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The Journal of Immunology, 1999, 162: 1974-1981.
Copyright © 1999 by The American Association of Immunologists

Dose-Dependent Induction of Distinct Anergic Phenotypes: Multiple Levels of T Cell Anergy1

Leonie S. Taams, Willem van Eden and Marca H. M. Wauben2

Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell anergy has been proposed as one of the mechanisms underlying peripheral T cell tolerance. In recent years, the functional relevance of T cell anergy has been studied extensively in vitro and in vivo, using different species, cell systems, and ways to induce anergy. Although these studies concurred about the induction of unresponsiveness, conflicting findings were obtained with respect to the function of anergic T cells and to the persistence of T cell anergy. In the present study, T cell anergy was induced through T-T presentation of the specific Ag by rat MHC class II+ T cells in the absence of professional APC. We show that, depending on the Ag dose with which T cells were incubated, distinct anergic phenotypes were induced. Incubation of T cell clones with a low (suboptimal) Ag dose induced hyporesponsiveness. Incubation with a higher (optimal) Ag dose induced an anergic state capable of exerting immunoregulatory effects. Incubation with a high (supraoptimal) Ag dose led to an anergic suppressive phenotype that was persistent and was not reversed by APC, Ag, and rIL-2. These findings demonstrate that T cell anergy is not confined to a single state of functional inactivation. Instead, multiple levels of T cell anergy exist. Thus, anergic T cells can contribute to the regulation of the immune response either in a persistent and active manner or in a passive manner, depending on their level of T cell anergy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Homeostasis of the immune system is maintained by mechanisms of central and peripheral tolerance. Whereas central tolerance is achieved through negative thymic selection, peripheral tolerance is characterized in various ways, including ignorance 1 , deletion 2 , active suppression/immunoregulation 3, 4, 5 , and T cell anergy 6, 7 .

T cell anergy is defined as a state in which a viable T lymphocyte fails to display certain functional responses, such as proliferation or IL-2 production, upon antigenic restimulation under otherwise stimulatory conditions 8 . Full stimulation of T cells occurs when T cells receive an antigenic stimulus through the TCR (signal 1) together with a second signal delivered by costimulatory molecules (signal 2). When T cells receive signal 1 in the absence of signal 2, a state of T cell anergy ensues 9, 10, 11 . T cell anergy can also be induced in the presence of costimulatory signals, in the case of altered peptide ligands presented by professional APC 12 , or can be induced by activated MHC class II+ T cells, which present peptides to other T cells (T-T presentation) 13, 14, 15, 16 . Although these different forms of T cell anergy are similar with respect to the induction of unresponsiveness and the subsequent block in IL-2 production, discrepancies are observed in other aspects. Some studies have shown that anergy was prevented or reversed by the addition of exogenous IL-2 17, 18 , whereas in other reports this was not the case 19, 20 . Similarly, we and others 15, 21 demonstrated that anergic T cells can suppress the proliferative response of other T cells, whereas in other studies this suppressive effect was absent 22 . These differences might be due to the different anergy-induction protocols or might be a consequence of the different species or cell types involved. Alternatively, the discrepancies found in the earlier studies could be caused by differences in the activation state of functionally anergic T cells 23 .

In the present study, we demonstrate that T cell anergy is not confined to a single state of functional inactivation. Anergy was induced through T-T presentation of specific Ag by MHC class II+ rat T cell clones in the absence of professional APC, a phenomenon that could occur under physiological circumstances. Unresponsiveness was readily induced not only when a supraoptimal Ag dose was used, but also when optimal or even suboptimal Ag doses were used. Interestingly, incubation of T cells with increasing Ag doses led to different anergic phenotypes ranging from Ag-specific unresponsiveness, to an anergic phenotype displaying suppressive effects, and to an anergic suppressive phenotype that was persistently present, i.e., nonreversible by APC, Ag, and rIL-2. We propose that these distinct functional phenotypes of anergic cells reflect different levels of T cell anergy, a concept analogous with the concept of multiple levels of peripheral tolerance 24 . Thus, depending on the anergy-inducing Ag dose, multiple levels of T cell anergy exist, resulting in T cells that can contribute in a persistent and active manner or in a passive way to the regulation of the immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
T cell clones and peptides

The isolation, maintenance and properties of the rat CD4+ T cell clones A2b and Z1a have been described previously 25, 26 . Briefly, the arthritogenic T cell clone A2b was derived from the draining lymph nodes of a Lewis rat that was immunized with Mycobacterium tuberculosis (Mt)3 in IFA. A2b recognizes the 180- to 188-amino acid sequence (TFGLQLELT) of the mycobacterial 65-kDa heat shock protein (hsp) 27 , and the longer variant 176- to 190-amino acid sequence (EESNTFGLQLELTEG) 28 in the context of RT1.BL 29 . The encephalitogenic T cell clone Z1a was derived from draining lymph nodes of a Lewis rat, immunized with guinea pig myelin basic protein (MBP) in CFA. Z1a recognizes amino acid sequence 72–85 (QKSQRSQDENPV) of guinea pig MBP, as well as the peptide analogue MBP72–85S79A (QKSQRAQDENPV), which has an increased RT1.BL binding affinity 30, 31 . Peptides were synthesized by standard solid-phase F-moc chemistry 32 , analyzed and purified by reversed-phase HPLC, and checked by fast atom bombardment mass spectrometry.

In vitro lymphocyte proliferation assay

Proliferation of T cell clones was measured in flat-bottom 96-well microtiter plates (Costar, Cambridge, MA) in triplicate cultures. Viable T cells (2 x 104 per well) were cultured in 200 µl of culture medium, in the presence of irradiated (3000 rad) syngeneic thymus-derived APC (1 x 106 per well), and a dose range of the specific Ag or human rIL-2 (10 U/ml) (PharMingen, San Diego, CA). In some experiments, APC (6 x 107/ml) were prepulsed for 2 h at 37°C with peptide at the indicated concentrations. T cells were cultured for 3 days and subsequently pulsed for 16–20 h with 0.4 µCi/well of [3H]TdR (sp. act. 1 Ci/mmol; Amersham, Arlington Heights, IL). Cells were harvested on fiberglass filters, and [3H]TdR incorporation was measured by a scintillation counter (Wallac Oy, Turku, Finland). Results are expressed as the mean cpm of triplicate cultures ± SD.

Anergy induction of rat T cell clones

T cell clones A2b and Z1a were kept in IL-2-containing medium in the absence of APC and Ag for at least 6 days before anergy induction. T cell anergy was induced as described previously 15 . In brief, T cells were incubated (3 x 106/ml) in the absence of professional APC with increasing concentrations of their respective stimulatory peptides, peptide 176–190, or peptide MBP72–85S79A. Incubation was performed for 20–24 h in 6-well plates (Costar) in culture medium (Iscove’s modified Dulbecco’s medium (Life Technologies, Gaithersburg, MD) supplemented with 2 mM L-glutamine, 50 µM 2-ME, 50 U/ml penicillin, 50 U/ml streptomycin, and 2% heat inactivated normal rat serum). Viable T cells were collected by Ficoll-Isopaque gradient centrifugation and cultured in culture medium for an additional 3–7 days at 4–8 x 105 cells/ml. Viable T cells were collected by Ficoll-Isopaque gradient centrifugation, and proliferative responses were measured in a T lymphocyte proliferation assay. For anergy reversal experiments (schematically shown in Fig. 6Go), A2b T cells were rendered anergic, rested for 3 days, and restimulated (4 x 105/ml) for 20–24 h with or without peptide 176–190 (1 µg/ml) and/or human rIL-2 (100 U/ml) in the presence of irradiated (3000 rad) thymus-derived APC (1 x 107/ml). Viable T cells were collected by Ficoll-Isopaque gradient centrifugation, cultured in culture medium for 3–6 days, and restimulated in a T lymphocyte proliferation assay with APC and a dose range of peptide 176–190 (or hsp65 or Mt, data not shown).



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  		FIGURE 6. Schematic representation of the anergy reversal protocol. T cells were preincubated for 20 h with the optimal (1 µg/ml) or supraoptimal (10 µg/ml) concentration of peptide 176–190. Viable cells were collected and rested for 3 days (first rest period). Next, viable A2b cells were restimulated overnight in the presence of APC + medium; APC + rIL-2 (50 or 100 U/ml); APC + Ag (peptide 176–190 (1 µg/ml) or Mt (5 µg/ml)); or APC + Ag + rIL-2 (first restimulation phase). After overnight incubation, cells were washed and viable T cells were collected. T cells were rested for 3–6 days (second rest period), and finally the proliferative responses of A2b cells were measured during a standard lymphocyte proliferation assay (second restimulation phase) in the presence of APC and Ag (peptide 176–190, hsp65, or Mt).

 
Coculture assay

Responder T cells (2 x 104 per well) were cocultured with (non) anergic T cells in 96-well plates in the presence of APC and Ag. The number of (non) anergic T cells varied (0, 2 x 104, or 6 x 104 cells per well), resulting in T:T ratios of 1:0, 1:1, and 1:3, respectively. After 3 days of culture, [3H]TdR was added to the wells for 16–20 h, and incorporation was measured. (Non) anergic T cells were not irradiated.

Flow cytometry

Cell surface immunofluorescence analysis of T cells was performed using a FACScan analyzer (Becton Dickinson). In brief, T cells (5–10 x 104 per sample) were incubated at 4°C for 30' with saturating amounts of anti-TCR{alpha}ß mAb R73 (a kind gift of Prof. T. Hünig) or the IgG1 isotype control UD15 (anti-chloramphenicol), washed, and further incubated at 4°C for 30' with FITC-conjugated goat-anti-mouse secondary Ab (Becton Dickinson). Blast formation was measured via forward scatter (FSC) analysis. FITC fluorescence was detected in the FL1 channel. Incubation with isotype control-matched mAb UD15 resulted in background staining (data not shown).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Dose-dependent induction of T cell anergy through T-T presentation of the specific Ag

Previously, Lamb et al. showed that in human T cell clones anergy was induced through T-T presentation of a supraoptimal dose of the specific Ag 13 . In the present study, we investigated whether T-T presentation-induced anergy can also occur using low doses of Ag. For this, rat CD4+ T cell clones A2b and Z1a were used. FACS analysis revealed high expression levels of MHC class II molecules (mean fluorescence intensity: 600–700) on these clones, and low expression levels of B7, as measured by staining with the CLTA4Ig fusion protein (mean fluorescence intensity: 30–50) (data not shown). T cell anergy was induced in vitro by incubating the T cell clones in the absence of professional APC for 20 h with increasing concentrations of their respective stimulatory peptides, peptide 176–190 or peptide MBP72–85S79A. Viable T cells were collected and rested for at least 3 days before restimulation in the presence of professional APC and Ag. Fig. 1GoA shows the proliferative response of A2b cells after preincubation with peptide. Cells preincubated without peptide showed a normal proliferative response to peptide 176–190 upon antigenic restimulation, with an optimal proliferation at a concentration of 1 µg/ml. In contrast, cells preincubated with increasing concentrations of peptide 176–190, ranging from suboptimal to supraoptimal Ag doses, displayed a dose-dependent anergic phenotype upon antigenic restimulation, which was already evident when a suboptimal (0.1 µg/ml) Ag dose was used during anergy induction. The Ag specific unresponsiveness coincided with rIL-2 hyperresponsiveness, which is a characteristic feature of anergic T cells 11, 15, 33 . Similar results were obtained for T cell clone Z1a (Fig. 1GoB). The optimal proliferative response of Z1a cells preincubated without peptide was measured at a concentration of 10 µg/ml of peptide MBP72–85S79A. Preincubation of Z1a T cells with increasing concentrations of peptide MBP72–85S79A induced dose-dependent T cell anergy in these cells, as well as hyperresponsiveness to rIL-2. These results indicate that T cell anergy through T-T presentation can be induced using not only a supraoptimal Ag dose, but also using optimal or even suboptimal Ag doses.



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  		FIGURE 1. Dose-dependent induction of T cell anergy. A2b (A) and Z1a (B) T cells were preincubated in the absence of professional APC with increasing concentrations of their respective stimulatory peptides 176–190 (0, 0.01, 0.1, 1, 10 µg/ml), or MBP72–85S79A (0, 1, 10, 50 µg/ml). Viable cells were collected, washed, and rested for 3 days before restimulation with professional APC and a dose-range of their stimulatory peptide or human rIL-2 (10 U/ml) in a standard proliferation assay. Proliferative responses were measured by [3H]TdR incorporation. Results are expressed as the mean cpm of triplicate cultures ± SD.

 
Dose-dependent TCR down-regulation after peptide preincubation

The effect of the anergy induction protocol on TCR expression was investigated by FACS analysis. TCR surface expression was measured directly after anergy induction and after 3 days of rest, i.e., the time at which the T cells were tested in a proliferation assay. Preincubation of A2b cells with increasing concentrations of peptide 176–190 in the absence of APC resulted in a clear blast formation for all Ag doses tested, as measured by an increase in FSC (Fig. 2Go, left panel). Moreover, overnight peptide preincubation led to a dose-dependent down-regulation of TCR surface expression (Fig. 2Go, middle panel). TCR down-regulation was most prominent when cells were preincubated with the optimal (1 µg/ml) or supraoptimal dose (10 µg/ml) of peptide. Interestingly, despite the fact that preincubation with a suboptimal Ag dose (0.1 µg/ml) induced only minor TCR down-regulation, clear blast formation was observed (Fig. 2Go, left panel). Moreover, upon antigenic restimulation these cells displayed a hyporesponsive phenotype (Fig. 1Go). This finding indicated that the signal transduced through the TCR was sufficient to induce an anergic state in these T cells without down-regulating the TCR. The TCR down-regulation observed with the optimal and supraoptimal Ag dose was a transient event, returning to basal levels after 3 days of rest (Fig. 2Go, right panel). Similar results were obtained with T cell clone Z1a (data not shown). Importantly, in these experiments complete peakshifts in FITC fluorescence were observed, indicating that the whole T cell population was affected by the anergy induction protocol. Preincubation with a high dose of an irrelevant RT1.BL-binding peptide did not induce TCR down-regulation (data not shown).



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  		FIGURE 2. TCR down-regulation after peptide preincubation. A2b T cells were preincubated for 20 h in the absence of professional APC without (open histograms) or with 0.1, 1 or 10 µg/ml of peptide 176–190 (solid histograms). Viable cells were collected and FSC (left panel) and TCR surface expression (middle and right panels) were measured. TCR surface expression was measured directly after peptide preincubation (middle panel) and after a subsequent 3 days rest period (right panel) using anti-TCR mAb R73, followed by incubation with FITC-conjugated goat-anti-mouse Ab. Incubation with isotype control UD15 resulted in background staining for all samples (data not shown).

 
The induction of T cell anergy is determined by Ag dose, not by incubation time

We investigated whether the Ag dose-dependent differences in (un)responsiveness and TCR expression could also be observed after incubation of T cells with a fixed Ag dose for different periods of time. In other words, does a short incubation time with a high Ag dose induce the same effects as a long incubation time with a low Ag dose? A2b cells were preincubated with 10 µg/ml of peptide 176–190 (the supraoptimal Ag dose that induced profound unresponsiveness) for various periods of time. Cells were washed, and rested for 3 days before rechallenge in a proliferation assay. Fig. 3Go shows that induction of T cell anergy using a supraoptimal Ag dose was an extremely rapid process. Preincubation with peptide for as short as half an hour was sufficient to induce a complete unresponsive state in T cells. The induction of anergy was preceded by a rapid but transient TCR down-regulation, as shown in Fig. 4Go. These experiments show that short-time (0.5 h) preincubation of T cells with a high (supraoptimal) Ag dose was more efficient in inducing TCR down-regulation and anergy (Figs. 3Go and 4Go) than long-term (20–24 h) preincubation with a low (suboptimal) Ag dose (Figs. 1Go and 2Go).



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  		FIGURE 3. Time-dependent induction of T cell anergy. A2b T cells were preincubated in the absence of professional APC with 10 µg/ml of peptide 176–190 for 0, 0.5, 1, 4, or 20 h. Viable cells were collected, washed, and rested for 3 days before restimulation with professional APC and a dose-range of the stimulatory peptide or human rIL-2 (10 U/ml) in a standard proliferation assay. Proliferative responses were measured by [3H]TdR incorporation. Results are expressed as the mean cpm of triplicate cultures ± SD.

 


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  		FIGURE 4. TCR down-regulation after preincubation with a supraoptimal Ag dose for various periods of time. A2b T cells were preincubated in the absence of professional APC with 10 µg/ml of peptide 176–190 for 0 h (open histograms) or 0.5, 1, 4, or 20 h (solid histograms). Viable cells were collected and TCR cell surface expression was measured either directly after peptide preincubation (left panel) or after 3 days of rest (right panel) using anti-TCR mAb R73, followed by incubation with FITC-conjugated goat-anti-mouse Ab. Incubation with isotype control UD15 resulted in background staining for all samples (data not shown).

 
Dose-dependent induction of T cell anergy is correlated with T cell suppression

Recently, it was shown that anergic T cells can mediate suppressive effects on other T cells 5, 15, 21, 34 . To investigate a possible correlation between the degree of unresponsiveness and the ability to exert suppression, T cell clones A2b and Z1a were preincubated with a dose-range of their respective stimulatory peptides 176–190 or MBP72–85S79A. After 3 days of rest, the preincubated T cells were tested for their proliferative response to APC and Ag (Fig. 5Go, A and B) and for their T cell suppressive capacity (Fig. 5Go, C and D). T cells preincubated with an optimal or supraoptimal Ag dose were unresponsive upon antigenic restimulation. Moreover, these anergic T cells displayed immunosuppressive effects when cocultured with responder T cells (Fig. 5Go, C and D). A2b cells that were preincubated with a suboptimal Ag dose were hyporesponsive. The addition of these hyporesponsive cells to the coculture led to neither an increase nor a decrease of the proliferative A2b response, even when added at a 1:3 ratio. Addition of nonanergic A2b or Z1a cells that were normally responsive did enhance the proliferative T cell responses in coculture. These results show that the degree of T cell suppression was correlated with the degree of unresponsiveness and was mediated most efficiently by T cells that were rendered profoundly anergic.



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  		FIGURE 5. T cell suppression by anergic T cells. A2b or Z1a T cells were preincubated in the absence of professional APC with their respective stimulatory peptides 176–190 (0, 0.1, 1, 10 µg/ml) or peptide MBP72–85S79A (0, 1, 10, 50 µg/ml). Viable cells were collected and rested for 3 days before restimulation (A and B) or coculture with responder T cells (C and D) in the presence of professional APC and Ag. A, A2b cells preincubated with 0, 0.1, 1, or 10 µg/ml of peptide 176–190 were restimulated with APC prepulsed with peptide 176–190 (10 µg/ml). B, Z1a cells preincubated with 0, 1, 10, or 50 µg/ml of peptide MBP72–85S79A were restimulated with APC and MBP (10 µg/ml). C, Responder A2b cells were cocultured without (black bars) or with A2b cells which were preincubated with 0, 0.1, 1, or 10 µg/ml of peptide 176–190, at a 1:1 (cross-hatched bars) or 1:3 (open bars) ratio, in the presence of peptide-pulsed APC. D, Responder Z1a cells were cocultured without (black bars) or with Z1a cells that were preincubated with 1, 10, or 50 µg/ml of peptide MBP72–85S79A, at a 1:1 (cross-hatched bars) or 1:3 (open bars) ratio, in the presence of APC and MBP (10 µg/ml). Proliferative responses were measured by [3H]TdR incorporation.

 
Persistence of T cell anergy is dependent on the dose with which T cell anergy is induced

We next investigated whether T cell anergy induced through T-T presentation was persistent. Therefore, we set up an "anergy reversal" protocol, which is schematically depicted in Fig. 6Go. During the preincubation phase, A2b cells were cultured for 20 h with the optimal (1 µg/ml) or supraoptimal (10 µg/ml) Ag dose, both of which induced complete anergy (Figs. 1Go and 5Go). Viable cells were collected and rested for 3 days (first rest period). Next, viable A2b cells were restimulated overnight in the presence of: APC plus medium (i.e., all costimulatory and adhesion molecules present); APC and a high concentration of exogenously added human rIL-2 (100 U/ml); APC and the optimal concentration of peptide 176–190 (1 µg/ml); or APC, peptide 176–190 (1 µg/ml), and exogenously added human rIL-2 (100 U/ml) (first restimulation phase). After overnight incubation, cells were washed, and viable T cells were collected. T cells were rested for 3–6 days (second rest period), and finally the proliferative responses of A2b cells were measured during a standard lymphocyte proliferation assay (second restimulation phase). Adding 5 µg/ml Mt instead of 1 µg/ml peptide 176–190, or varying the amount of rIL-2 (50 U/ml instead of 100 U/ml) during the first restimulation phase yielded similar results (data not shown). T cells preincubated with 1 or 10 µg/ml peptide were still unresponsive following incubation with APC plus medium during the first restimulation phase, showing that anergy persisted for at least 9 days (Fig. 7GoA). Incubation with APC plus rIL-2 and subsequent antigenic restimulation in the proliferation assay showed that the optimal Ag dose-incubated cells became hyporesponsive, whereas the supraoptimal Ag dose-incubated cells were still anergic (Fig. 7GoB). When anergic T cells were cultured with APC plus Ag during the first restimulation phase, cells that were preincubated with the supraoptimal Ag dose remained unresponsive as measured during the second restimulation phase, whereas cells preincubated with the optimal dose were partially reversed in their anergic state (Fig. 7GoC). Strikingly, when anergic T cells were cultured with APC plus Ag plus rIL-2 during the first restimulation phase, i.e., the most optimal stimulatory condition, those preincubated with the optimal Ag dose reverted to full responder cells upon subsequent restimulation. In contrast, T cells that were preincubated with the supraoptimal Ag dose remained fully anergic (Fig. 7GoD).



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  		FIGURE 7. Persistence of T cell anergy. A2b T cells were preincubated with 0 µg/ml (solid circles), 1 µg/ml (solid triangles), or 10 µg/ml (open squares) of peptide 176–190. After a 3-day rest period, T cells were restimulated overnight with APC + medium (A); APC + rIL-2 (100 U/ml) (B); APC + peptide 176–190 (1 µg/ml) (C); and APC + rIL-2 (100 U/ml) + peptide 176–190 (1 µg/ml) (D). Viable cells were collected, rested for 5 days, and subsequently restimulated with APC and peptide 176–190 in a standard proliferation assay. Proliferative responses were measured by [3H]TdR incorporation.

 
T cell anergy is maintained despite an increase in absolute cell number

Fig. 7Go showed that T cells that were preincubated with the supraoptimal Ag dose and subsequently restimulated with APC, rIL-2, and Ag maintained their anergic state upon subsequent antigenic restimulation. Interestingly, we observed that such anergic T cells were metabolically active after rIL-2 stimulation, i.e., during the second rest period, and that blast formation and cluster formation occurred. Therefore, we investigated whether an actual increase in total cell number of anergic T cells occurred during the second rest period using trypan blue staining. The results of two different experiments are shown in Table IGo. Following the protocol described in Fig. 6Go, A2b cells were preincubated with 0 or 10 µg/ml of peptide 176–190. Viable cells were rested for 3 days, restimulated overnight with APC plus rIL-2 or APC plus Ag plus rIL-2, and rested again (second rest period) for 3 days (Expt. I) or 6 days (Expt. II). The number of viable cells was counted at the end of the second rest period (before the second restimulation) and related to the number of cells at the beginning of the second rest period (% of input). Table IGo shows that during the second rest period under all conditions an increase in total cell number was observed for the peptide-preincubated T cells (% of input > 150%, shown in bold in Table IGo). Interestingly, when the peptide-precultured cells, including these newly generated cells, were subsequently restimulated during the second restimulation phase, still a complete anergic phenotype was found (proliferation < 15% as compared with medium-preincubated T cells). Thus, despite the fact that anergic T cells divided due to stimulation with rIL-2, none of these cells regained their Ag-specific responsiveness.


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  		Table I. Relative increase in cell numbers of anergic A2b cells after restimulation with rIL-2

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several in vivo studies using different systems have suggested a role for T cell anergy in peripheral tolerance 2, 7, 35, 36, 37, 38, 39 . In addition, various methods have been employed to study the induction of T cell anergy in CD4+ T cells in vitro (reviewed in 8 . An in vitro method for anergy induction that might reflect physiological circumstances is Ag presentation by MHC class II+ nonprofessional APC, such as activated T cells (T-T presentation) 13, 14, 15, 16 . It was suggested that T-T presentation-induced anergy required supraoptimal Ag doses 13 . However, in the present study using two different rat CD4+ MHC class II+ T cell clones, we clearly demonstrated that T-T presentation of optimal and even suboptimal Ag doses was sufficient to induce T cell anergy.

Previously it was proposed that T cell anergy was a consequence of TCR down-regulation 2, 40, 41 . In our study, we observed a dose-dependent but transient TCR down-regulation after T-T presentation of Ag, indicating that the observed unresponsiveness was not directly caused by a lack of available TCR. Interestingly, preincubation with a suboptimal Ag dose induced only marginal TCR down-regulation, whereas these cells were hyporesponsive upon antigenic restimulation. This indicated that TCR down-regulation was correlated with, but was not required for the induction of unresponsiveness. Shorter (2 h) or longer (2 days) preincubation periods with the suboptimal Ag dose did not significantly affect TCR expression either (data not shown). In contrast, short-time (0.5 h) incubation with a supraoptimal Ag dose induced a strong TCR down-regulation and complete unresponsiveness. This demonstrated that long-term, low Ag dose incubation is not equivalent to short-time incubation with a high Ag dose. A similar conclusion was recently reported for T cell activation in vivo, using TCR transgenic mice 42 . Our findings suggest that it is not the persistence of Ag, but the initial "hit" with a certain ligand density displayed by MHC class II+ T cells that determines the quality of the anergic state.

FACS analysis revealed complete peak shifts in FSC, and TCR expression after peptide preincubation, indicating that the whole cell population was affected by the anergy induction protocol. This implies that the hyporesponsive state observed after preincubation with the suboptimal Ag dose could not be attributed to the outgrowth of a subpopulation of nonanergic T cells, but rather reflected the response of an entire hyporesponsive T cell population.

Recently, it was shown that T cells at various degrees of activation can be functionally anergic 23 . In the present study, the dose-dependent levels of TCR down-regulation might reflect such different degrees of (initial) activation. Therefore, the question was raised whether T-T presentation of different Ag doses would induce distinct anergic phenotypes in T cells. Previously, it was shown that anergic T cells can function as immunoregulatory cells 5, 15, 21, 34 . Now we show that T cells rendered anergic with either optimal or supraoptimal Ag doses could suppress the responses of other T cells in coculture, while addition of T cells rendered hyporesponsive after preincubation with a suboptimal Ag dose did not display this suppressive effect. Thus, depending on their anergic state, T cells can suppress responses of other T cells, and can therefore contribute either actively or passively to immunoregulation. The observed suppression does not appear to be due to peptide carry-over and subsequent T-T presentation by the peptide-preincubated T cells, as we have shown previously that anergic T cells can also suppress T cell responses to additional epitopes presented by APC, provided the presence of their cognate ligand on the APC 15 .

A matter of debate for a possible role for T cell anergy in vivo is the issue of anergy persistence. It has been proposed that T cell anergy is a consequence of the inability of a T cell to produce IL-2, and subsequently to divide, following stimulation through the TCR 18, 43 . In support of this hypothesis, it has been shown using different anergy models that anergy was prevented and/or reversed by the addition of exogenous IL-2 17, 18, 43, 44 or IL-12 45 . In contrast, other studies showed that anergy was not reversed by addition of exogenous IL-2 19, 20 or IL-12 19, 46 . In the present study, we showed that reversal of T-T presentation-induced anergy is dependent on the Ag dose with which anergy was induced. The anergic state in supraoptimal Ag dose-preincubated T cells persisted at least 10 days despite attempts to reverse anergy in these T cells by restimulation with APC, Ag, and/or rIL-2. In contrast, cells rendered fully anergic with the optimal Ag dose were more susceptible to these reversal conditions. Such cells eventually regained their Ag specific responsiveness after incubation with APC, Ag, and rIL-2.

The persistence of anergy after incubation with rIL-2 is an intriguing phenomenon, especially because anergic T cells are hyperresponsive to rIL-2 (Fig. 1Go). Indeed, anergic T cells were metabolically active upon incubation with rIL-2, as assessed by blast and cluster formation, and an increase in total cell number was observed (Table IGo). Despite the generation of these new T cells, no Ag specific proliferative response was observed when these cells were restimulated in a proliferation assay, indicating that neither the anergic T cells nor the newly generated T cells were susceptible to antigenic stimulation. An explanation for the persistence of anergy could be that the anergic T cells suppressed the Ag specific proliferative response of the newly generated T cell population or that T cell anergy had spread to the new cell population, a phenomenon called infectious tolerance or spreading anergy 3, 47 . Alternatively, one cannot exclude the possibility that the anergic state of a cell is inherited by its progeny.

Taken together, our data demonstrate that anergy is not an all-or-none phenomenon but exists at multiple levels depending on the anergy-inducing Ag dose (summarized in Table IIGo). Low (suboptimal) Ag doses induce hyporesponsiveness, which is preceded by marginal TCR down-regulation. At higher (optimal) Ag doses, profound but transient TCR down-regulation is observed, and upon antigenic restimulation these cells are completely unresponsive. Moreover, these cells display an immunoregulatory phenotype in the sense that they exert suppressive effects to other T cells. High (supraoptimal) Ag doses induce a similar phenotype in T cells, which is not reversible by restimulation with APC, Ag, and rIL-2. Our concept of multiple levels of T cell anergy is analogous with the concept of the multistep system of T cell tolerance 24, 41 . In these studies using transgenic mice, Arnold and co-workers demonstrated that, depending on the amount of transgene expressed, different levels of T cell tolerance in vivo existed, ranging from TCR/CD8 down-regulation through anergy to deletion. The present study shows that anergy is a multistep Ag dose-dependent system as well, in which the functional outcome of the anergic cell is determined by the initial ligand density displayed on activated MHC class II+ T cells. An important question to answer is how and when T-T presentation occurs in vivo. It has been described previously that activated T cells can actively acquire MHC/peptide complexes from APC 48 . Furthermore, it has been shown that upon activation T cells internalize their TCR, which can subsequently be processed and presented in an MHC class II-restricted manner to TCR peptide-specific T cells 49 . Indeed, it has been demonstrated that such TCR peptide-specific T cells do exist in vivo and can play a regulatory role in autoimmunity 50, 51, 52 . Alternatively, it has been hypothesized that self-hsp molecules, which are up-regulated in the event of stress or inflammation, can be presented in he context of MHC class II on activated T cells, leading to the induction of regulatory hsp-specific T cells 53, 54 . In this respect, it is of interest to know that T-T presentation-induced anergy could occur even in the presence of professional APC, as it is not due to a lack of costimulation 16, 55 . The ligand density of the MHC/peptide complexes displayed on activated T cells will vary depending on the amount of TCR internalization or stress-induced hsp up-regulation. Strong activation or stress signals will lead to high ligand densities, and the subsequent induction of regulatory and persistent anergic T cells. In contrast, when low ligand densities are presented, due to weak activation or stress signals, the induction of regulatory cells is less required. As such, the induction of multiple levels of T cell anergy could serve as a fine-tuning mechanism for the regulation of the immune response.


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  		Table II. Multiple levels of T cell anergy

 


   Acknowledgments
 
We thank A. Besseling for technical support, Dr. R. Van der Zee and A. Noordzij for peptide purification, and C. Versluis for FAB-MS peptide analysis (Biomolecular Mass Spectrometry, Bijvoet Center, Utrecht University, Utrecht, The Netherlands). We also thank Prof. I. R. Cohen for the use of T cell clones A2b and Z1a.


   Footnotes
 
1 This work was supported by N. V. Organon, The Netherlands. The research of M.H.M.W. has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. Back

2 Address correspondence and reprint requests to Dr. M. H. M. Wauben, Institute of Infectious Diseases and Immunology, Department of Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508 TD Utrecht, The Netherlands. E-mail address: Back

3 Abbreviations used in this paper: Mt, Mycobacterium tuberculosis; MBP, myelin basic protein; hsp, heat shock protein; FSC, forward scatter. Back

Received for publication June 15, 1998. Accepted for publication November 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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