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The Journal of Immunology, 1999, 163: 5219-5227.
Copyright © 1999 by The American Association of Immunologists

Differential Requirements for NF-{kappa}B and AP-1 trans-Activation in Response to Minimal TCR Engagement by a Partial Agonist in Naive CD8 T Cells1

Nathalie Auphan2,*, Sankar Ghosh{dagger}, Richard A. Flavell{dagger} and Anne-Marie Schmitt-Verhulst*

* Centre d’Immunologie, Institut National de la Santé et de la Recherche Médicale- Centre National de la Recherche Scientifique (INSERM-CNRS) de Marseille-Luminy, Marseille, France; and {dagger} Section of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06517


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the basis for partial reactivity of naive CD8 T cells expressing an alloreactive transgenic TCR in response to a mutant alloantigen. When unstimulated APCs were used, IFN-{gamma} as well as IL-2 and cell proliferation were observed in response to wild-type Ag, whereas mutant Ag induced only IFN-{gamma}. DNA binding and reporter gene assays showed that the response to mutant Ag involved NF-{kappa}B, but not AP-1 activation, whereas wild-type Ag activated both transcription factors. Increasing the contribution of costimulatory signals by using LPS-activated APCs partially corrected the activation by mutant Ag, because proliferation and weak IL-2 production could be measured. This also led to AP-1 activation, albeit with delayed kinetics, in response to mutant Ag. To explain how engagement of the same TCR by distinct ligands results in different T cell responses, it may be proposed, in line with models stressing the importance of the kinetics of Ag/TCR interaction, that two types of signals be distinguished: a "fast" short-lived signal is sufficient to activate NF-{kappa}B; whereas a "slow" signal obtained after prolonged TCR engagement is required for AP-1 activation. Failure to activate AP-1 in limiting conditions (unstimulated mutant APC) was partially corrected by increasing costimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcells recognize through their specific TCR short antigenic peptides in association with MHC molecules (1, 2, 3). More recently, it has become clear that a particular TCR can interact with more than one ligand, especially those displaying subtle amino acid substitutions as compared with the initial agonist (4). These small changes in the peptide ligand can alter responses of the T cells. Indeed, responses to these altered peptide ligands (APL)3 range from stimulation of some, but not all, of the TCR-induced functions, corresponding to partial activation (5), to abrogation of T cell responses, exemplified by anergy induction (6) or TCR antagonism (7). Several studies have indicated that the pattern of T cell responses toward APLs is a consequence of differential TCR-mediated signaling. This included altered TCR-{zeta} phosphorylation which was arrested at an intermediate stage (8) and a subsequent abrogation of signal transduction including phosphorylation of ZAP-70 (9, 10) and LAT (11). In addition to these qualitative differences, quantitative parameters such as the degree of TCR engagement must be considered. This notion came from studies arguing that T cells counted the number of engaged TCRs and responded when a certain threshold was reached (12). To determine this activation threshold, not only the number of engaged TCRs but also the duration of this engagement was shown to be important (13). In this context, both coreceptors (CD4 (14) and CD8 (15, 16)) and costimulating molecules (CD28 (12, 17)) participated in lowering TCR requirements to sustain the activation threshold.

The outcome of APL recognition is the induction of a differential functional T cell program that relies on a selective transcriptional activation of a particular set of lymphokine (18)- or cytotoxic mediator (19, 20)-encoding genes. These observations raise the question of the nature of the differential induction of transcription factor activities by partial agonists. One of the genes that has been shown to be negatively regulated in anergized T cell clones is the IL-2 gene (21). Its transcription is controlled by a number of factors, including NF-{kappa}B and AP-1 (22). AP-1 is composed mainly of members of the Fos and Jun families of bZip proteins, but newly described bZip proteins can associate with those AP-1 components (reviewed in Ref. 23), increasing the number of possible dimers capable of interacting with AP-1 sites. The complexity of transcriptional regulation mediated by AP-1 is further increased by the independent modulation of DNA-binding and transcriptional activities. In in vitro-derived unresponsive T cell clones, AP-1 activation has been shown to be defective (24). However, the molecular mechanism underlying unresponsiveness or partial reactivity of naive T lymphocytes has not been addressed.

In the present study, we compared trans-activation in naive CD8+ CTL precursors responding to either a full or a partial agonist. We found that inefficient IL-2 synthesis in response to a partial agonist correlated with an abortive AP-1 activation. Moreover, our work showed that a minimal TCR engagement, not sufficient to induce detectable TCR down-modulation, may be capable of stimulating cytotoxic effector differentiation, IFN-{gamma} production, and NF-{kappa}B trans-activation, whereas this activation threshold must be supplemented by costimulatory components to allow IL-2 production, up-regulation of activation markers, and AP-1 trans-activation. Therefore, our study suggests for the first time that a differential transcriptional control may account for the distinct functional pattern induced in naive CD8+ T cells by a full vs a partial agonist.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Mice transgenic for the BM3.3 TCR (25) on the CBA/Ca background (tgTCR), C57BL/6 (B6), and C57BL/6.C-H-2bm8 (bm8) mice were bred in the Centre d’Immunologie, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique de Marseille-Luminy animal facility. Mice transgenic for AP-1-luciferase (26) and NF-{kappa}B -luciferase (R. J. Phillips, M. Rincon, R. A. Flavell, and S. Ghosh, manuscript in preparation), backcrossed on the B10.BR strain, were crossed with the tgTCR mice at the Centre d’Immunologie, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique (INSERM-CNRS) de Marseille-Luminy animal facility (16).

Flow cytometric analyses

Reagents used for immunofluorescence staining were: biotin-mAb Ti98, an anticlonotypic mAb specific for the BM3.3 TCR (27) and FITC-anti-CD69 (H1.2F3 (28)), both conjugated in the laboratory, FITC-anti-CD25 and APC-anti-CD8{alpha} (PharMingen, Becton Dickinson, Mountain View, CA). After staining, 2 x 104 viable cells in each sample were analyzed on a FACScan cytofluorometer (Becton Dickinson).

Cell purification, culture conditions, and functional assays

CD8+ cells were purified from lymph nodes of tgTCR mice by negative selection using rat anti-CD4 mAb supernatant (H129.19.6 (29)) and a mix of both anti-mouse and anti-rat IgGs Dynabeads (Dynal, Oslo, Norway). In all experiments, CD8+ T cells represented 90–98% of the enriched population. These CD8+ tgTCR+ cells were plated in microplates at 105 cells/well together with 2 x 105 irradiated stimulating cells, in triplicates. [3H]Thymidine incorporation was assessed after a pulse with 1 µCi/well. To determine lymphokine production, 100 µl of culture supernatant were harvested 48 h after stimulation and used to measure IL-2 secretion by evaluating its ability to sustain the proliferation of an IL-2 dependent T cell line (CTL.L) as described (30). To measure tgTCR, CD25, and CD69 cell surface modulation, 2 x 105 purified CD8+ cells were cultured in duplicates with 4 x 105 APCs for the indicated time. Immunofluorescence staining was then performed directly in the microplates. When indicated, in vitro cultures were conducted in the presence of one of the following rat mAb: a neutralizing anti-IL-2 (JES6, PharMingen, 4 µg/ml), anti-CD4 (H129.19.6 (29), 4 µg/ml), anti-ICAM-1 (BE29G1 (31), 10 µg/ml), anti-B7.2 (GL1 (32), 5 µg/ml), anti-B7.1 (1G10, PharMingen, 5 µg/ml) or of CTLA4-Ig (5 µg/ml) produced as described (33). Activated APCs were obtained by stimulating splenocytes for 24 h with 15 µg/ml LPS (Sigma, St. Louis, MO).

5(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) and propidium iodide stainings

Determination of number of T cell divisions was done by flow cytometry as described (34) using the cytoplasmic dye CFSE, that was shown to exhibit sequential halving of intracellular fluorescence intensity at each division step (34). Purified CD8+ tgTCR+ cells were incubated for 10 min at 37°C with 5 µM CFSE (Molecular Probes, Eugene, OR). After two washes, labeled cells were stimulated in duplicates for 48 h as described above. Cell cycle determination was done by flow cytometry after staining with propidium iodide. A 2 µg/ml solution of propidium iodide (Sigma) was used to resuspend ethanol-fixed cells.

Luciferase activity analysis

Lymph node cells were recovered from double tg (TCR x AP-1/luciferase) or (TCR x NF-{kappa}B/luciferase) mice, and 106 cells were cultured with 2 x 106 stimulating cells as indicated or with PMA, 10 ng/ml, and ionomycin, 200 ng/ml, for the indicated time period in duplicates. At each time, cells were harvested and lysed in lysis buffer (luciferase assay, Promega, Madison, WI), and luciferase activity was measured with the luciferase reagent (Promega) and a luminometer (Lumat LB96P, Berthold, Nashua, NH).

Cell extract preparation and electrophoretic mobility shift assay (EMSA)

Cells (4 x 106) were cultured with 8 x 106 stimulating cells in 6 ml culture medium for different periods of time. To remove APCs, cell suspensions were first incubated with anti-I-Ak (when CBA APCs were used) or anti-H-2Kb (when B6 or bm8 APCs were used) mAb, in PBS supplemented with 1% FCS and 5 mM EDTA, and secondly with anti-mouse IgG Dynabeads. Cell extracts were prepared as previously reported (35) and 5-µg protein samples were incubated with AP-1 or NF-{kappa}B binding site probes (see sequences in Ref. 36). Protein-DNA binding was analyzed by EMSA as described (36). For supershift, Abs specific for Jun or Fos proteins were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lack of proliferation after stimulation by a partial agonist can be to some extent restored by costimulatory components

We have previously shown that for CD8+ T cells expressing an anti-H-2Kb tgTCR, H-2Kbm8 behaves like a partial agonist, efficient for the induction of cytolytic function and IFN-{gamma} production but inefficient for the triggering of cell proliferation in vitro due to a lack of IL-2 secretion (see Ref. 16 and Table IGo). H-2Kbm8 presents four amino acid substitutions in the ß-pleated sheets of the {alpha}1 domain of H-2Kb, that essentially affect MHC class I-peptide interactions (37). Whether these mutations in the peptide groove of H-2Kbm8 may affect the presentation of the same endogenous peptide which also binds to H-2Kb (A. Guimezanes et al., manuscript in preparation) or lead to presentation of a distinct endogenous peptide that forms a complex with H-2Kbm8, for which the tgTCR is cross-reactive, is currently being investigated.


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  		Table I. Restored proliferation induced by bm8 ligand on activated APCs is inhibited by blocking reagents toward costimulatory components

 
In our previous study (16), we used unstimulated splenocytes as APCs, which poorly expressed costimulating (B7.1 and B7.2) and adhesion (ICAM-1) molecules (see Fig. 1Go). Here, we wondered whether costimulatory components may supplement the defect in T cell activation mediated by the recognition of a partial agonist. For this purpose, we used as stimulating cells, LPS-activated splenocytes that have up-regulated both B7.2 and ICAM-1 surface expression (Fig. 1Go). Such activated bm8 APCs efficiently induced tgTCR+ CD8+ cell proliferation at a level comparable with that observed with unstimulated B6 APCs (Fig. 2GoA). To evaluate whether the up-regulated B7 and/or ICAM-1 molecules contribute to this proliferative response to the partial agonist, relevant Abs or the soluble ligand (CTLA4-Ig) of B7.1 and B7.2 molecules were tested for potential blocking during tgTCR proliferation in response to activated bm8 APCs (Table IGo). The results showed that either ICAM-1 or B7.1/B7.2 blocking alone only partially inhibited proliferation, which was totally obliterated, however, when reagents blocking all three molecules were present. In the same conditions, blocking of ICAM-1 and B7.1/B7.2 only partially inhibited the response to the agonist on activated APCs (Table IGo). These results suggest that in limiting conditions of TCR stimulation (partial agonist), either ICAM-1 or B7.1/B7.2 molecules could contribute to the proliferation inducing signal.



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  		FIGURE 1. LPS-activated splenocytes showed up-regulated levels of costimulating (B7.2)-, adhesion (ICAM-1)-, and MHC class I molecules. Immunofluorescence staining of bm8 splenocytes activated by LPS for 24 h. The same phenotype was observed for splenocytes of the different mouse strains.

 


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  		FIGURE 2. Presentation of the partial agonist by activated APCs restored CD8+tgTCR+ T cell proliferation and entry into the S phase of the cell cycle. Proliferation (A) and cell cycle progression (B) by tgTCR+CD8+ T cells were assessed after in vitro stimulation by either unstimulated or LPS-activated (*) APCs of H-2b (B6), H-2bm8 (bm8), or H-2k (B10. BR) haplotype. A, Cultures were conducted in the absence of any mAb ({blacksquare}), or in the presence of an anti-IL-2 mAb ({blacktriangleup}), or of an isotype control mAb (anti-CD4) ({square}).

 
The difference in proliferation rates induced by the partial agonist bm8, in the context of unstimulated vs activated APCs, was further analyzed. First, the number of cell divisions was estimated with the use of the fluorescent cytoplasmic dye CFSE (see Materials and Methods), and results shown in Table IIGo confirmed the lack of proliferation on encounter of the partial agonist bm8 presented by unstimulated APCs. However, this altered ligand was able to trigger tgTCR+ cell division in the context of activated APCs, albeit with a slightly weaker efficiency than the native Ag H-2Kb, that may reflect differences in the kinetics of the response (see below). Second, the progression in cell cycle was monitored after DNA labeling with propidium iodide (Fig. 2GoB), and revealed that the agonist H-2Kb was driving entry into the S phase as early as 25 h after initiation of the stimulation. In contrast, during 48 h tgTCR+ cells remained in the G0/G1 phase when cultured with unstimulated bm8 APCs, and only a moderate proportion of tgTCR+ cells entered the S phase when activated bm8 APCs were used. However, when secreted IL-2 was measured in the supernatant of the same in vitro culture, no excess of IL-2 was detected (Table IIGo), suggesting either 1) that all the secreted IL-2 was consumed by proliferating tgTCR+ cells and that in contrast to the agonist stimulation (B6), the partial agonist (bm8) still induced a limited production of IL-2, or 2) that the restored proliferation was IL-2 independent. To discriminate these two hypotheses, thymidine incorporation assays were performed in the presence of neutralizing anti-IL-2 Ab. Calibration with the IL-2-dependent T cell line CTL.L allowed us to determine that 100% inhibition of proliferation was obtained when up to 6 U/ml recombinant IL-2 were used to sustain CTL.L growth (data not shown). When the anti-IL-2 Ab was added to in vitro cultures of tgTCR+ cells, it was able to partially inhibit the restored proliferation observed with the altered ligand bm8 on activated APCs (Fig. 2GoA), suggesting that this cell growth went at least partially through an IL-2-dependent pathway. Consistent with this, assays measuring IL-2 promoter trans-activation in transgenic mice showed a measurable trans-activation in response to bm8-activated APCs, albeit 50-fold lower than in response to B6 counterparts (results not shown). However, the possible implication of another cytokine responsible for the remaining proliferation has to be further analyzed. In the same way, proliferation induced by the agonist H-2Kb was also partially inhibited by the anti-IL-2 Ab.


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  		Table II. Inefficiency of a partial agonist to drive entry into cell cycle, thymidine incorporation, and cell division is partially corrected by costimulation

 
Failure of a partial agonist to up-regulate activation markers may be partially compensated by costimulatory components

Since H-2Kbm8 presented on unstimulated APCs failed to induce tgTCR+ cell proliferation but remained efficient for the induction of cytolytic function and IFN-{gamma} production (16), we wondered whether such partially activated tgTCR+ cells would up-regulate markers that are considered as a hallmark of activation such as CD69, CD44, and CD25. As shown in Fig. 3GoA, stimulation by the full agonist induced a rapid up-regulation of CD69 on nearly 100% of tgTCR+ cells at 20 h. In this case, the level of CD69 is further increased when H-2Kb is presented on activated APCs (in Fig. 3Go, means of fluorescent intensity are reported in parentheses). The partial agonist did not induce either CD69 expression when unstimulated APCs were used (as measured up to 20 h) or CD44 (data not shown). However, when tgTCR+ T cells were stimulated for a longer period of time (48 h), ~30% of them had slightly up-regulated CD69 (data not shown). When activated bm8 APCs were used, the activation of tgTCR+ cells was more efficient because 60% were CD69+ as early as 12 h after the initiation of the stimulation. Nevertheless, this percentage never exceeded 60% even when stimulation continued for longer periods.



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  		FIGURE 3. CD69 and CD25 expression were slightly induced on stimulation by the partial agonist expressed on activated APCs. The percentages of CD69 (A)- and CD25 (B)-positive cells were measured by immunofluorescence staining before (hatched diamond) or after in vitro stimulation with unstimulated (closed symbols) or activated (*) (open symbols) APCs of H-2b (B6, circles), H-2bm8 (bm8, squares) or H-2k (B10. BR, triangles) haplotype. For each point, the mean of fluorescence intensity is reported in parentheses. C, Cultures were conducted in the presence of an anti-IL-2 mAb (open bars) or of an isotype control mAb (anti-CD4) (hatched bars).

 
It was also interesting to define the conditions required for the up-regulation of the {alpha}-chain of the IL-2 receptor (CD25), as it is known to be regulated synergistically by TCR- and by IL-2-signaling pathways (38). Whereas stimulation with the agonist (B6) rapidly led to the up-regulation of CD25 on nearly all the CD8+tgTCR+ cells, <30% of them expressed CD25 on presentation of the partial agonist bm8 by unstimulated APCs, and this level never exceeded 60% with activated bm8 APCs (Fig. 3GoB). Thus, whereas in the absence of costimulation, the partial agonist bm8 barely induced the up-regulation of CD69 and CD25, it appeared to trigger more efficiently the induction of these activation markers when expressed on activated APCs.

In addition to an enhanced expression of costimulating and adhesion molecules, LPS-activated APCs also expressed increased levels of MHC class I molecules (Fig. 1Go) which may consequently increase the number of specific MHC/peptide complexes. Both parameters may play a role, individually or synergistically, to partially compensate for the defect in IL-2 induction and T cell activation by a partial agonist.

Inability of a partial agonist to induce tgTCR down-modulation

TCR down-modulation has been suggested to reflect the number of engaged TCRs and therefore to be an appropriate way to measure signal 1 (39). We thus compared tgTCR surface expression after triggering with full or partial agonists (see Fig. 4Go). Because APCs used in this study present the endogenously generated peptide, i.e., at low concentration, late kinetics of TCR down-modulation were analyzed first. Data in Fig. 4GoA show that a clear down-modulation of the TCR was observed after 12 and 24 h in response to B6 whether APCs were activated or not, but not in response to bm8. At shorter time points and when TCR resynthesis was prevented by the protein synthesis inhibitor cycloheximide, no TCR down-modulation was observed in response to bm8 APCs, whereas it was significant in response to B6 APCs (Fig. 4GoB). Therefore, even in the presence of activated APCs which up-regulated surface expression of MHC molecules, TCR engagement mediated by the partial agonist H-2Kbm8 seemed to be minimal. This result argues that the partial recovery of tgTCR+ cell proliferation observed on stimulation by activated bm8 APCs is more likely due to costimulation pathways that may lower the number of engaged TCRs required to reach the activation threshold rather than to an increase in the number of TCRs interacting with the up-regulated MHC class I/peptide-specific partners. This is also consistent with the inhibition of proliferation by agents blocking costimulatory events (Table IGo).



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  		FIGURE 4. Inability of the partial agonist to induce tgTCR down-modulation. A and B, tgTCR surface expression was measured by immunofluorescence staining with an anti-clonotypic mAb, after an in vitro culture in medium alone (open diamond) or with unstimulated (closed symbols) or activated (*) (open symbols) APCs of H-2b (B6), H-2bm8 (bm8), or H-2k (B10. BR) haplotype. B, The entire experiment was conducted in the presence of 10 µg/ml cycloheximide.

 
Different requirements for NF-{kappa}B and AP-1 trans-activation in response to partial agonist

The lack of IL-2 secretion after triggering by the partial agonist H-2Kbm8, together with efficient IFN-{gamma} induction, suggested that specific regulation may occur at the transcriptional level and prompted us to analyze the ability of such altered ligand to activate transcriptional factors such as AP-1 and NF-{kappa}B. To this end, we used mice transgenic for a luciferase reporter under the control of two DNA binding sites for AP-1 or NF-{kappa}B, that we crossed with the tgTCR mice. Peripheral CD8+ T cells were purified from these double tg mice and stimulated in vitro. Whereas the full agonist readily induced AP-1 trans-activation when unstimulated B6 splenocytes were used as APCs, the bm8 counterparts were inefficient. When the agonist was expressed at the surface of activated APCs, the kinetics of the AP-1-mediated response was unchanged, but the level of trans-activation was markedly enhanced. However, when the response to the partial agonist was tested in the same conditions, we observed an efficient but delayed AP-1 trans-activation stimulated by activated APCs (Fig. 5GoA). In contrast, NF-{kappa}B -mediated transcription was activated by both full or partial agonists, even presented by unstimulated APCs (Fig. 5GoB). The prolonged activation of NF-{kappa}B in response to the partial agonist is compatible with a slight delay in the response to the partial agonist as compared with the agonist (as observed at the 24-h point; results not shown). trans-Activation by AP-1 and NF-{kappa}B can be influenced by the state of phosphorylation of, respectively, c-Jun (23) and p65 (40), which may not affect DNA binding of these transcription factors. It was thus possible that transcription factor DNA binding could occur in the absence of detectable trans-activation. For this reason, we further analyzed the level of AP-1- and NF-{kappa}B-DNA binding after T cell activation (Fig. 6GoA). We visualized a reduced AP-1 binding after bm8 as compared with B6 activation, whereas both types of stimulation induced a comparable NF-{kappa}B-DNA binding. However, an efficient but delayed AP-1-DNA binding could be observed when bm8-stimulated APCs were used (Fig. 7GoA), these kinetics being compatible with the results reported above at the transcriptional level. The nature of the AP-1 components induced by an efficient antigenic stimulation (B6) was analyzed in supershift experiments (Fig. 6GoB) and revealed that all AP-1 binding complexes contained a Fos family member as shown by the abrogation of AP-1 binding on addition of a pan anti-Fos mAb. In contrast, only a slight decrease and supershift of AP-1 binding was seen after incubation with a pan anti-Jun mAb. This observation should be qualified by the fact that the latter reagent has a weaker activity in supershift experiments than the former (A. K. Simon and N. A., unpublished data). Nevertheless, the use of specific anti-c-Jun, -JunB, and -JunD mAb revealed that c-Jun and JunB were less represented in AP-1 binding complexes than JunD. Therefore, the lack of AP-1 binding after triggering by the partial agonist in conditions of limiting costimulatory components may reflect a defect in the induction of Fos and Jun members, as also reported for anergic CD4+ T cells (41, 42). AP-1 complexes detected after 42 h in response to bm8 on activated APCs also contained Fos and JunD (Fig. 7GoB). Furthermore, it appeared that a band that does not contain proteins of the Fos family may be more represented in the extract from bm8-stimulated tgTCR T cells. Whether this lower band corresponds to complexes containing only Jun family members or additional components and whether it has any functional relevance requires further investigation.



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  		FIGURE 5. Different requirements for NF-{kappa}B or AP-1 trans-activation in response to partial agonist. CD8+ T cells from double tg mice expressing both the tgTCR and the luciferase reporter gene under the control of either AP-1 (A) or NF-{kappa}B (B) were cultured in vitro with unstimulated (B and closed symbols in A) or activated (open symbols in A) APCs of H-2b (B6, circles in A), H-2bm8 (bm8, squares in A) or H-2k (CBA, triangles in A) haplotype. For AP-1, a polyclonal stimulation by ionomycin + PMA was included (hatched triangles in A).

 


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  		FIGURE 6. Different requirements for induction of NF-{kappa}B or AP-1 DNA binding in response to partial agonist. DNA binding was analyzed by EMSA with the use of NF-{kappa}B (A) and AP-1 (A, B) specific oligonucleotides. CD8+tgTCR+ cells were cultured with unstimulated APCs for 24 h, and supershifts using anti-Fos or Jun mAb are shown in B. Numbers on graphs correspond, respectively, to: 1, CBA; 2, B6; 3, bm8 unstimulated APC; and 4, ionomycin + PMA stimulation. Full arrow, position of the NF-{kappa}B or AP-1 complexes; stippled arrows, position of the mAb supershifted complexes.

 


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  		FIGURE 7. Contribution of costimulatory components to induction of AP-1 DNA-binding activity in response to partial agonist. A, Kinetics of AP-1 induction after stimulation of tgTCR+CD8+ T cells with LPS-activated APCs; B, supershifts using anti-Fos or Jun mAb in extracts of such T cells after 42 h stimulation. DNA binding was analyzed by EMSA using AP-1-specific oligonucleotides. Numbers on graphs correspond, respectively, to: 1, CBA*; 2, B6*; 3, bm8* LPS stimulated APC; and 4, ionomycin + PMA stimulation. Arrows are as in Fig. 6Go.

 
Most importantly, the point developed in this study is that in limiting conditions of costimulation, a situation of partial activation can lead to the selective trans-activation by NF-{kappa}B in the absence of AP-1 that is correlated with the DNA binding of these two factors.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results extended our earlier studies identifying H-2Kbm8 presented on unstimulated APCs as a partial agonist, efficient to induce IFN-{gamma} secretion and cytolytic function (16), but unable to drive entry into the cell cycle and subsequent proliferation for BM3.3 tgTCR-naive CD8+ T cells. When expressed by activated APCs with increased B7.2 and ICAM-1 surface expression, H-2Kbm8 seemed to behave as a moderate agonist inducing a delayed entry into cell cycle and a partial recovery of IL-2-dependent proliferation (Tables I and II; Fig. 2Go). This is consistent with previous data showing that both ICAM-1 and B7.1 costimulation can increase proliferation and IL-2 production of naive CD8+ T cells (43, 44, 45).

The observed lack of TCR down-regulation after triggering by a partial agonist agrees with some (46, 47, 48), but not all (39) previous reports. However, the discrepancy with the results reported by the latter group may be explained by the fact that we analyzed the TCR behavior after encounter with an endogenously expressed partial agonist without supplementation with high concentration of exogenously added peptide. Moreover, whereas the independence of cytotoxicity and IFN-{gamma} secretion from TCR modulation has already been observed (36), here we report a situation where proliferation can also occur independently of the down-regulation of TCR surface expression. Thus, our results suggest that the number of internalized TCRs does not correlate with the global efficiency of T cell activation as previously proposed (12, 17) but rather reflects the TCR-mediated signal 1, the outcome of T cell activation being determined by the conjunction of both signal 1 and signal 2 (44).

In a particular T cell lineage, CD8lowtgTCRhigh that failed to produce IL-2 on Ag stimulation, we have previously observed that this impaired activity correlated with a lack of AP-1-mediated trans-activation (16). Here, this correlation between production of IL-2 and AP-1 trans-activation can be extended to naive CD8+ CTL precursors. The expression of costimulating molecules on APCs for the partial agonist H-2Kbm8 allowed partial recovery of TCR- induced IL-2 production which correlated with an efficient but delayed AP-1 trans-activation. Whether this time shift in AP-1 trans-activity was due to the induction of a different set of Fos and Jun members is presently being tested at the mRNA level. Furthermore, these results suggest that there was a convergence of signaling cascades initiated by the TCR and by costimulating molecules at the level of AP-1 activation. One of the possible points for the integration of these two types of signaling may be JNK, the protein kinase responsible both for posttranslational activation of AP-1 (26, 49, 50) and for stabilization of IL-2 mRNA (51). Additionally, a role of CD28 at enhancing protein tyrosine kinases activation through raft microdomain reorganization has recently been proposed, which led to increased downstream signaling including the ERK/MAPK and JNK pathways (52, 53).

Interestingly, the different requirements for NF-{kappa}B and AP-1 trans-activation as illustrated in this study after triggering by a partial agonist may reflect distinct kinetics in the signaling cascade initiated at the TCR level (54, 55, 56). The former (NF-{kappa}B) may respond to a signal delivered very rapidly after TCR engagement and therefore be less dependent on the duration of TCR triggering, whereas the latter (AP-1) may require a longer engagement to allow the generation of a slower signaling event. Thus, the short interaction time between TCR and altered ligand due to a rapid dissociation rate, may not be sufficient to induce AP-1 activity. This assumption is consistent with the mechanisms that lead to activation of NF-{kappa}B and AP-1, respectively. Indeed, NF-{kappa}B is constitutively present in the cytoplasm and its activation, which is probably dependent on a signaling cascade involving a mitogen-activated protein 3-kinase (57) and the {alpha} and ß forms of the I{kappa}B kinases, leads to the phosphorylation and the subsequent ubiquitination and proteasomal degradation of its inhibitor I{kappa}B (58). In contrast, AP-1 activity is achieved after neosynthesis of Fos and Jun members and their further posttranslational modifications (23). In addition, our results suggest that in naive CD8 T cells, activation of NF-{kappa}B is less dependent on costimulatory signals than that of AP-1.

Expression of both CD69 and CD44 Ags has been shown to be regulated by AP-1 (59, 60). In the present study, we showed a parallel between CD69 and CD44 expression, IL-2 production and AP-1 trans-activation, especially concerning the influence of costimulatory components during the course of induction by a partial agonist. Besides, minimal TCR engagement by a partial agonist seemed to weakly induce CD25, the expression of which was more pronounced in the presence of costimulation. However, this synergy between signals 1 and 2 seemed rather due to the secondary recovery of IL-2 secretion than to a stronger TCR engagement, because the totality of the upgraded CD25 expression was abrogated in the presence of an anti-IL-2 mAb (Fig. 3GoC). Furthermore, it should be noted 1) that both the level of CD69 and CD25 induced by bm8 in conditions of costimulation never reached the one observed with the full agonist, suggesting that a lower activation threshold was reached by the partial agonist, and 2) that only 60% of the tgTCR+ cells were responsive to bm8, indicating that only a fraction of tgTCR+ T cells responded to partial agonist encounter. Together these observations may explain the fact that stimulation with activated bm8 APCs leads to only partial recovery of IL-2 production, but it also raises the question of why a "monoclonal" tgTCR+ CD8+ population shows an heterogeneous response to a specific stimulus. One possibility would be that the expression of specific H-2Kbm8/peptide complexes is so weak that not all tgTCR+ cells/APC encounters lead to productive engagement of TCRs. This observation is reminiscent of a previous example of heterogeneity in cytokine responses of monoclonal CD4+ T cells (61).

We have shown that a minimal TCR engagement (signal 1) by a partial agonist which did not induce down-regulation of cell surface expressed TCRs (this study), still triggered sufficient signaling to complete differentiation from naive to effector T cells and IFN-{gamma} production (16). However, both IL-2 synthesis and up-regulation of activation markers failed to be stimulated in such conditions, and we propose that this is due to an inefficient activation of the AP-1 transcription factor. When expression of costimulating molecules was increased on APCs (signal 2), the partial agonist induced limited IL-2 secretion and up-regulation of activation markers as well as a recovery of AP-1 trans-activation. Altogether, these observations allowed the establishment of a hierarchy between T cell functions in terms of their sensitivity to endogenously expressed APL. It also showed that costimulation may be particularly important in the course of an immune response toward weakly stimulatory ligands. Moreover, these data showed that both TCR- and costimulatory molecule-mediated signals converge toward the AP-1 transcription factor. The particular situation of AP-1 at the crossroads of both signal 1 and signal 2 may explain why it appeared to be the target for inactivation in anergic T cells (21) and designated it as a key modulator of T cell responses that one can try to specifically target by either immunostimulatory or immunosuppressive designed strategies. In such approaches, it will be important to keep in mind the dissociation of signals leading to NF-{kappa}B and AP-1 trans-activation as well as their differential sensitivity to T cell activation thresholds.


   Acknowledgments
 
We thank C. Boyer, S. Guerder, L. Leserman, and B. Malissen for critical reading of the manuscript; M. Pophillat for transgenic mouse screening; C. Marra and G. Warcollier for animal care; and C. Béziers La Fosse for help with graphics.


   Footnotes
 
1 This work was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique and by grants from Association pour la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer (LNFCC), and the LNFCC-Comité des Bouches du Rhône. Back

2 Address correspondence and reprint requests to Dr. Nathalie Auphan, Centre d’Immunologie, Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, 13288 Marseille, Cedex 9, France. E-mail address: Back

3 Abbreviations used in this paper: APL, altered peptide ligands; CFSE, 5(and -6)-carboxyfluorescein diacetate succinimidyl ester; EMSA, electrophoretic mobility shift assay. Back

Received for publication May 3, 1999. Accepted for publication August 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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