeated with F17 clone with the same results. b, The treatment of DC with anti-CD40 antagonistic Ab favors suppressive phenomena. Responder T cell clone F17 (5 × 103 cells) was cultured with 5 × 103 DRB1*1101+ mature DC pulsed with HA 307–319, in the presence of 1.5 × 104 F17 anergic cells (▨) or in the presence of 1.5 × 104 PMA plus I-stimulated F17 T cells (□). DC were either untreated or pretreated with the antagonistic anti-CD95 Ab (M3 mAb) or the antagonistic anti-CD40 Ab. T cell proliferation was measured after 72 h of culture. Results are expressed as percentage of suppression of [3H]TdR incorporation, and derive by one of two separate experiments. These experiments were repeated with the G12 clone with the same results.

FIGURE 7.

CD40-CD154 interaction protects DC from cell death. a, Stimulation via CD40 protects DC from death induced by anergic T cells. A total of 9 × 103 anergic G12 T cells was cultured in the presence of 3 × 103 51Cr-labeled DRB1*0101+ mature DC either untreated or treated with M3 mAb or cross-linked with anti-CD40 mAb (G28-5 mAb). The results are expressed as the percentage of specific 51Cr release and are representative of two experiments. These experiments were repeated with F17 clone with the same results. b, The treatment of DC with anti-CD40 antagonistic Ab favors suppressive phenomena. Responder T cell clone F17 (5 × 103 cells) was cultured with 5 × 103 DRB1*1101+ mature DC pulsed with HA 307–319, in the presence of 1.5 × 104 F17 anergic cells (▨) or in the presence of 1.5 × 104 PMA plus I-stimulated F17 T cells (□). DC were either untreated or pretreated with the antagonistic anti-CD95 Ab (M3 mAb) or the antagonistic anti-CD40 Ab. T cell proliferation was measured after 72 h of culture. Results are expressed as percentage of suppression of [3H]TdR incorporation, and derive by one of two separate experiments. These experiments were repeated with the G12 clone with the same results.

Close modal

The data of this study present the first evidence that the maturational state of DC can influence the quality of immunosuppressive signals mediated by anergic CD4+ T cells. The observations that allowed us to reach this conclusion were that anergic T cells: 1) inhibited the proliferation of responder T cells stimulated by immature DC by antagonizing the activating effect of responder T cells on the DC function, whereas 2) they induced apoptosis of DC (and, in turn of responder T cells) when fully mature DC were used as APC. The reason for the differential susceptibility of immature and mature DC to CD95/CD95L-mediated apoptotic signals depends on the fact that these cells became CD95+ only at advanced states of their maturation. In addition, we found that apoptosis of mature DC in the presence of anergic T cells depends on the lack of ligation of CD40 expressed on DC. This is explained by the fact that, differently from correctly activated T cells, anergic T cells, either treated with OKT3 Ab or following coculture with cognate RTEC, become CD95L+ in the absence of significant up-regulation of CD154. Moreover, we present the evidence that the defect of CD40 triggering by anergic cells is associated with lack of up-regulation of MHC and CD86 molecules on immature DC.

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.

This study is dedicated to the memory of F. Tatò. We are grateful to the Avis (Bergamo, Italy) for providing the blood. We thank Dr. Cristina Gagliardi for her helpful suggestions in DC preparation, and Dr. Federica Marelli-Berg for her critical reading of this manuscript.

1

This work was supported by grants from University of Rome “La Sapienza,” Ateneo Project, the Ministry for the University and Scientific and Technological Research (MURST Cofinanziamento; 2000), and the National Council of Research (Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie).

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.

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