Death by neglect as a deletional mechanism of peripheral tolerance

Patrick Bertolino2, Marie-Claude Trescol-Biémont, Joëlle Thomas, Barbara Fazekas de St Groth1, Maria Pihlgren, Jacqueline Marvel and Chantal Rabourdin-Combe

Ecole Normale Supérieure de Lyon, INSERM U98, 46 Allée d'Italie, 69364 Lyon Cedex 07, France
1 Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW 2042, Australia

Correspondence to: P. Bertolino


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In contrast to most organs, the anatomy of the liver may allow naive CD8+ T cells to make direct contact with liver parenchymal cells. We have previously shown, using a combination of TCR transgenic T cells specific for H-2 Kb and hepatocytes expressing a transgenic H-2 Kb molecule, that hepatocytes can induce antigen-specific activation and proliferation of naive CD8+ T cells independently of CD28 co-stimulation. However, T cell activation by hepatocytes leads to premature T cell death and tolerance, bothin vivo andin vitro. In this study, we investigated the mechanisms of T cell death induced by hepatocytesin vitro using primary hepatocytes to activate purified CD8+ T cells. Neither Fas nor tumor necrosis factor receptor were involved, indicating that hepatocyte- induced death was distinct from activation-induced cell death. Before they started to divide, T cells activated by hepatocytes expressed lower levels of thebcl-xL survival gene and 30 times less IL-2 mRNA than CD8+ cells activated by splenic antigen-presenting cells. Since CD28 co-stimulation increases both IL-2 andbcl-xL expression, this suggests that hepatocyte-activated T cells die by neglect because they fail to receive effective co-stimulatory signals. In agreement with this model, premature death promoted by hepatocytes could be prevented by cross-linking CD28. Survival after CD28 cross-linking correlated with increased IL-2 andbcl-xL expression, and sustained T cell proliferation, while cytotoxic T lymphocyte activity was prolonged as compared with cells stimulated without CD28 co-stimulation. This study confirms that high- affinity TCR transgenic antigen-specific CD8+ T cells can be activated to proliferate and differentiate into cytotoxic effector cells. However, prolonged T cell survival and cytotoxicity required CD28 co-stimulation as well. To our knowledge, this is the first report suggesting that tolerance in the context of lack of CD28 co-stimulation can result from Fas-independent peripheral deletion rather than from anergy.

Keywords: activation-induced cell death, co-stimulation, hepatocytes, T lymphocytes, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Effective activation of resting CD8+ T cells requires interaction of the TCR with specific peptide–MHC complexes, in addition to triggering of co-stimulatory pathways. One of the most effective co-stimulatory stimuli is the interaction of CD28 with its natural antigen-presenting cell (APC) ligands CD80 and CD86. Co- ordinate triggering of TCR and CD28 has been shown to induce cytokine secretion, T cell proliferation and cytotoxic T lymphocyte (CTL) activity (13). CD28 co-stimulation acts by enhancing both the rate of transcription and half-life of IL-2 and othercytokine mRNAs (46), in addition to up-regulating the expression of survival genes such asbcl-xL (7). Cytokines and survival factors are both important in ensuring T cell survival and a sustained immune response (7,8).

Despite its importance in enhancing T cell activation, the role of co-stimulation in self-tolerance is not clear. Some reports, based on studies using CD4 T cell clones, suggest that T cell activation in the absence of co-stimulation induces unresponsiveness or anergy (9). This concept has also been applied to naive T cells, although the experimental evidence is not entirely consistent with this hypothesis. Thus, several recent reports suggest that although co- stimulation is important to increase immune response, its absence does not always lead to anergy(1014).In vitro studies using naive CD8+ T cells further suggest that the importance of co-stimulation in mounting an effective CD8+ T cell response may result from lack of T cell survival, rather than from induction of anergy (7,15).

To address thein vivo fate of autoreactive CD8+ T cells able to recognize a specific antigen presented by APC which cannot provide CD28 co- stimulation, several groups have used a combination of TCR transgenic mice and transgenic mice expressing a known antigen in parenchymal cells which do not normally express CD80 and CD86. Extrathymic antigen expression resulted in induction of peripheral tolerance in multiple models (1618). However, the fate of auto-reactive CD8+ T cells depended on a number of factors, including activation state, frequency, and accessibility and nature of the tissue APC. Parenchymal cells such as pancreatic islets are not easily accessible to naive T cells, presumably due to the effectiveness of the physical barrier formed by a combination of endothelial cells, basement membrane and epithelial cells. Thus, when expression of the allo-MHC class I H-2 Kb molecule was restricted to the ß islets of the pancreas, autoreactive CD8+ T cells ignored the antigen (19), unless previously primed (20), or unless the islets co-expressed transgenic CD80 (21) or IL-2 (19). Similar results have been obtained in other transgenic systems (2224).

In contrast to the pancreas, the liver not only lacks an effective basement membrane, but possesses a loose fenestrated endothelial barrier, allowing naive T cells to make direct contact with hepatocytes (2528). In agreement with this observation, we showed previously that in transgenic mice expressing theH-2 Kb molecule in the liver, autoreactive CD8+ T cells infiltrated the liver lobules, were rapidly activated by hepatocytes, proliferated, expressed cytotoxic activity and then died by apoptosis (17). Recently, we established anin vitro system using purified primary hepatocytes and naive CD8+ T cells, confirming thein vivo observations (29). Ourin vitro results clearly showed that primary T cell activation by hepatocytes does not lead to anergy, since proliferating T cells acquired normal cytolytic activity in the absence of exogenously added IL-2. However, unlike T cells activated by splenic dendritic cells, hepatocyte-activated cells underwent premature cell death (29).

Death of activated T cells can be either active or passive.In vitro, active death can occur upon TCR re-cross-linking and is known as activation-induced cell death (AICD). AICD is thought to maintain the homeostasis of the immune system and results from the expression of members of the tumor necrosis factor (TNF)/TNF receptor (TNFR) family on the surface of activated T cells. Ligation of Fas (CD95) by Fas ligand (FasL) is responsible for AICD in most CD4+ T cells (3032) and some CD8+ T cellsin vitro (33), whereas a death signal transmitted by TNF binding to TNFR-1/TNFR-2 has been implicated forsome CD8+ T cells (3436). The expression of the TNFR family molecules and their ligands is induced during activation, resulting in an autocrine or paracrine activation of apoptosis. Fas-dependent death of activated CD4+ (30) or CD8+ (37) T cells has also been shown to occurin vivo in TCR transgenic models and has been suggested to be mediated by AICD. However, there are some discrepancies betweenin vitro andin vivo results (33,38), and there is no evidence that TCR re-cross-linking and T–T cell contact is required for Fas- dependent deathin vivo. IL-2 seems to promote rather than inhibit AICDin vitro by driving activated cells into S phase, during which they are more sensitive to apoptosis (8,39,40). AICD mediated via Fas but not TNFR-2 seems to be insensitive to the expression ofbcl-xL orbcl-2 (33,36,4143). In contrast to AICD, passive death or death by neglect results from growth factor deprivation of recently activated cells (44). This form of programmed cell death appears to be independent of the expression of members of the TNF/TNFR family, but can be prevented or delayed by IL-2 or expression of members of thebcl-2 family includingbcl-xL (43,45,46).

In the present study, we explored the mechanisms leading to premature death of CD8+ T cells activated by antigen-bearing hepatocytes. We show that neither Fas nor TNFR is involved, but that limited IL-2 production and low expression of thebcl-xL andbcl-2 survival genes correlates with susceptibility to premature death. Thus, premature death could be prevented by supplying exogenous IL-2 or by providing CD28 co-stimulation, suggesting that the failure of hepatocytes to express CD28 ligands may have been responsible for premature death of activated CD8+ T cells. Our results are consistent with a model in which T cells activated by hepatocytes die by neglect rather than AICD. This mechanism would allow self-reactive CD8+ T cells to die prematurely before they could become harmful to the body. Together with our previousin vivo data, this report is the first physiological evidence suggesting that death by neglect can function as a deletional mechanism, inducing peripheral tolerance in naive CD8+ T cells activated by class-I restricted antigens expressed by parenchymal tissue APC rather than professional APC.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Transgenic mice, B10.BR (H-2k) and C57BL/6 (H-2b) mice were bred at the Ecole Normale Supérieure de Lyon. Des-TCR transgenic mice expressing a Kb-specific TCR (16) identifiable by a clonotypic antibody, `Désiré' (47), were kindly provided by Drs B. Arnold, G. Schönrich and G. J. Hämmerling (Deutsches Krebsforschungszentrum, Heidelberg, Germany). The Des-TCR line was maintained by mating with B10.BR mice and the progeny screened by flow cytometry.

Preparation of stimulator cells
Spleens were removed aseptically and transferred into supplemented DMEM containing 6% FCS (referred to as DMEM culture medium). Splenocyte suspensions were made by pressing the tissue through an 80 gauge stainless steel mesh and washing twice with the same medium. Hepatocytes and spleen dendritic cells were purified as previously described (29). Hepatocytes were incubated overnight in RPMI culture medium [RPMI 1629 (Life Technologies, Paris, France) supplemented with 2 mML-glutamine (Life Technologies), 40 µg/ml gentamycin (Life Technologies), 10% FCS (TechGen, Paris, France), 10 mM HEPES and 50 µM 2-ß mercaptoethanol]. T cells were added to hepatocyte cultures after 1 day.

Splenocytes were irradiated (1500 rad). When required, splenocytes were incubated for 2 h at 37°C on plastic plates to enrich for adherent cells. Non-adherent cells were removed with three successive washes with RPMI culture medium.

All assays described in this paper used either 24- or 96-well flat-bottom plates. When 24-well plates were used, 5x105 purified CD8+ T cells were co-cultured in 2 ml with 5x104 hepatocytes or 5x106 splenocytes. When 96-well plates were used, 5x104 purified CD8+ T cells were co-cultured in 200 µl with 5x103 hepatocytes or 5x105 splenocytes.

Preparation of responder cells
Single-cell suspensions of lymph nodes were prepared as for splenocytes (see above). Cells were incubated for 2 h at 37°C and 7% CO2 in RPMI culture medium in Petri dishes to remove adherent cells. Non-adherent cells were then carefully harvested and incubated for 30 min at 4°C in a mixture of anti-mouse CD4 [GK1.5 (48)]-, anti-B220 [RA36B2 (49)]-, anti-I-Ab [M5/114 (50)]-, anti-Mac1 [M1/70 (51)]- and anti-GR1 [RB68C5 (52)]-specific mAb. Cells were washed 3 times and incubated for 30 min at 4°C with goat anti-rat IgG (H + L)-coupled beads (Biomag; PerSeptive Diagnostics, Warrington, UK) at a ratio of 10 beads per cell. Cells attached to the beads were removed by a magnet and the remaining cells were washed once before being added to culture. The cell preparation obtained using this protocol contained high and reproducible yields of CD8+ T cells (90–98%) as determined by flow cytometry analysis.

Proliferation assays
All proliferation assays were performed in RPMI culture medium in 96-well flat-bottom plates at 37°C and 7% CO2. Purified CD8+ lymph node responder cells (5x104) from Des-TCR mice were co-cultured with different concentrations of stimulator cells for the required time at 37°C. Proliferation was measured by adding 0.5 µCi of [3H]thymidine to each well for 8 h at 37°C before harvesting and counting using a Microbeta Trilux counter (Wallac, Evry, France).

Cytotoxic assays
Purified CD8+ lymph node cells (5x105) from Des-TCR mice were co-cultured in RPMI culture medium at 37°C in 24-well plates with either 2x106 irradiated C57BL/6 splenocytes or 5x104 C57BL/6 hepatocytes precultured overnight. After 2–3 days of co-culture, effector cells were purified over Ficoll-Hypaque (Cedarlane, Ontario, Canada). Target cells (106 P815 or P815-Kb cells) were incubated with 50 µCi of51Cr for 1 h at 37°C and 3000 cells were added to different concentrations of effector cells in 96- well U-bottom plates. Cells were incubated for 4 h at 37°C. Assays were performed in duplicates and counted with a Microbeta Trilux counter (Wallac).

CTLL-2 assays
The amount of IL-2 present in the supernatant of hepatocyte- or splenocyte-activated T cells was measured by proliferation of the IL-2-dependent cell line CTLL-2 using a standard protocol (53). Supernatants were half-diluted with culture medium for the test. Proliferation of CTLL-2 cells was evaluated with a MTT colorimetric assay as described elsewhere (53). Assays were performed in triplicate.

T cell death assays
To induce AICD, lymph node T cells from Des-TCR mice were first activated for 3 days by co-culture with C57BL/6 splenocytes in 24-well plates. Activated cells were purified over Ficoll-Hypaque, cultured for a further 2 days in the presence of IL-2 and re-purified over Ficoll-Hypaque. AICD was performed as previously described (34) by culturing 2x105 activated cells (in 200 µl) for 24–48 h in 96-well flat-bottom plates precoated with 4 µg/ml of anti-TCR (H57) in the presence or absence of titered anti-mouse TNF-{alpha} mAb (G281-2626; PharMingen, CliniSciences, Montrouge, France), anti-mFasL.1 (Kay-10; PharMingen, CliniSciences), rat IgG1 isotype-control for anti-TNF-{alpha} or mouse IgG2b isotype-control for anti-mFasL.1. Cells incubated in uncoated plates were used as a control.

T cell death in response to hepatocytes was tested by co-culturing C57BL/6 hepatocytes with purified CD8+ T cells from Des-TCR mice in the presence or absence of a panel of blocking or control antibodies described above. To ensure that saturating concentrations of blocking antibody were available, low numbers of CD8+ T cells (5x103 cells/well) were used in these assays. Antibodies were added during the second day of co-culture before activated cells started to die. Co-cultures were allowed to proceed for a further 2 days before harvesting.

In both assays, cells were stained with propidium iodide (PI), and for expression of CD8 and Des- TCR. The percentage of PI+ CD8+ Des-TCR+ (apoptotic cells) was assessed using a FACScan and analyzed using CellQuest software.

Semi-quantitative RT-PCR assays
Naive or activated CD8+ T cells from Des-TCR mice were purified over Ficoll-Hypaque and lysed with RNA Now reagent (Biogentex, Ozyme, Montigny-le-bix, France). RNA was extracted with chloroform, washed with ethanol and quantified by spectrophotometry. Three-fold dilutions were made to give 300, 100, 30 and 10 ng of sample RNA per tube. To quantify the amplified products, a standard competitor RNA was transcribed from the pMus3 plasmid containing multiple cytokine primer sequences, a gift of Dr D Shire (54). A constant amount of standard RNA (100 fg) was added to each tube, and both sample and standard RNAs were hybridized with oligo(dT) primers (Boerhinger Mannheim, Meylan, France). mRNAs were reverse-transcribed for 1 h at 37°C by using 200U of MuMLV reverse transcriptase (Gibco/BRL, Paris, France), 20 U of RNasin (Promega, Lyon, France), 10 mM DTT (Gibco/BRL), 0.5 mM dNTP mix (Promega) and reverse transcriptase first-strand buffer (Gibco/BRL). Reversetranscribed cDNAs were amplified by PCR, using cytokine primers (IL-2, IL-4, IL-5, IFN- {gamma} and TNF-{alpha}) or ß2-microglobulin primers as an internal control. The primer sequences and PCR conditions have been described elsewhere (54). Amplifications were performed in the GeneAmp PCR System 9600 thermocycler (Perkin-Elmer Cetus, Norwalk, CT). The PCR products were detected by ethidium bromide staining after electrophoresis and quantified using ImageQuant software. The band corresponding to the competitor RNA has a different size from that of the band amplified from the cytokine mRNA. Both bands were quantified individually and used to calculate a standard/cytokine ratio that allowed estimation of the level of cytokine expression.

RNA preparation and northern blot analysis
T cell total RNA was isolated by the RNA Now method (Biogentex). Total RNA (10 µg) was separated on 1% agarose formaldehyde gel, transferred to Hybond N (Amersham) nylon membrane, hybridized overnight at 42°C with32P-labeled probes and washed using a standard protocol. Blots were successively hybridized with different probes. The murinebcl-xL probe was a 0.8 kbSacI–XhoI fragment and the murinebax probe was a 0.6 kb fragment, both containing the complete coding region (a kind gift of Dr D Loh). The murinebcl-2 probe was a 0.6 kbBamHI–HindIII fragment containing the first coding exon (kind gift of Dr G. Nunez). The murine actin probe was a 0.4 kbKpnI–XbaI fragment.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Premature death of CD8+T cells activated by hepatocytes does not involve Fas or TNFR
We previously reported that primary hepatocytes can induce the proliferation of naive CD8+ T cells from Des-TCR mice that express an H-2Kb-specific transgenic TCR. Hepatocyte co-cultures generate equivalent numbers of T cells to splenocyte co-cultures during the first 2 days of co-culture and CD8+ T cells activated by hepatocytes acquire CTL activity at the peak of the proliferative response, but die sooner than T cells activated by spleen APC (29). This is not due to failure of hepatocytes to provide long-term stimulation since hepatocytes can survive >2 weeks in culture without losing their stimulatory properties. In this study, we defined the relationship between stimulatory cell number, time of culture and proliferation ( Fig.1Go). At early time points (42 h), hepatocytes induced higher T cell proliferation than an equivalent number of purified spleen dendritic cells. However, hepatocyte-mediated T cell proliferation dropped after 42 h and reached background levels by 90 h. In contrast, T cells activated by spleen dendritic cells were still proliferating very effectively at 90 h ( Fig. 1Go). These results suggest that T cell fate is not due to a quantitative difference in the level of APC- derived signals but rather reflects an intrinsic difference between the two types of APC in inducing T cell activation. Moreover, activated T cells survived if co-cultured with both hepatocytes and splenocytes (29), indicating that premature T cell death was not due to exhaustion of nutrients or toxicity mediated by hepatocytes. We therefore investigated the mechanisms of premature death of T cells activated by hepatocytes by comparing them to the previously described mechanisms that induce death of T cells activated by splenic APCin vitro, a phenomenon termed AICD.



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Fig. 1. Proliferation of CD8+ T cells activated by hepatocytes peaks earlier than T cell proliferation induced by dendritic cells. Purified lymph node CD8+ T cells (5x105 cells in 200 µl) isolated from Des-TCR mice were cultured with titered numbers of purified C57BL/6 spleen dendritic cells or purified C57BL/6 hepatocytes in 96-well plates. Proliferation was measured at different time points by adding [3H]thymidine for 8 h before harvest. Proliferation of Des-TCR CD8+ T cells in response to syngeneic B10BR hepatocytes or dendritic cells was negligible (data not shown). When error bars are not apparent, they are too small to be visible.

 
Death of splenocyte-activated Des-TCR CD8+ T cells in response to TCR re-cross-linking (AICD) could be specifically inhibited by blocking FasL ( Fig. 2AGo). The level of inhibition was comparable to that observed for anti-FasL antibody-dependent AICD of C57BL/6 CD4+ T cells (data not shown) and was also consistent with the quantitative difference between the level of AICD in cultures of CD4+ T cells fromlpr orgld versus wild-type mice (34,43 and data not shown). This suggests that the use of anti-FasL blocking antibody was effective in totally inhibiting death mediated by Fas, but that other death molecules are also involved in the death of activated CD8+ T cellsin vivo. Such molecules do not include TNFR, since no inhibition was observed when TNF-{alpha} was blocked ( Fig. 2AGo) using antibody concentrations capable of inhibiting TNF-mediated cytotoxicity of the TNF-sensitive WEHI 164 cell line (data not shown). These results suggested that splenocyte-induced AICD of Des-TCR CD8+ T cells was partly mediated via Fas but was TNF independent.



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Fig. 2. Premature death of hepatocyte-activated T cells is not due to AICD. (A) AICD of Des-TCR CD8+ T cells is partially mediated by Fas but not TNFR. Lymph node T cells from Des-TCR mice were activated for 3 days with C57BL/6 splenocytes, then for a further 2 days with additional IL-2. AICD was induced as described in Methods, by adding activated cells to anti-TCR-coated plates for 24 h with saturating amounts of IL-2 in the presence or absence of anti-mFasL, anti-TNF-{alpha} or isotype-matched control mAb. The percentage of dead CD8+ cells 48 h after receptor ligation was measured by FACS using PI and anti-CD8 antibody. The percentage of dead cells in cultures from uncoated plates is shown as a control in the right panel. (B) Fas and TNFR are not involved in the premature death of T cells activated by hepatocytes. In the left panel, purified Des-TCR CD8+ lymph node T cells (5x103/well) were incubated for 4 days with hepatocytes from C57BL/6 mice (5x103/well) in 96-well plates in the presence or the absence of anti-Fas-L, anti-TNF-{alpha} or control isotype-matched mAb. Survival was assayed by PI staining as for (A). The right panel shows the percentage of dead CD8+ T cells in 4 day co-cultures of Des-TCR T cells with hepatocytes, hepatocytes plus IL-2 or splenocytes.

 
Blocking antibodies were also used to investigate the premature death of T cells activated by hepatocytes. Concentrations of anti-FasL mAb which were effective in blocking classical AICD of Des-TCR CD8+ T cells failed to inhibit the premature death of T cells co-cultured with C57BL/6 hepatocytes ( Fig. 2BGo). Likewise, addition of blocking anti-TNF-{alpha} mAb had no effect on T cell death ( Fig. 2BGo). Since these experiments made use of smaller numbers of T cells than in the AICD assays mentioned above, lack of inhibition was not due to insufficient blocking antibody. These results show that T cell death induced by hepatocytes is not an active process involving members of the TNFR family, suggesting that it is distinct from AICD. Addition of IL-2 prevented premature death of cultured CD8+ T cells activated by hepatocytes ( Fig. 2BGo), whereas AICD has been reported to be increased by addition of exogenous IL-2 (8,39,40). Taken together, these data indicate that hepatocyte-activated Des-TCR T cells are unlikely to die by AICD mediated via either Fas or TNFR.

CD8+T cells activated by hepatocytes express limited amounts of cytokines
In addition to apoptosis mediated via TNFR family members, activated T cells are known to be susceptible to death as a result of cytokine deprivation and/or low levels of survival gene expression(5557). Since premature death of T cells activated by hepatocytes could be abrogated by IL-2 ( Fig. 2BGo), T cells may have been dying due to insufficient IL-2 production. To measure the level of cytokine expression by activated T cells, we used semi-quantitative RT-PCR. T cells from Des-TCR mice were activated by hepatocytes or spleen adherent cells from C57BL/6 mice and harvested at different time points to prepare RNA for RT-PCR. None of the Th2-type cytokines tested (IL-4 and IL-5) was detected in any samples (data not shown), but both co- cultures induced expression of Th1-type cytokines (IL-2, IFN-{gamma} and TNF-{alpha}) as early as 12 h after activation ( Fig. 3BGo). Th1 cytokine production required T cell activation since no expression was detected in CD8+ Des-TCR+ T cells co-cultured with control syngeneic spleen adherent cells ( Fig. 3BGo). mRNA cytokine expression peaked 24 h after activation and decreased to basal levels by 72 h. However, despite similar kinetics of expression, quantitative differences were seen between the two types of cultures. At the peak of mRNA cytokine synthesis (24 h), CD8+ T cells activated by hepatocytes expressed 30 times less IL-2 mRNA and 5 times less IFN- {gamma} and TNF-{alpha} mRNA than splenocyte-activated T cells ( Fig. 3AGo), despite proliferation in hepatocyte co-cultures being several fold higher than in DC co-cultures between 24 and 48 h ( Fig. 1Go). By 48 h, the IL-2 RT PCR band was no longer detectable for hepatocyte-activated T cells but could still be detected for T cells activated by splenic APC ( Fig. 3BGo). For IL-2, this difference was confirmed at the protein level using a CTLL-2 bioassay ( Fig. 3CGo) which indicated that bioactive IL-2 secreted by hepatocyte-activated CD8+ T cells reached maximal levels 24 h after activation but was undetectable by 48 h (Fig.3CGo). In contrast, IL-2 secreted by CD8+ T cells activated by spleen adherent cells could be detected 72 h after activation ( Fig. 3CGo). Since IL-2 mRNA could not be detected by RT-PCR at 72 h, it is likely that the functional IL-2 activity detected at this time point resulted from the large amount of IL-2 synthesized in the early phase of the response.



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Fig. 3. CD8+ T cells activated by hepatocytes express lower levels of IL-2, IFN-{gamma} and TNF-{alpha} cytokine mRNAs than those activated by adherent spleen APC. Purified lymph node Des-TCR CD8+ T cells activated by co-culture in 24-well plates with C57BL/6 hepatocytes or spleen adherent cells were harvested at different times, purified by Ficoll-Hypaque and lysed to prepare RNA. RT PCR was performed with IL-2, IFN-{gamma}, TNF-{alpha} or ß2-microglobulin primers on sequential 3-fold dilutions of RNA samples mixed with 100 fg of standard RNA as competitor. (A) RT-PCR bands in naive purified CD8+ T cells or T cells activated for 24 h in 24-well plates with either C57BL/6 hepatocytes or spleen adherent cells. ß2-Microglobulin was used as a positive control. The amplified band corresponding to the standard competitor mRNA has a different size and is not shown. These results are representative of three independent experiments. (B) Kinetics of cytokine mRNA expression. All RNA amounts were calculated by comparing the band intensity of the cytokine with that of the competitor RNA template in the same assay. (C) CTLL-2 proliferation assay using hepatocyte or splenocyte culture supernatants at a final concentration of 1:2. Error bars indicate the SD of triplicate samples. When error bars are not apparent, they are too small to be visible.

 
These results showed that CD8+ T cells activated by hepatocytes synthesized IL-2 and other Th1-type cytokines during the early phase of the proliferative response. However, their production was far lower than that observed in T cells activated by adherent splenic APC. Thus, IL-2 deprivation may have been responsible for the premature death of hepatocyte-activated T cells.

CD8+T cells activated by hepatocytes express lower levels of bcl-xLand bcl-2 than T cells activated by splenocytes
Since triggering of the IL-2 receptor (IL-2R) {gamma} chain has been shown to influence expression of anti-apoptotic genes such asbcl-xL andbcl-2 (46), we examined expression ofbcl-xL andbcl-2 as well as the pro-apoptotic genebax (58) in T cells activated by hepatocytes or splenocytes. Purified Des-TCR CD8+ T cells co-cultured with either C57BL/6 hepatocytes or splenocytes were harvested, purified over Ficoll-Hypaque and lysed to extract RNA. Northern blots were hybridized with probes specific forbcl-xL, bcl-2 orbax. As shown in Fig. 4Go,bcl-xL mRNA expression was low in unactivated cells but was induced during the first 18 h of co-culture with either hepatocytes or splenocytes, and decreased at later time points. However, the peak level ofbcl-xL expression in T cells activated by hepatocytes was only half that detected in T cells activated by splenocytes. This 2-fold difference was maintained at later time points. By day 3 of co-culture, i.e. when hepatocyte-activated T cells started dying, the level ofbcl-xL expression in such cells had decreased to the basal level detected in naive T cells. Likewise,bcl-2 mRNA expression peaked at 66 h in T cells activated by splenic APC but was not induced in T cells activated by hepatocytes ( Fig. 4Go). In contrast tobcl-xL andbcl-2, mRNA expression of the pro-apoptotic molecule Bax was induced with similar kinetics and at similar levels in hepatocyte and splenocyte co-cultures, the peak being seen at 18 h ( Fig. 4Go). However at later times, the level ofbax mRNA in splenocyte co-cultures was higher ( Fig. 4Go).



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Fig. 4. Premature death of CD8+ T cells activated by hepatocytes correlates with poor induction of thebcl-xL andbcl-2. Purified lymph node Des-TCR CD8+ T cells activated in 24 well plates with C57BL/6 splenocytes (open circles), hepatocytes (filled squares), hepatocytes plus 10 µg/ml of anti-CD28 mAb (open squares) or hepatocytes plus IL-2 (open triangles) were harvested at different times, purified over Ficoll-Hypaque and lysed to prepare RNA. Northern blotting was performed using 10 µg of RNA per sample. The blot was hybridized successively with probes specific forbcl-xL,bcl-2,bax and mouse ß-actin. Specific bands were quantified using ImageQuant software and the ratio of specific band to actin band calculated for each sample. All time points except 0 and 18 h represent the mean ± SD of the ratios calculated from two independent experiments. Except 0 and 18 h time points, when error bars are not apparent, they are too small to be visible.

 
These results showed that T cells activated by hepatocytes induced lower early expression ofbcl-xL andbcl-2 than splenocyte-activated T cells, Therefore, despite their ability to provide the signals required to initiate early T cell responses, hepatocytes appear not to induce sufficient expression of cytokine and survival genes to ensure survival and sustained T cell proliferation.

CD28 co-stimulation up-regulates IL-2 and survival gene expression and prevents premature T cell death in hepatocyte co-cultures
Expression of both IL-2 andbcl-xL genes is up-regulated when CD28 is cross-linked with CD3 (13,7). Co-culture with anti-CD28 mAb resulted in increased expression levels ofbcl-xL mRNA ( Fig. 4Go). Althoughbcl-xL expression in T cells activated by hepatocytes in the presence of anti-CD28 mAb was not as high as that induced by splenic APC, two independent experiments confirmed that the level was significantly higher than that of T cells activated by hepatocytes only. Anti-CD28 mAb also slightly increasedbcl-2 mRNA expression between 40 and 90 h but did not significantly affectbax mRNA levels ( Fig. 4Go). The presence of exogenous IL-2 in hepatocyte co-cultures did not increase early expression ofbcl-xL mRNA (18 h after activation) but appeared to induce further late expression ( Fig. 4Go). IL-2 also induced high levels ofbcl-2 as well asbcl-xL mRNA at late time points ( Fig. 4Go). In contrast,bax mRNA levels were not affected by IL-2 ( Fig. 4Go). Since exogenous IL-2 had no effect on up-regulation ofbcl-xL mRNA during the first 18 h of activation, these results suggest that triggering of the IL-2R common {gamma} chain does not augment the early induction ofbcl-xL, but is important for its expression at later time points. Thus, CD28 co-stimulation appeared to up- regulatebcl-xL expression independently of positive feedback from IL-2.

CD28 co-stimulation also enhanced cytokine expression. Cytokine mRNA expression, assayed by RT-PCR at 48 h, was present in T cells activated by splenocytes but not hepatocytes ( Fig. 5Go). In contrast, T cells activated by hepatocytes in the presence of CD28 co-stimulation expressed levels of IL-2 and IFN-{gamma} mRNA at 48 h comparable to T cells activated by splenic adherent cells ( Fig. 5Go).



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Fig. 5. CD28 co-stimulation leads to enhanced expression of IL-2, IFN-{gamma} and TNF-{alpha} cytokine mRNAs. RNA was isolated from Des-TCR CD8+ T cells activated by co-culture for 48 h in 24-well plates with either spleen adherent cells or hepatocytes in the presence or the absence of 10 µg/ml of anti-CD28 mAb. RT-PCR was performed as described in Fig. 2Go. ß2 -Microglobulin was used as a positive control. The amplified band corresponding to the standard competitor mRNA has a different size and is not shown. The experiment was performed twice with similar results.

 
To investigate whether CD28 co-stimulation could protect T cells activated by hepatocytes from premature death, purified CD8+ lymph node T cells from Des-TCR mice were co-cultured with C57BL/6 hepatocytes in the presence of titered concentrations of CD28 mAb. As shown in Fig. 6Go, anti-CD28 antibodies prevented T cell death on day 3 of the response, a plateau being reached at concentrations >1 µg/ml. No T cell activation was seen in the presence of anti-CD28 antibody alone (data not shown).



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Fig. 6. T cells activated by hepatocytes can be rescued from death by CD28 cross-linking. Purified C57BL/6 hepatocytes were co-cultured for 3 days in 24-well plates with CD8+ lymph node T cells from Des-TCR mice in the presence or absence of different concentrations of anti-CD28 mAb. Cells were harvested and their survival assayed by PI staining. Error bars indicate the SD of triplicate samples.

 
Correlating with increased survival at day 3, the T cell proliferative response induced by hepatocytes in the presence of anti-CD28 mAb paralleled that of T cells activated by purified spleen dendritic cells, peaking at day 3 ( Fig. 7Go). We have previously shown that T cells activated by hepatocytes acquire CTL activity at day 2, suggesting that they are not anergic after activation by hepatocytes (29 and Fig. 8Go). However, in contrast to T cells activated by splenic APC, this activity is only transient in hepatocyte-activated T cells and is lost by day 3, presumably due to the initiation of death within the cultures (29). In agreement with these results, CTL activity was maintained at day 3 when death was prevented by the presence of anti-CD28 mAb or IL-2 ( Fig. 8Go).



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Fig. 7. Addition of anti-CD28 mAb to hepatocyte co-cultures results in sustained T cell proliferation with kinetics paralleling the response induced by splenocytes. Purified CD8+ T cells from Des-TCR mice were added to 24-well culture plates containing either C57BL/6 hepatocytes in the presence or the absence of 10 µg/ml of anti-CD28 mAb or C57BL/6 adherent splenocytes. Proliferation was measured at different time points by adding [3H]thymidine for 8 h before harvest. These results are representative of three independent experiments.

 


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Fig. 8. T cells activated by hepatocytes in the presence of anti-CD28 mAb retain cytotoxic activity after 3 days of co-culture. Purified CD8+ T cells were co-cultured for 2–3 days in 24-well plates containing either C57BL/6 splenocytes, hepatocytes or hepatocytes plus IL-2 or 10 µg/ml of anti-CD28 mAb as described in the legend to Fig. 7Go. Viable cells were purified on Ficoll-Hypaque and tested for their ability to lyse P815-Kb or control P815 target cells. CTL activity of non-activated T cells (freshly isolated from Des-TCR mice) cells was also measured as a control (left panel). These results are representative of three independent experiments.

 
These results show that addition of exogenous IL-2 or CD28 co-stimulation prevented the premature death of T cells activated by hepatocytes, increasing their proliferative response and maintaining their CTL activity during the late phase of the response. Collectively, these results suggest that T cells activated by hepatocytes die by neglect due to insufficient autocrine IL-2 production and poor survival gene expression, and that activation in the absence of effective co-stimulation is the primary cause of death by neglect.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we have investigated the mechanisms leading to the premature death of CD8+ T cells activated by hepatocytes. Our results suggest that T cells activated by hepatocytes do not undergo active death involving Fas or TNFR molecules. Instead, death results from the failure of hepatocytes to trigger co-stimulatory pathways that are responsible for effective cytokine production and expression of survival genes.

AICD is thought to be responsible for the death of cells activated upon TCR re-cross-linking (43), in particular dendritic cells. For CD4+ T cells fratricide via FasL expression appears to be the mechanism of AICD (3032). Although some studies have suggested that CD8+ T cells are resistant to Fas-mediated death upon repeated stimulationin vitro, being instead more sensitive to death induced via the TNFR pathway (34,36), it has been shown that, like AICD of CD4+ T cells, AICD in CD8+ T cellsin vitro (33) can be Fas dependent but TNF independent. Our data demonstrate that although AICD of Des- TCR T cells co-cultured with splenocytes was TNFR independent but Fas dependent, only part of the apoptotic response could be prevented by anti-FasL mAb, suggesting that molecules other than Fas or TNFR were involved. These results are consistent with previousin vitro data (34,43). AICD of both CD4+ and CD8+ T cells has been shown to occur late in the response, allowing T cells to proliferate, produce cytokines and/or acquire cytolytic activity before dying (43,59). It is now well documented that most T cells express high levels of Fas and FasL on their surface within 2 days of activation, but genes which have recently been identified (60,61) regulate Fas-mediated cell death so that cells become sensitive to apoptosis only 3–4 days after activation (62,63). Confirming these results, we found that T cells activated by splenocytes or purified C57BL/6 spleen dendritic cells up-regulated Fas and FasL between days 1 and 2 but did not die during the first 5 days of co-culture (29 and unpublished data).

In contrast to T cells activated by splenocytes, T cells activated by hepatocytes underwent death as early as 3 days after activation, despite lower surface expression of Fas (data not shown). In our previous report, we showed that premature death of T cells activated by hepatocytes was not caused by a dominant-positive death signal or by depletion of nutrients in the co-culture, since T cells activated in the presence of a combination of both hepatocytes and splenocytes survived as well as those activated by splenocytes alone (29). Thus hepatocyte-mediated death was unlikely to be an active process involving members of the TNFR family. In the present study, addition of anti-FasL or anti-TNF blocking mAb to co-cultures failed to prevent death ( Fig. 2Go), confirming these results. Moreover, IL-2 prevented the premature death of T cells activated by hepatocytes, whereas it has been shown to augment AICD ( 8,39,40). Collectively, these experiments suggested that the mechanisms involved in the premature death of hepatocyte-activated T cells are distinct from those involved in AICD.

The survival of a cell has been shown to depend upon a subtle balance in the expression of genes which either promote or prevent apoptosis. Up-regulation ofbcl-2 and its related genebcl-xL has been shown to inhibit passive cell death (death by neglect) when cells are deprived of growth factors (55,64,65). Death by neglect resulting from withdrawal of IL-2 from an IL-2-dependent cell line has been shown to be preceded by down-regulation ofbcl-xL but notbcl-2 (57), suggesting that the level ofbcl-xL expression may determine the survival of activated T cells. As shown here ( Fig. 4Go), naive T cells expressed low levels ofbcl-xL but rapidly up-regulated it after activation by either hepatocytes or splenocytes, reaching a peak at 24 h in both cases (55,57). It is still unclear whether down-regulation ofbcl-xL, which occurs 2–3 days after primary activation, plays a role in Fas-mediated AICD by increasing the sensitivity of the cell to the Fas pathway (7,8,36,41,42,66).

CD28 co-stimulation has been shown to be important for T cell survival. The mechanism of its action is thought to be via up-regulation ofbcl-xL expression and increased production of cytokines, in particular IL-2 (67). Hepatocytes express neither CD80 nor CD86 and therefore cannot provide CD28 co-stimulation to T cells (29). In agreement with this model, T cells activated by hepatocytes expressed only limited amounts of IL-2 andbcl-xL mRNA compared with T cells stimulated by splenic APC ( Figs 3 and 4GoGo), and died prematurely. Furthermore, adding anti-CD28 mAb to the co-cultures increased IL-2 production as well as survival gene expression and promoted T cell survival. The correlation between kinetics, levels ofbcl-xL expression and T cell survival in hepatocyte co-cultures supplemented with anti-CD28 and in splenocyte co-cultures suggests that lack of CD28 co-stimulation is indeed responsible for premature death in T cells activated via hepatocytes alone. Moreover, in contrast to exogenous IL-2, which increasedbcl-xL mRNA expression with kinetics quite distinct from those in splenocyte co-cultures, stimulation with hepatocytes plus anti-CD28 mAb closely mimicked the kinetics of the splenocyte co-cultures.bcl-xL expression is known to be up-regulated by triggering via CD28 (67) or the common {gamma} chain of several cytokine receptors including IL-2R (46). The independence of these two pathways is difficult to assess since CD28 co-stimulation also increases IL-2 production. However, our results suggest that CD28 and the IL-2R common {gamma} chain up-regulatebcl-xL independently of each other, since the kinetics ofbcl-xL expression were different in hepatocyte co-cultures supplemented with anti-CD28 mAb versus IL-2. Thus, CD28 co-stimulation increased the early peak ofbcl-xL expression, whereas the effect of exogenous IL-2 is not apparent until later time points. Whether the effect of IL-2 onbcl-xL mRNA levels is via increases in stability or transcription remains to be established.

T cell activation in the absence of CD28 cross-linking induced sufficient expression of IL-2 to initiate early events of activation including proliferation, suggesting the involvement of other co-stimulatory pathways such as those mediated by LFA-1/ICAM-1, which has previously been shown to be involved in T cell activation and proliferation induced by hepatocytes (29). Nonetheless, levels of IL-2 andbcl-xL were insufficient to ensure survival beyond 3 days of culture. These results confirm recently published reports showing thatin vitro activation involving LFA-1/ICAM-1 interaction in the absence of CD28 ligation also resulted in premature death due to IL-2 deprivation (6870). Our previously publishedin vivo data demonstrated that such abortive activation of CD8+ T cells can also take place in a physiological manner and is therefore relevant to peripheral tolerance. In addition, stimulation in the absence of co-stimulation can induce deletion rather than anergy.

CD4+ T cells have been shown to play an important role in CD8+ T cell activation and survival by providing help to CD8+ T cells. However, the requirement for cytokines secreted by CD4+ T cells depends on the affinity of the TCR. It is likely that CD8+ T cells bearing a low-affinity TCR need help to initiate proliferation and to survive. In contrast, we showed here that high-affinity CD8+ T cells do not require help during primary activationin vitro, and start to proliferate earlier than CD4+ T cells bothin vitro andin vivo (P. Bertolino, unpublished data). Our results suggest that in addition to the TCR avidity, the short- term fate of high-affinity activated CD8+ T cells is dependent on the APC involved in the primary activation. If CD8+ T cells are activated by dendritic cells, they receive proper co-stimulation and induce high levels of survival genes which allow them to survive long enough to receive help from CD4+ T cells. Help is perhaps also important for long-term survival of CTL. In contrast, if CD8+ T cells are activated by parenchymal cells (via ICAM-1/LFA-1 and other adhesion molecules), they die prematurely before they can receive help from CD4+ T cells. Therefore, death by neglect, resulting from activation without effective co-stimulation, could be a major tolerance-inducing mechanism for CD8 responses that do not require CD4 help for their initiation. This mechanism would allow T cells to die prematurely before becoming fully cytolytic and causing autoimmune damage. We have previously shown that tolerance to the H-2Kb molecule can be induced in chimeric transgenic mice in which expression of the H-2Kb molecule is limited to hepatocytes (17). Heavy infiltration of the liver by Des-TCR T cells was seen both in bone marrow chimeras and in adoptive transfer models. In such cases, infiltrating cells proliferated actively, but rapidly diedin situ, inducing only limited damage to hepatocytes. Ourin vitro results are consistent with thesein vivo observations and suggest that CD8+ T cells die by neglectin vivo as a result of activation in the absence of co-stimulation. Although this form of programmed cell death has been well described in growth factor-dependent cell lines and in T cells activated by anti- CD3 mAb, this is, to our knowledge, the first clear evidence showing that this mechanism is both physiologically relevant to tolerancein vivo and distinct from induction of anergy. These results are in agreement with the model proposed by P. Matzinger suggesting that self-reactive T cells can be deleted as a result of stimulation by parenchymal APC (26). However, in her model, only T cells previously activated by professional APC in peripheral lymphoid organs have access to tissue APC. We suggest that naive T cells can also be tolerized by parenchymal APC in those organs which are accessible to circulating naive T cells, e.g. the liver and the lung. It is possible that parenchymal APC isolated from other organs also have the ability to induce apoptosis following activation. However, if such APC are not accessible to naive CD8+ T cells, their physiological relevance in tolerance induction remains unclear.

In conclusion, the results presented in this paper are consistent with a model in which CD8+ T cells activated by hepatocytes die by neglect due to poor production of IL-2 andbcl-xL. Given its easy accessibility to naive CD8+ T cells, this would suggest that the liver has unique properties favoring induction of tolerance in the CD8 compartment. Such a mechanism could account, at least in part, for the striking ability of liver grafts to induce tolerance (71,72).


    Acknowledgments
 
We wish to thank Drs Bernd Arnold, Gunther Schönrich and Gunther Hämmerling for the Des-TCR transgenic mice, Dr Anne-Marie Schmitt-Verhulst for the Désiré-1 mAb, Drs David Shire, Gabriel Nunez and Dennis Loh for DNA reagents, Dr Phil Hodgkin for the isotype- control antibodies, and Dr Woon-Puay Koh for her help in the final corrections of the paper. We are also grateful to Dr Patrice Dubois for very helpful discussions and critical reading of this manuscript. P. B. and M. P. were supported by a fellowship `Association pour la Recherche sur le Cancer'. J. T. was supported by a fellowship `Fondation Marcel Mérieux'. This work was supported by institutional grants from the Centre National de la Recherche Scientifique and the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, and by additional support from the Association Franciaise contre les Myopathies.


    Abbreviations
 
AICDactivation-induced cell death
APCantigen-presenting cell
CTLcytotoxic T lymphocyte
FasLFas ligand
IL-2RIL-2 receptor
PIpropidium iodide
TNFtumor necrosis factor
TNFRTNF receptor

    Notes
 
Transmitting editor: D. Tarlinton

2 Present address: Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW 2042, Australia Back

Received 27 November 1998, accepted 16 April 1999.


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