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The Journal of Immunology, 1999, 163: 5929-5936.
Copyright © 1999 by The American Association of Immunologists

Antigen-Experienced T Cells Undergo a Transient Phase of Unresponsiveness Following Optimal Stimulation1

Fabrizio De Mattia, Susanna Chomez, François Van Laethem, Véronique Moulin, Jacques Urbain, Muriel Moser, Oberdan Leo and Fabienne Andris2

Laboratoire de Physiologie Animale, Université Libre de Bruxelles, Gosselies, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interaction of the Ag-specific receptor of T lymphocytes with its Ag/MHC ligand can lead either to cell activation or to a state of unresponsiveness often referred to as anergy. It has been generally assumed that anergy develops as a consequence of inadequate stimulation, such as in response to altered peptide ligands or to agonists presented by costimulatory-deficient accessory cells. The present study uncovers an alternative way of inducing an unresponsive state in T cells. Indeed, we demonstrate herein that Ag-stimulation of murine CD4+ Th clones induces cellular activation, characterized by cytokine production and cell proliferation, followed by a state of transient (lasting up to 6 days) unresponsiveness to further antigenic stimulation. This state of activation-induced unresponsiveness 1) is not a consequence of inadequate costimulation, as it occurs when cells are stimulated in the presence of dendritic cells or anti-CD28 Abs; 2) develops after an optimal response to Ag; 3) is not due to cell death/apoptosis or CTLA-4 engagement; 4) down-regulates the proliferation and cytokine production of both Th1- and Th2-like clones; and 5) does not affect the early steps of signal transduction. Finally, naive T cells are not sensitive to this novel form of unresponsiveness, but become gradually susceptible to activation-induced unresponsiveness upon Ag stimulation. Collectively, these data suggest that activation-induced T cell unresponsiveness may represent a regulatory mechanism limiting the clonal expansion and effector cell function of Ag-experienced T cells, thus contributing to the homeostasis of an immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Triggering of the TCR can either lead to cellular activation (causing cell proliferation and/or expression of effector function) or to an abortive response followed by anergy (1). Numerous studies, mostly performed in vitro with clonal populations, have demonstrated that T cell anergy is induced following an inadequate TCR stimulation, such as observed in response to costimulatory deficient APC (2) or to peptide Ags unable to evoke a complete TCR signaling (3).

However, the study of in vivo models of T cell unresponsiveness has revealed that in many experimental settings, a transient T cell activation precedes induction of tolerance/unresponsiveness. Exposure of mature T cell in vivo to Mls Ags (4, 5), allo-MHC (6), bacterial exotoxins (7), peptide Ags (8), or mitogenic anti-CD3 Abs (9) leads to T cell activation (characterized by cytokine production and/or clonal amplification) followed by hyporesponsiveness. Similarly, several studies using established cell lines and clones have shown that unresponsiveness can develop following a productive in vitro stimulation (10, 11). Notably, these observations are in keeping with the procedure widely used to grow T cell lines and clones in vitro, in which cells are allowed to rest 7–14 days after each antigenic stimulation. Collectively, these data challenge the simple idea that anergy is only a consequence of inappropriate stimulation and suggest that some forms of T cell unresponsiveness can be induced following a productive stimulation. In most of the aforementioned studies, it is not clear whether activation and anergy are confined to different T cell populations or whether the same cell can become transiently unresponsive following a productive activation by Ag.

To approach this question, we have analyzed the functional consequences of Ag stimulation on a panel of murine T cell clones producing distinct sets of cytokines. In this study, we demonstrate that following a productive stimulation by Ag presented by adequate accessory cells, murine CD4+ clones undergo a transient phase of unresponsiveness to any further Ag stimulation. In contrast to classical anergy, this activation-induced refractory phase cannot be overcome by the addition of exogenous cosignals. Investigations of the mechanisms responsible for this hyporeactivity suggest that they involve a novel pathway of T cell nonresponsiveness. Finally, naive T cells were found to be insensitive to this form of unresponsiveness, suggesting that it may represent a mechanism for selectively down-regulating the response of Ag-experienced T cells in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and reagents

The medium used in all experiments was RPMI 1640 supplemented with 5% FCS, penicillin-streptomycin, glutamine, nonessential amino acids, and 5 x 10-5 M 2-ME. The hamster mAb 37.51 to murine CD28 and control mAb F531, kindly provided by J. Allison (University of California, Berkeley, CA) (12), were used at 1/10,000 dilution of ascitic fluids. The hamster mAb 4F10 to CTLA-4 (13) and the mouse mAb KJ1.26 against the clonotypic DO11.10 TCR (14) were kindly provided by J. Bluestone and J. Kappler, respectively. The biotinylated anti-CD44 mAb was purchased from Leinco Technology (Balwin, MO).

Cell lines and mice

Six- to 8-wk-old BALB/c mice were purchased from Charles River Wiga (Sulzfeld, Germany) and A/J and B10.A mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in pathogen-free conditions in our animal facility.

The A.E7 Th1 clone, specific for pigeon cytochrome c (PCC)3 plus IEk (15), was a kind gift of R. H. Schwartz and maintained in culture as described (16). The HG-4 Th2 clone was generated from the draining nodes of A/J (H-2a) mice primed with 300 µg human {gamma} globulin (tail and foot pads, s.c. in CFA). T cell clones (106/well) were restimulated biweekly in a 24-well plate with 107 irradiated syngeneic splenocytes/well and 1 mg/ml human {gamma} globulin. At 48 h, the cells were expanded 5-fold into medium containing 10 U/ml of rIL-2 and rested for at least 7 days. The HG-4 clone was found to secrete IL-4, IL-5, and IL-10 but not IL-2 or IFN-{gamma} in response to Ag stimulation and was therefore referred to as Th2. The OVA peptide (323–339)-specific Th clones were derived as described (17). Briefly, spleen cells from naive DO11.10 mice (5 x 106) were cultured in a 24-well plate with 500 ng/ml of OVA (323–339) in the presence of rIL-12 and 10 µg/ml anti-IL-4 (clone 11B11) to promote the development of Th1-like cells or 200 U/ml IL-4 and 50 ng/ml anti-IL-12 (clone C17.8) to favor the differentiation toward a Th2 phenotype. The cytokine secretion profiles of the clones used throughout this study are summarized in Table IGo. APC populations (spleen cells, Sephadex G10-depleted splenocytes, and dendritic cells (DC)) were purified as described (18, 19).


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  		Table I. Lymphokine secretion patterns of Th clones cultured with splenic APC and Ag1

 
Induction of T cell unresponsiveness

Th clones or purified CD4+ spleen cells from naive DO11.10 mice (106/ml) were preincubated for 3 days in complete medium in the presence of the specific Ag and APC (5 x 106 to 107/ml). Control cells were cultured in the presence of APC and medium alone. Then, 100 µl of the culture supernatants were harvested and tested for cytokine content 48 h later. Viable cells were recovered after the primary activation by centrifugation on lympholyte M solution (Cedarlane, Ontario, Canada), rested 1–8 days in medium alone, and restimulated (106/ml in 200-µl 96-well plates) in the presence of freshly isolated irradiated syngeneic feeder cells (5 x 106/ml) and serial dilutions of Ag. Then, 50 µl of culture supernatants were tested 24 or 48 h later for IL-4, IL-5, and IFN-{gamma} content by ELISA, as described (20, 21). Proliferation was assessed 48 h after the initiation of culture by [3H]thymidine incorporation.

Flow cytometry

Specific cell-surface staining were performed using standard procedures and analyzed with a FACScan cytometer (Becton Dickinson, Mountain View, CA). Cells were labeled with carboxyfluorescein succinimidil ester (CFSE) as described (22). Briefly, T cells were resuspended at 107/ml in complete medium in the presence of 10 mM CFSE and incubated for 10 min at 37°C. The reaction was stopped by adding five volumes of ice-cold medium and, after two washes, CFSE-labeled cells were used for in vitro cultures and FACS analysis.

Immunoprecipitation and immunoblotting

Cloned T cells (4 x 107/ml) were incubated for 5 min with anti-CD3 mAbs (clone 7D6 (23), 4 µg/ml) and then cross-linked with rabbit anti-mouse (20 µl of serum/ml) for 90 s at 37°C. T cells were lysed in 1% Brij buffer (200 mM boric acid, 150 mM NaCl, pH 8.0) containing 2 mM PMSF, 5 mM EDTA, 1 mM sodium orthovanadate, and 5 mM NaF. Postnuclear lysates were immunoprecipitated overnight at 4°C with anti-CD3 {epsilon}-chain mAbs (145-2C11) (24) coupled to cyanogen bromide-activated Sepharose beads (Amersham-Pharmacia, Little Chalfont, U.K.), washed four times with cold lysis buffer, and boiled in Laemmli sample buffer. Immunoprecipitates were electrophoresed on 14% SDS-PAGE gels (Novex, San Diego, CA), and the fractionated proteins were transfered to a Hybond enhanced chemiluminescence nitrocellulose membrane. Blots were then incubated with the 4G10 anti-phosphotyrosine mAb (Upstate Biotechnology, Lake Placid, NY), followed by HRP-conjugated protein A (Sigma, St. Louis, MO). Protein detection was performed by enhanced chemiluminescence (Amersham-Pharmacia) following the manufacturer’s instructions. Membranes were stripped of bound Ab and reprobed with a monoclonal anti-TCR {zeta}-chain mAb (clone H146-968), kindly provided by R. Kubo (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of T cell clones induces a transient refractory phase to further antigenic stimulation

The purpose of this study was to investigate the experimental conditions leading to T cell unresponsiveness following TCR engagement by an antigenic ligand. In particular, we wished to examine the functional consequences of Ag stimulation of a prototype Th1 murine clone (the I-Ek/PCC-specific A.E7 clone) in the presence or absence of costimulatory-bearing APC. Costimulatory-deficient splenic APC were generated by removal of the Sephadex G10-adherent cells. As expected from previous studies (26), G10-depleted spleen cells (comprising mostly small resting T and B cells (18)) failed to stimulate the A.E7 clone (Fig. 1GoA). Abs to CD28 restored the stimulating properties of this accessory cell population, suggesting that the cells had retained the ability to generate an appropriate TCR ligand and were defective in the delivery of costimulatory signals (Fig. 1GoB).



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  		FIGURE 1. Comparison of accessory cell functions of splenic and G10-nonadherent splenic APC. The A.E7 clone (5 x 104 cells/well) was cultured (A) with unfractionated (•) or G10-passed ({circ}) spleen cells (5 x 105 cells/well) and serial dilutions of PCC and (B) in the presence or absence of 2 µM PCC and Abs to CD28 (clone 37.51) or control Abs (clone F531). Proliferation was assayed on day 2 by [3H]thymidine incorporation.

 
To study the functional outcome of a productive vs an inadequate Ag-stimulation, A.E7 T cell clones were stimulated in vitro by Ag presented by syngeneic unfractionated or G10-passed spleen cells. Control cells were left untreated. At the end of a 3-day culture period, Ag-exposed and control cells were harvested, allowed to rest in fresh medium for 0, 3, or 6 days, and exposed to a secondary stimulation by serial dilutions of the Ag in the presence of freshly isolated, costimulatory-bearing, splenic APC. As expected (27), T cells stimulated with APC lacking costimulatory functions were found unresponsive to antigenic challenge for at least 6–8 days, the latest experimental time tested (Fig. 2GoA, B, and C). Unexpectedly, we observed that cells prestimulated with Ag presented by adequate APC were hyporesponsive to Ag when tested on day 0 (at the end of the primary response; Fig. 2GoA) or after a 3-day resting period (Fig. 2GoB). Six days after the end of a productive, primary sensitization, A.E7 clones recovered the ability to proliferate in response to PCC, although with sometime reduced Ag sensitivity (note the shift in the dose-response curve in Fig. 2GoC).



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  		FIGURE 2. Functional consequences of adequate and inadequate primary T cell stimulation. A.E7 clones (106 cells/well in 24-well plates) were cultured from day -3 to day 0 with irradiated, unfractionated, or G10-passed spleen cells as a source of APC (107 cells/well) and PCC (1 µM) or medium. At day 0, viable cells were recovered. Aliquots of cells were restimulated in 96-well plates after a resting period ranging from a few hours to several days in fresh medium. Restimulations were performed 3 h (A), 3 days (B), or 6 days (C) after the end of the primary stimulation. A.E7 cells (5 x 104) recovered from each primary culture were cultured with freshly isolated syngeneic spleen cells (5 x 105/well) and serial dilutions of PCC. Proliferation was assessed on day 2 by the addition of [3H]thymidine for an additional 16 h.

 
To establish whether this unresponsive phase was a general characteristic of clonal populations, we derived four other T cell clones from two different strains of mice, as described in Material and Methods. Thus, a total of five clones, comprising Th0-, Th1-, and Th2-like cells, were stimulated by Ag and adequate accessory cells, rested for one day in complete media, and challenged for a secondary response.

As shown in Fig. 3Go, A–E, all clonal populations, irrespectively of their Th1/Th2 phenotype, were found to be Ag unresponsive when tested 1 day after the end of a primary stimulation. Th cell unresponsiveness was not a consequence of TCR down-modulation, as revealed by flow cytometry (data not shown), and unresponsive cells retained the ability to proliferate in response to rIL-2, indicating that the cells were viable and possessed a functional enzymatic machinery for cell division (Fig. 3Go, F–I). All tested clones spontaneously recovered their proliferative response to Ag 6–8 days after a primary stimulation (data not shown). Therefore, we refer to this refractory state as "activation-induced unresponsiveness."



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  		FIGURE 3. Unresponsive Th clones retain the ability to proliferate in response to IL-2. Th clones were preincubated for 3 days with total splenic APC in the presence or absence of Ag. The cytokine production during the primary sensitization of the OVA (323–339)-specific clones was: CLOVA 1.1 cells, 133 U/ml of IFN-{gamma}; CLOVA 1.4 cells, 133 U/ml of IFN-{gamma}; CLOVA 2.9 cells, 4701 U/ml of IL-5. Cytokine production of control cells was below the detection limits of the assay (6 U/ml and 1 U/ml for IFN-{gamma} and IL-5, respectively). Ag-exposed and control cells were restimulated 1 or 2 days (CLOVA cells and clones AE.7 and HG.4, respectively) after the end of the primary stimulation with (A–E) syngeneic APC and serial dilution of the respective Ag or (F–I) serial dilution of rIL-2. Proliferation was assessed on day 2 by the addition of [3H]thymidine for an additional 16 h.

 
Studies performed with anergic cells in vitro have shown that IL-2 production is extremely sensitive to the induction of unresponsiveness, while other cytokines (including IFN-{gamma} and Th2-derived cytokines) appear to be less affected (2, 28). Therefore, we wished to analyze the cytokine secretion levels in control and unresponsive Th cell clones. Ag-stimulation of the CLOVA 1.4 and CLOVA 2.9 clones led to a state of unresponsiveness characterized by reduced expression of all the cytokines tested (IL-4, IL-5, and IFN-{gamma}, see Fig. 4Go). Similarly, experiments performed with the A.E7 clone showed that the short-term unresponsive phase induced by optimal Ag-stimulation resulted in a severe reduction of IFN-{gamma} production (data not shown).



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  		FIGURE 4. Cytokine production by unresponsive Th clones. IL-5 (A and C), IFN-{gamma} (B), and IL-4 (D) production by control or preactivated CLOVA1.4 (Th0) (A and B) and CLOVA2.9 (Th2) clones (C and D). Cytokine contents were determined by ELISA and are expressed as mean of U/ml ± SD of duplicate cultures. ND, Below detection levels (13.99 U/ml for IL-5 and 1.2 U/ml for IFN-{gamma}).

 
Costimulatory signals prevent the induction of a long-term Th cell anergy but do not affect the activation-induced refractory phase

The A.E7 clone was stimulated with purified splenic DC, known to express high levels of the costimulatory molecules CD80 and CD86. As a control for anergy induction, the A.E7 clone was stimulated by Ag presented by purified, resting B cells. Fig. 5GoA shows that a primary stimulation with highly purified DC or with resting B cells led to Ag unresponsiveness, suggesting that activation-induced refractoriness could not be prevented by stimulation with APCs expressing high levels of costimulatory molecules. As previously shown, T cell clones stimulated with DC recovered the ability to proliferate in response to PCC on day 6, while clones stimulated by resting B cell displayed an anergic phenotype. In a second set of experiments, the A.E7 clone was stimulated with G10-depleted spleen cells in the presence of exogenous anti-CD28 mAbs. As expected from published reports (29) and data presented in Fig. 1Go, Abs to CD28 enhanced the clonal primary response, as judged by increased blastogenesis, IL-2 receptor expression, and proliferation (data not shown). However, anti-CD28 Abs added during the primary stimulation failed to restore a proliferative response when preactivated Th clones were rechallenged shortly after the primary stimulation (see Fig. 5GoB for one representative experiment of four). As expected, T cell clones pretreated with G10-passed APC and anti-CD28 mAbs were found to be immunocompetent on day 6 following a primary response, suggesting that anti-CD28 Abs were effective in preventing anergy induction in our experimental setting.



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  		FIGURE 5. T cell clone unresponsiveness is induced following antigenic stimulation despite the presence of costimulatory signals. A, A.E7 clones (2 x 104 cells/well in 96-well plates) were stimulated with purified DC (104 cells/ml) or resting B cells (2 x 105 cells/ml) and PCC peptide (81–104) 0.3 µg/ml. B, A.E7 clones were preactivated in the presence of PCC, unfractionated or G10-passed splenic APC, and anti-CD28 or control mAb as indicated. Control cells were cultured with splenic APC and control mAb. After a 1-day or 6-day resting period, the A.E7 clones were restimulated with splenic APC and 0.4 µM of PCC. Results are expressed as percent of untreated cell proliferative responses. The control cell response (100%) were (A) day 1, 87,075 cpm; day 6, 92,683 cpm; (B) day 1, 12,529 cpm; day 6, 68,393 cpm. The background proliferation (in the absence of Ag) was <1000 cpm in all populations.

 
To confirm at the clonal level that Th cells undergo an unresponsive phase following a productive response to Ag, cells of the CLOVA 1.4 clone were labeled with the fluorescent dye CFSE, which allows the tracking of cell divisions using flow cytometry (22), and stimulated in culture as described above. Cells were harvested after a primary response and a 1-day resting period, assayed in vitro for immunocompetence, and analyzed by flow cytometry. This analysis performed at the time of secondary stimulation revealed that the vast majority of cells present in the secondary cultures had undergone blastogenesis and cellular division (over 84%, as judged by cell size increase and reduced CFSE fluorescence, respectively) while displaying an unresponsive phenotype (Fig. 6Go).



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  		FIGURE 6. Induction of unresponsiveness follows cell divisions in vitro. CLOVA1.4 were loaded with CFSE and preincubated with irradiated splenic APC from BALB/c mice and 0.1 µM of OVA (323–339) or media. After 1 day of rest in fresh media, the control (A) and preactivated cells (B) were tested for their fluorescence content and cellular proliferation in response to serial dilutions of Ag (C).

 
T cell unresponsiveness is not a consequence of activation-induced cell death (AICD), defective TCR signaling, or CTLA-4 engagement

Lack of cytokine and proliferative responses by unresponsive Th cells could be the consequence of Ag-induced apoptosis/cell death during the course of the secondary stimulation. To test this hypothesis, untreated and preactivated A.E7 clones were exposed in vitro to PCC and splenic APC for 24 h. Following this culture period, wells containing the antigenic stimulus were supplemented with rIL-2. As shown in Fig. 7Go, unresponsive cells that had failed to respond to TCR stimulation were able to proliferate in response to IL-2 added 24 h following the TCR agonist, demonstrating that the secondary antigenic stimulation did not cause massive cell death. Accordingly, no decrease in cell viability was observed in secondary cultures upon Ag stimulation (<2% increase in propidium iodide staining in secondary cultures stimulated with Ag when compared with nonstimulated control cultures, data not shown).



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  		FIGURE 7. T cell clone unresponsiveness is not a consequence of cell death. Control and preactivated A.E7 clones were incubated in vitro with 0.1 µM of PCC or medium. After 24 h, rIL-2 (5 U/ml) was added in some cultures and proliferation was assayed at the end of a 48-h culture period as described.

 
Tyrosine phosphorylation of the TCR {zeta}-chain is one of the first intracellular events in response to TCR stimulation. We examined whether TCR stimulation resulted in phosphorylation of the TCR {zeta}-chain in unresponsive T cell clones. For this purpose, the CLOVA1.1 clone was stimulated by anti-CD3 mAbs. The TCR-CD3 complex was immunoprecipitated using an anti-CD3{epsilon}-chain mAb, resolved on reducing SDS-PAGE gel, and immunoblotted by antiphosphotyrosine mAb. Similar phosphorylation of the TCR {zeta}-chain occurred after anti-CD3 complex stimulation both in control and unresponsive cells (Fig. 8Go). Of note, anti-CD3 mAb-mediated stimulation of the pretreated CLOVA1.1 clones showed a marked inhibition in their proliferative response (data not shown). Moreover, the association between the TCR {zeta}-chain and the ZAP-70 tyrosine kinase and the mobilization of intracellular calcium occurred normally in unresponsive cells (data not shown). Collectively, these observations suggest that this form of T cell unresponsiveness is not simply the consequence of receptor desensitization.



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  		FIGURE 8. Normal TCR {zeta}-chain phosphorylation occurs in unresponsive T cell clones. Control and preactivated CLOVA 1.1 clones (4 x 107cells/ml) were stimulated for 90 s in the presence of 4 µg/ml of anti-CD3 mAbs and rabbit anti-mouse antiserum, as described in Materials and Methods. Cell lysates were immunoprecipitated with anti-CD3{epsilon} mAb. A, Immunoprecipitates were subjected to SDS-PAGE under reducing conditions and immunoblotted by the 4G10 antiphosphotyrosine mAb. B, Membranes were stripped and reblotted with anti-TCR {zeta}-chain Abs. Results are representative of three different experiments.

 
As T cell clone restimulations were performed in the presence of B7 expressing-APC, the possible involvement of CTLA-4 signaling in their postactivation-refractoriness was investigated. This was tested by adding saturating doses of anti-CTLA-4 mAbs during the rechallenge of the unresponsive CLOVA1.1 clones. As shown in Fig. 9Go, no significant increase in T cell responsiveness was observed in CLOVA1.1 clones restimulated with anti-CTLA-4 mAbs. Of note, the same preparation of the anti-CTLA-4 mAb did augment the proliferative response of naive T cells in a primary MLR (Fig. 9GoB). Therefore, these data suggest that signaling through the CTLA-4 receptor is not responsible for the induction of T cell clone unresponsiveness in this experimental system.



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  		FIGURE 9. Activation induced unresponsiveness is not due to CTLA-4 engagement. A, Control and preactivated CLOVA 1.1 clones were incubated in vitro with splenic APC, 0.1 µM of OVA (323–339), and 100 µg/ml of control Ab or anti-CTLA-4 mAb (clone 4F10). Proliferation was assayed at the end of a 48-h culture period as described. B, A total of 2 x 105 spleen cells from BALB/c mice (effector cells) were incubated in vitro with 2 x 105 irradiated T-depleted spleen cells from CBA mice (stimulator cells) in MLR in the presence of 100 µg/ml of 4F10 mAb or control Ab. Results represent the mean ± SD of triplicate cultures.

 
Memory, but not naive, T cells are sensitive to Ag-induced unresponsiveness

To test whether all T cell populations were sensitive to this form of Ag-induced unresponsiveness, naive CD4+ cells isolated from DO11.10 transgenic mice were stimulated by the OVA peptide in the presence of splenic APC. After a 3-day culture period, viable CD4+ T cells were recovered, rested 1 day in fresh medium, and rechallenged in vitro with serial dilutions of OVA and freshly purified syngeneic APC. In marked contrast to previously characterized clonal populations, Ag-stimulated naive T cells displayed a vigorous response upon secondary Ag exposure in vitro (Fig. 10GoA). This enhanced secondary response by Ag preactivated cells could not be simply explained by an increase in the frequency of cells bearing the transgenic receptor because the proliferative response was normalized according to the frequency of OVA-specific TCR transgenic-bearing cells present in both groups (Fig. 10GoB). Moreover, cell-labeling experiments using CFSE demonstrated that the vast majority of OVA-reactive cells had undergone cell division during the primary response (as shown by increased cell size and decreased cell fluorescence of activated cells, Fig. 10GoD, compared with unstimulated cells, Fig. 10GoC), suggesting that the enhanced secondary response was mediated by cells activated during the primary culture. To test whether CD4+ cells sensitive to Ag-induced unresponsiveness could be generated by a short-term culture, naive DO11.10 cells were stimulated in vitro by the OVA peptide and APC to induce their differentiation in memory-type cells. After three rounds of biweekly stimulations, DO11.10-derived cells were found to express higher levels of the CD44 memory marker (Fig. 10GoF) and to produce both Th1- and Th2-type cytokines upon stimulation (data not shown). In vitro stimulation of these cells according to the protocol used throughout this study revealed that this memory-type T cell line was sensitive to the postactivation refractory phase (Fig. 10GoE). Collectively, those results suggest that only memory-type helper cells undergo a transient unresponsive phase upon productive Ag stimulation.



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  		FIGURE 10. Secondary stimulations in naive and in vitro-derived memory Th cells. A and B, Purified T cells from DO11.10 transgenic mice were cultured for 3 days with splenic APC and 0.1 µM of OVA (323–339) and rested for 1 day in fresh medium. Aliquots of viable cells recovered from the primary culture and freshly isolated purified T cells were stained with the KJ1.26 mAb and anti-CD4 mAb to estimate the number of the clonotype-expressing Th cells in the cultures. The cells were restimulated (105/well) with freshly isolated syngeneic spleen cells (5 x 105/well) and serial dilutions of OVA (323–339). A, Total proliferative response of the naive and prestimulated populations. B, Proliferative response corrected for 1000 KJ1.26+ T cells. C and D, Spleen cells from DO11.10 transgenic mice were loaded with CFSE and stimulated with 0.25 µM OVA (323–339) (D) or media (C). Results represent the flow cytometric analysis (CFSE fluorescence vs forward scatter) of KJ1.26+ cells. Ag-primed cells displayed an enhanced secondary proliferative response when compared with naive cells (not shown). E, Memory-type CD4+, KJ1.26+ cell line was derived from naive DO11.10 splenocytes following three biweekly in vitro stimulations in the presence of APC and Ag. Viable cells were recovered and prestimulated for 3 days with splenic APC alone or in the presence of 0.25 µM of OVA (323–339). Restimulations were performed on day 1 as for the Th clones. F, Cell-surface staining of naive and in vitro-derived memory-type Th cells with FITC labeled anti-CD44 mAbs. Solid line, naive T cells; short dots, in vitro-derived memory T cells; long dots, T cells stained with an isotype-matched control Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous studies have suggested that excessive immune responses, although directed at nonself-Ags, represent potential threats to the organism and therefore need to be regulated in an appropriate fashion. Deregulated, immune-mediated destruction of healthy host tissues can indeed occur during infections with virus (30, 31) or in response to bacterial superantigens (32). The present study demonstrates that Ag-experienced T lymphocytes cannot respond to Ag in a repetitive fashion. Indeed, stimulation of murine Th clones and lines by Ag presented by adequate accessory cells induces T cell activation (characterized by cell proliferation and lymphokine production), which is followed by a transient state of unresponsiveness to Ag. This unresponsive state, which we have tentatively termed "activation-induced unresponsiveness," may represent a mechanism contributing to the homeostasis of the immune system.

This form of unresponsiveness is not readily accommodated within the framework of previously described in vitro models of T cell anergy. Indeed, first, T cell clones become refractory to Ag stimulation following a proliferative in vitro response to an agonist stimulus (Fig. 6Go). Unresponsiveness in this model is not a consequence of inadequate, costimulatory-deficient stimulation, as neither highly purified DC nor the addition of saturating amounts of anti-CD28 mAbs were able to counteract the development of hyporesponsiveness (Fig. 5Go). Second, the activation-induced refractory state is characterized by defective IFN-{gamma} production, while secretion of this cytokine is only marginally affected in anergic cells (2). Third, unresponsiveness secondary to optimal activation is characterized by the down-regulation of both Th1 and Th2 cytokines secretion (Fig. 4Go), whereas anergy results in a selective reduction in IL-2 production, often leaving Th2-type cytokines secretion unaffected (28). Finally, T cells stimulated in a productive fashion recover Ag responsiveness after a rest period of 3–6 days, while partial activation generally leads to long-term unresponsiveness and/or apoptosis. Collectively, these observations indicate that activation-induced unresponsiveness and anergy represent distinct mechanisms for down-regulating T cell reactivity to Ag.

Under appropriate experimental settings, stimulation of proliferating T cells has been shown to result in cell death by apoptosis, a process referred to as AICD (33). Care was taken to exclude the possibility that the lack of proliferation seen upon restimulation of T cell clones in this system was due to the induction of cell death. No increase in apoptotic cells was seen upon Ag restimulation, (our unpublished observations), and unresponsive cells were found to respond to IL-2, even if this cytokine was added 24 h after Ag rechallenge (Fig. 7Go). In keeping with our observations, it has been shown that the A.E7 clone is resistant to AICD induced by Ag stimulation, and that Ag-induced cell apoptosis is only observed in cells that were pretreated with high doses of exogenous IL-2 before Ag exposure. In particular, it has been established that IL-2 produced by the A.E7 clone in response to Ag and APCs does not program T lymphocytes for apoptosis (34). Finally, and in contrast to AICD, Ag-induced unresponsiveness 1) affects both Th1- and Th2-like cells; 2) is a transient phenomenon, as unresponsive cells reacquire full immunocompetence a few days after the Ag withdrawal; and 3) is not associated with altered tyrosine phosphorylation of TCR/CD3 subunits (35).

The activation-induced refractory phase does not appear to be a consequence of CTLA-4 ligation, as the addition of an adequate concentration of blocking anti-CTLA-4 Abs (known to augment the level of proliferation of naive T cells to alloantigen) did not antagonize the development of hyporesponsiveness (Fig. 9Go).

The unresponsive cells expressed comparable levels of TCR, CD3, and CD4 molecules, suggesting that unresponsiveness in this model could not be attributed to an altered expression of these molecules. Although the characterization of the molecular defect associated with this form of unresponsiveness was beyond the scope of this study, our observations suggest that the early steps of TCR signal transduction were not affected (Fig. 8Go and data not shown). Further analysis will be required to determine whether unresponsive cells share or not with anergic clones increased Fyn kinase activity (36), high Rap1-GTP content (37), and/or impaired TCR-mediated activation of extracellular signal-related kinase and c-Jun NH2-terminal kinase (38, 39).

A major observation from this study is that naive T cells are not sensitive to activation-induced T cell unresponsiveness, as naive T cells from TCR transgenic mice proliferate vigorously to a secondary Ag-stimulation (Fig. 10Go). The observation that the majority of clonotype-positive cells proliferate during primary sensitization and the fact that the secondary response is of stronger avidity/magnitude (note both the shift in the doses-response curve and the increase thymidine incorporation at high Ag doses) strongly argue against the possibility that secondary responses are mediated by cells that failed to respond during primary Ag exposure. Although we cannot exclude that enhanced secondary responses in this model reflect the loss of a cell population with regulatory properties, these data demonstrate that primary lymphocytes do not become unresponsive following in vitro stimulation. The present findings are compatible with numerous publications describing in vitro alloreactive secondary responses. Indeed, lymphocytes primed to alloantigens in mixed cultured (MLR), display a strong secondary response upon re-exposure to the same alloantigen in vitro (see as an example Ref. 40). However, in keeping with the behavior of clonal cell lines, Ag-experienced cells become gradually susceptible to activation-induced unresponsiveness following in vitro Ag stimulation (see Fig. 10GoE). This suggests that activation-induced unresponsiveness does not simply reflect cellular desensitization, but rather represents a regulatory mechanism that constrains memory T cell proliferation to repeated Ag exposure.

The interest in this phenomenon lies in its possible in vivo relevance. The immune system depends greatly on clonal expansion for amplification of any response to become effective. Ag-specific naive T cells, present at low frequency in unprimed animals, may therefore need to undergo multiple rounds of division in response to Ag to generate an adequate number of regulatory/effector cells. In contrast, the proliferation to Ag of memory/effector cells may need to be tightly regulated to avoid excessive in vivo secondary responses, possibly leading to inflammation-related self-injury. In support of this conclusion, induction of in vivo T cell unresponsiveness often requires repetitive administration of soluble Ag (41).

Therefore, we propose that T cell unresponsiveness secondary to Ag-stimulation in the presence of costimulatory functions represent an intrinsic feedback regulatory mechanisms limiting the expansion of memory/effector T cells in the continuous presence of Ag. Further studies are required to help elucidate the biochemical basis of this form of unresponsiveness.


   Acknowledgments
 
We thank G. Dewasme, P. Veirman, and M. Swaenepoel for technical assistance and E. Baus for critical review of the manuscript.


   Footnotes
 
1 This work was supported by the Belgian Program in Interuniversity Poles of Attraction initiated by Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsibility is assumed by its authors. F.D. and F.V.L. are supported by the Fonds pour la Recherche dans l’Industrie et l’Agriculture (Belgium), M.M. is supported by the Fonds National pour la Recherche Scientifique (Belgium). Back

2 Address correspondence and reprint requests to Dr. Fabienne Andris, Laboratoire de Physiologie Animale, Université Libre de Bruxelles, Rue Pr. Jeener et Brachet 12, 6041 Gosselies, Belgium. E-mail address: Back

3 Abbreviations used in this paper: PCC, pigeon cytochrome c; DC, dendritic cell; CFSE, carboxyfluorescein succinimidil ester; AICD, activation-induced cell death. Back

Received for publication April 9, 1999. Accepted for publication September 17, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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