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The Journal of Immunology, 1998, 160: 4810-4821.
Copyright © 1998 by The American Association of Immunologists

Consequences of Intrathymic TCR Engagement by Partial Agonist on Selection Events and Peripheral T Cell Activation Program1

Nathalie Auphan2,*, Anna Katharina Simon*, Hélène Asnagli*, Roderick J. Phillips{dagger}, Mercedes Rincon3,{dagger}, Sankar Ghosh{dagger},{ddagger}, Richard A. Flavell{dagger},{ddagger} and Anne-Marie Schmitt-Verhulst*

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Functions elicited from mature T cells depend on the nature of the Ag. Thus, an agonist induces a larger set of cytokine responses than a partial agonist. Additionally, Ags present in the thymus influence both the selection of TCRs generated by gene rearrangement and the potential functional program of developing thymocytes. This can be approached by analysing the development of T cells in mice expressing the same transgenic TCR (tgTCR) under different conditions of intrathymic selection. H-2Kbm8 was found to act as a partial agonist for CD8+ T cells expressing a tgTCR specific for the H-2Kb alloantigen. Intrathymic exposure to full or to partial agonist affected the development of thymocytes at different stages, consistent with the respective CD8-independent and -dependent characteristic of the tgTCR/Ag interaction. The presence of the partial agonist led to the accumulation of a major population of thymocytes (tgTCRhighCD4-CD8low) originating from TCR engagement at the immature single-positive CD8low stage as evidenced by: 1) results from reaggregated thymic organ culture in the presence of H-2k/bm8 thymic stromal cells; 2) the absence of CD4+ thymocytes, the development of which depends on rearrangements of endogenous TCR{alpha} genes; and 3) the identification of the CD8low thymocytes as cycling cells. Peripheral CD8low T cells selected in an H-2k/bm8 thymus expressed a partial functional program in response to H-2Kb, akin to the response of CD8high T cells to a partial agonist. The analysis of the molecular bases for partial reactivity revealed a correlation with inefficient AP-1, but efficient NF-{kappa}B transactivation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
It has been well established that T cells recognize short peptides bound to MHC molecules (1) and recently TCR and MHC + peptide have been cocrystallized (2). Studies using variants of antigenic peptides have shown that TCRs respond selectively to subtle changes in ligands, rather than responding in an "all or none" fashion to unique antigenic determinants. Moreover, interaction with variants of antigenic peptides can stimulate a subset of T cell effector functions without inducing cell proliferation: in this case, altered peptide ligands are referred to as partial agonists (reviewed in Refs. 3 and 4). Recent experiments have demonstrated that partial agonists generate altered intracellular signals (reviewed in 4 compared with agonist-induced T cell activation. Several studies have also suggested that recognition of an altered peptide ligand may be dependent on the coreceptors CD4 or CD8 (5, 6). Taken together, these data are compatible with the notion that different activation thresholds may be required for the induction of different T cell functions (7, 8). These may or may not be reached upon encounter of a full agonist or a partial agonist, since the overall avidity of such interaction will depend not only on the level of TCR and Ag expression but also on the coreceptors. Additional qualitative differences in the nature of signal transduction required for distinct functional programs have also to be considered.

The main pathway for TCR{alpha}ß+ cells involves their intrathymic differentiation from CD4-8-CD3/TCR- (double-negative (DN))4 immature precursors to CD4+8+ CD3/TCR+ (double-positive (DP)) thymocytes, the major stage for thymic selection events. Indeed, upon appropriate self-selection, DP thymocytes further differentiate in CD8+TCR+ or CD4+TCR+ single-positive (SP) cells, restricted by MHC class I and class II products and generally endowed with a cytotoxic and helper function, respectively. Several maturation stages can be identified during the transition from DN to DP, the transition from one stage to another being controlled by several checkpoints including TCRß gene rearrangements and selection of the TCR ß-chain upon its expression in association with the pT{alpha} (reviewed in Refs. 9 and 10). Along this pathway, thymic precursors up-regulate the expression of the CD8 coreceptor, passing through a discrete stage of immature SP (ISP) CD8low precursors, which precedes the initiation of TCR{alpha} gene rearrangements and CD4 expression necessary to reach the DP stage.

Analysis of thymocytes developing in tgTCR mice expressing both {alpha} and ß rearranged TCR genes under different conditions of intrathymic selection is an approach to establishing the role of the latter in T cell program acquisition. During intrathymic T cell maturation, the levels of TCR and coreceptors are tightly regulated and one may consider that, as in the periphery, interaction with self + peptide complexes will have different outcomes depending on the avidity of such recognition; thus, by using either a full or a partial agonist, we were interested in defining particular differentiation stages in which a high avidity signal, which normally induced negative selection, could be changed to a lower avidity interaction possibly mediating positive selection for the same tgTCR. As in several models of tgTCR mice, the tgTCR is expressed early in T cell development (11, 12); these may reveal selection rules that can occur upon TCR/CD3 engagement at stages that appear hidden or minor in normal mice.

In the present study, we first defined the H-2Kbm8 mutant as a partial agonist for a H-2Kb alloreactive tgTCR, since it efficiently induced CTL effectors from naive precursors but induced an altered pattern of cytokine secretion. Second, taking advantage of the fact that the tgTCR recognized endogenous peptide(s) in association with H-2Kb (13) or H-2Kbm8, we analyzed the consequences of intrathymic expression of the partial agonist on in vivo differentiation of tgTCR+ thymic precursors. We found that the presence of the partial agonist induced the emergence of CD8lowTCR+ cells, which exit the thymus at the ISP stage. This event blocked any further thymocyte differentiation as well as the development of CD4+ T cells expressing endogenous TCR{alpha} (TCR{alpha}e) (14). Based on this and previous studies (15), we propose that at each step of differentiation, a block in the development of tgTCR+ precursors can be induced depending on the overall avidity of the interaction between the thymocytes and the thymic stromal cells, which depends not only on the TCR and the Ag but also on the level of the coreceptors.

Interestingly, the CD8lowtgTCR+ cells that were thus selected in the context of H-2Kbm8 and migrated to the periphery appeared tolerant to H-2Kbm8 but were partially reactive to H-2Kb, thereby 1) indicating that selection at the ISP stage was accompanied by the acquisition of a functional gene expression program and 2) supporting the notion that natural partial agonistic peptides of potential tumor Ags may influence the efficiency of T cell immunosurveillance. We further analyzed the molecular bases for partial reactivity and established for the first time a correlation with a selective defect in AP-1 transactivation for naive T cells.


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

Mice transgenic for the BM3.3 TCR on the CBA/Ca background (tgTCR) have been described (16). They were crossed with either CBA/Ca (initially obtained from the National Institute for Medical Research, Mill Hill, London, U.K.), C57BL/6 (B6), or C57BL/6.C-H-2bm8 (bm8) in our animal facilities. Offspring were of the H-2k/k, H-2k/b, or H-2k/bm8 haplotypes, respectively. tgTCR Rag-1-/- H-2k/k mice were obtained as described (14). For reaggregated thymic organ culture (RTOC) experiments, CBA/Ca, (CBA/J x C57BL/6)F1 (abbreviated as CBAxB6) and (CBA/J x C57BL/6.C-H-2bm8)F1 (abbreviated as CBAxbm8) were bred in our animal facilities to obtain day 15 embryos.

Mice transgenic for AP-1-luciferase (17) and NF-{kappa}B-luciferase (R.J.P. and S.G., unpublished observations), obtained from R.A.F., were crossed with the tgTCR mice in our animal facility. The NF-{kappa}B-luciferase reporter mice were made using the pBIIX-luciferase construct with two copies of the {kappa}B sequence from the Ig{kappa} intronic enhancer (18).

Flow cytometric analyses

Reagents used for immunofluorescence staining were biotin-anti-TCR (BM3.3) mAb Ti98 (19) and FITC-anti-CD8{alpha} mAb (H59.101.2) (20), both conjugated in the laboratory, and phycoerythrin-anti-CD4 mAb (Caltag, San Francisco, CA). After staining, 2.5 x 104 viable cells in each sample were analyzed using a FACScan cytofluorometer (Becton Dickinson, Mountain View, CA).

DNA biosynthetic labeling

Bromodeoxyuridine (BrdU) was given in two i.p. injections (1 mg each) either at a 30-min or an 8-h interval, thymuses being harvested, respectively, 1.5 h and 24 h after the first injection. After surface staining (see preceding section), the cells were fixed in 70% ethanol-50 mM NaCl for 1 h and then in PBS containing 1% paraformaldehyde and 0.01% Tween 20 overnight. After treatment with DNase I, BrdU detection was performed with a FITC-coupled anti-BrdU mAb (Becton Dickinson).

Culture conditions and functional assays

Spleen or lymph node cells (106) from tgTCR mice were cultured in 1 ml of 5% FCS-supplemented RPMI 1640 with 3 x 106 irradiated cells per Costar well. In cytolytic assays performed at day 3 after stimulation, target cells were either RMA (H-2b) tumor cells or its variant, RMA-S, as a negative control. Targets (104), after labeling with 51Cr (sodium chromate; New England Nuclear, Boston, MA), were incubated with effector cells for 4 h at 37°C.

For assays presented in Figure 2Go, 105 lymph node cells from tgTCR mice were cultured with different dilutions of T cell-depleted stimulating cells in round-bottom microplates for 4 days. To determine lymphokine production, 100 µl of culture supernatant were harvested 48 h after stimulation and used to measured both IFN-{gamma} production by ELISA as described (21) and IL-2 secretion by measuring the ability of this supernatant to sustain the proliferation of an IL-2-dependent T cell line (CTL.L) as described (22). At day 4, cytotoxicity was measured by adding 50 µl of labeled targets directly to the microplates.



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  		FIGURE 2. Cytolytic function and IL secretion of T cells from tgTCR Rag-1-/- H-2k/k (A) or tgTCR H-2k/bm8 (B) mice responding to H-2Kb (B6) or H-2Kbm8 (bm8). Lymph node cells (105) isolated from either tgTCR Rag-1-/- H-2k/k (A) or tgTCR H-2k/bm8 (B) mice were cultured with decreasing doses (ranging from 3 x 105 to 4.5 x 103) of B6 or bm8 T cell-depleted splenocytes. Lymphokine secretion was assessed after 48 h of culture, and CTL assay was performed at day 4. Note that IL-2 was not detected in response to bm8 also after 24 h of culture (A).

 
Reaggregate thymic organ culture (RTOC)

Thymocytes were obtained from newborn tgTCR H-2k/k mice by teasing apart isolated lobes. DN thymocytes were prepared by immunomagnetic negative selection using anti-CD4 and anti-CD8 mAbs. Stromal cell suspensions were prepared from day 15 fetal thymic lobes as described elsewhere (23) from either (CBAxB6)F1 or (CBAxbm8)F1 or CBA mice with the following modifications. Trypsinized lobes were subjected to two rounds of immunomagnetic selection with anti-CD45 (H129.16.3) mAb. Less than 5% thymocytes are found in the enriched stromal cell population as estimated by microscopal analysis for cell morphology.

RTOCs were prepared by mixing together thymocytes and stromal cells at a ratio of 2:1. Mixed suspensions were pelleted by centrifugation and placed as a standing drop on the surface of a nucleopore filter in organ culture (24). They were allowed to develop for 24 to 96 h and then analyzed by cytometry.

Luciferase activity analysis

Lymph node cells were recovered from double tg (TCR x AP-1/luciferase) or (TCR x NF-{kappa}B/luciferase), and 106 cells were cultured with 2 x 106 stimulating cells, as indicated, or with 1 µg/ml Con A for 24, 48, and 72 h in duplicates. Each time cells were harvested, they were lyzed in lysis buffer (Luciferase Assay, Promega, Madison, WI) and luciferase activity was measured using the Luciferase Reagent (Promega) and a luminometer (Lumat LB96P, EG&G Berthold, France).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
For a CD8-independent anti- H-2Kb TCR, H-2Kbm8 behaves as a CD8-dependent stimulus

The BM3.3 tgTCR recognizes endogenous peptide(s) in association with H-2Kb and can be triggered by the Ag independently of CD8 (14, 16). It was interesting, therefore, to determine how MHC class I mutations that may affect peptide presentation will influence recognition by CD8+tgTCR+ cells, as well as the subsequent activation of their functional program. H-2Kbm8 presents four amino acid substitutions in the ß-pleated sheets of the {alpha}1 domain of H-2Kb that essentially affect class I-peptide interactions (25). Indeed, mutations in the peptide groove of H-2Kbm8 molecules may either 1) affect the presentation of the same endogenous peptide that also binds to H-2Kb; or 2) lead to presentation of a distinct endogenous peptide that forms a complex with H-2Kbm8 for which the tgTCR is cross-reactive. We first analyzed the phenotype of cells expanded after stimulation with B6 or bm8 APCs. We previously showed (14) that peripheral T cells from H-2k tgTCR mice contained tgTCR+ CD8+, but also tgTCR+ CD4+ T cells, and that both T cell populations developed into CTL in response to in vitro stimulation with H-2Kb APCs. In tgTCR mice deficient for the Rag-1 gene, in which no endogenous TCR (TCRe) gene rearrangements can occur, only CD8+ T cells were present, and we previously showed that CD4+ T cells present in tgTCR Rag-1+/+ mice were selected by their expression of TCR{alpha}e in addition to the TCR{alpha}tg chain (14). Thus, as expected from previous studies (14, 16), both CD8+ and CD4+ T cells were recovered after a 3-day stimulation of tgTCR peripheral T cells with B6 APC. In contrast, cells recovered after stimulation with bm8 APC were mainly CD8+ (Fig. 1GoA). To exclude any reactivity to H-2Kbm8 due to the expression of endogenously rearranged TCR{alpha}e genes, CD8+ T cells from tgTCR Rag-1-/- mice were shown to expand in response to bm8 (Fig. 1GoA) and to develop into H-2Kb-specific CTL after stimulation with either H-2Kb or H-2Kbm8 (Fig. 2GoA).



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  		FIGURE 1. Phenotype (A) and cytolytic function (B) of cells from tgTCR and tgTCR Rag-1-/- mice responding to H-2Kb (B6) or H-2Kbm8 (bm8) after a 3-day in vitro culture. A, Immunofluorescence staining using anti-CD8 and anti-CD4 mAb. B, During the 3-day in vitro culture, T cells from tgTCR mice were stimulated as indicated (B6 or bm8) either in the presence of anti-CD8 or anti-CD4 or without mAb. Cytotoxic activity was measured on an H-2Kb target cell, RMA. Percentage of lysis on RMA-S control cells never exceeded 5% (not shown).

 
The observation that only CD8+tgTCR+ cells expanded after bm8 stimulation raised the possibility that the CD8 coreceptor might play a role in the activation by the mutated Ag. Indeed, the differentiation into CTL effectors upon H-2Kbm8 stimulation was completely abolished if an anti-CD8 mAb was added during the 3-day in vitro culture, whereas CTL maturation induced by H-2Kb was not affected either by anti-CD8 or by anti-CD4 mAbs (Fig. 1GoB), as previously described (16). Lysis of bm8 target cells was not affected by the presence of anti-CD8 mAb, however, whether CTL precursors had been stimulated with B6 or bm8 (results not shown). This indicated a selective sensitivity to inhibition by anti-CD8 mAb for the response to H-2Kbm8 in the differentiation from CTL precursors into effectors, as compared with the cytolysis by fully competent CTL, as also had been previously observed in a different system (26). Thus, for CTL precursors expressing the same TCR, activation by the full agonist was CD8- independent, whereas activation by an altered ligand was dependent on CD8.

Several studies have documented the influence of CD8 on the fine specificity of MHC class I-restricted TCR, showing that high expression of CD8{alpha}{alpha} (27) or CD8{alpha}ß (5) dimers could confer recognition of MHC class I mutants or weak agonist peptide variants, respectively. These observations have been further confirmed by the demonstration that the avidity of the TCR for its specific ligand is enhanced in the presence of CD8 dimers (28, 29). Finally, the recently reported crystal structure of a CD8{alpha}{alpha} molecule complexed to HLA-A2 is also consistent with an avidity-based contribution of the CD8 coreceptor for MHC-TCR binding (30).

H-2Kbm8 behaves as a partial agonist

In bulk culture, H-2Kbm8 was able to stimulate the differentiation of tgTCR CD8+ precursors into CTL effectors, and the lytic activity was quite similar after B6 or bm8 stimulation for a given number of effector cells (81 ± 13% for bm8 as compared with 100% for B6 in four experiments). However, when we tried to estimate the frequency of CTL precursors reactive to H-2Kb or H-2Kbm8, fivefold fewer tgTCR+ precursors were stimulated by H-2Kbm8 (unpublished data). The superior stimulating capacity of H-2Kb was also apparent in proliferation assays (unpublished data) or when CTL activity, generated from an initial number of 105 CD8 T cells in microculture wells in the presence of different numbers of B6 or bm8 APCs, was assayed directly in the culture wells (Fig. 2GoA). Altogether, these results indicated that tgTCR+ cells were poorly stimulated to proliferate after exposure to H-2Kbm8 as compared with H-2Kb APC, but that once activated the potency of CTL effectors was equivalent. Thus, we examined the pattern of lymphokine secretion induced by mutant or wild-type Ags. For these assays, we used effectors from tgTCR+Rag-1-/- mice to consider cytokines produced by CD8+ cells exclusively and to avoid any differential secretion due to the H-2Kb stimulation of CD4+ T cells. As shown in Figure 2GoA, IL-2 was produced in response to B6, but not in response to bm8, upon stimulation of tgTCR+CD8+ T cells. In contrast, H-2Kbm8 was as efficient as H-2Kb at inducing IFN-{gamma} production by the same T cells. Thus, H-2Kbm8 behaved as a partial agonist in comparison with the full agonist H-2Kb, which triggered all of these functions.

Little is known about differentiation events determining the functional programming of T lymphocytes. However, the discrepancy between activation programs triggered by recognition of H-2Kb vs H-2Kbm8 cannot be explained solely by their respective independence of and dependence upon CD8. Indeed, we previously found that a full agonist was able to induce the same effector functions, and in particular IL-2 production, for CTL precursors expressing either CD8--independent or CD8--dependent TCRs (16, 31). It seems more likely that either a quantitative critical threshold or additional qualitative differences may be involved in the elicitation of different functions by a full agonist vs a partial agonist. It has already been shown in other models that partial agonists can trigger cytolysis but not cell proliferation, suggesting that an activation threshold was reached that was sufficient to trigger the CTL function (7) but was not sufficient to induce a function, such as cytokine production (IFN-{gamma}), that required a higher TCR occupancy. In other cases, however, perforin-dependent killing could not be triggered, although IFN-{gamma} could, in the response of CTL clones to self peptides that varied in one amino acid with the antigenic peptide (32, 33, 34). The fact that the nature of the effector function, which is selectively affected by stimulation with partial agonists, varies in different systems suggests that signals vary qualitatively and that not only thresholds are involved.

Consequences of the recognition of a partial agonist on intrathymic T cell differentiation: emergence of CD8lowtgTCRhigh cells

Since H-2Kbm8 partially activated peripheral CD8+tgTCR+ cells, we wondered if such mutated Ag would also induce partial signaling in thymic precursors, thus influencing the nature of thymic selection events. To address this question, we crossed the tgTCR mice either with B6 or bm8 mice to obtain H-2k/b and H-2k/bm8 F1 mice, respectively. We have previously shown that intrathymic expression of the full agonist H-2Kb (in H-2k/b F1) leads to a drastic reduction of thymic cellularity (~25-fold; see Table IGo). The few DP that remained (Fig. 3Go) did not express the tgTCR but a combination of TCR{alpha}e/TCRßtg, including precursors of CD4+ cells (35). In mice expressing H-2Kbm8, the number of cells recovered in the thymus was also reduced (~9-fold; see Fig. 3Go and Table IGo), but to a lesser extent than in H-2k/b mice. Interestingly, the proportion of the DP and CD4+ populations was strongly reduced, suggesting that H-2Kbm8 induced either a deletion of these subpopulations or a block in their maturation. However, a significant proportion of CD8+ thymocytes could be identified that expressed a low level of the CD8 coreceptor in comparison with the CD8high cells, which are positively selected in a H-2k/k background (Fig. 3Go). Nevertheless, both the CD8low cells differentiated in H-2k/bm8 mice and the CD8high cells found in H-2k/k controls express high and homogenous levels of tgTCR. Further immunofluorescence analysis showed that this CD8lowtgTCR+ population expressed neither CD44 nor CD25, but was partially HSA+ (unpublished data). Thus, intrathymic interaction of thymocyte precursors with a partial agonist led to enrichment in a particular CD8lowtgTCR+ thymocyte lineage and relative depletion of DP thymocytes. Thus, a lower proportion of DP and SP CD4+ thymocytes expressing a combination of TCR{alpha}e/TCRßtg were present in the k/bm8 as compared with the k/b mice. Altogether, if expression of the partial agonist H-2Kbm8 induced a moderate decrease in the total thymic cellularity as compared with the full agonist H-2Kb, the former seemed to be less permissive in view of the relative absence of DP thymocytes. This suggested that in the presence of the partial agonist a blockade occurred during thymic maturation at a stage distinct from that resulting from the presence of the full agonist (15). Because of the early tgTCR expression (11, 12), engagement of the tgTCR by the partial agonist could occur either before or after the DP stage.


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  		Table I. Representation of thymocyte subpopulations in the different tgTCR and normal mice1

 


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  		FIGURE 3. Phenotype of thymocytes recovered from tgTCR mice of H-2k/k or H-2k/bm8 or H-2k/b haplotypes: presence of CD8lowtgTCRhigh thymocytes in tgTCR H-2k/bm8 mice. On the left, double immunofluorescence staining with anti-CD8 and anti-CD4 mAb. On the right, tgTCR level detected on CD4-8- and CD8+ populations are reported; the CD8+ fraction represented is CD8high (gate R1) for the H-2k/k and H-2k/b mice, CD8low (gate R5) for the H-2k/bm8 mice. Absolute numbers of thymocytes recovered in each mouse are indicated.

 
CD8lowtgTCR+ cells correspond to the immature SP CD8low precursors

Two hypotheses could account for the phenotype of the CD8lowtgTCR+ cells: 1) these cells differentiate from DN to DP along the normal TCR{alpha}ß pathway and then to CD8+ SP, with CD8 down-modulation occurring at the mature SP stage; or 2) these cells correspond to the ISP precursors, an intermediate stage identified during the transition from DN to DP, this second hypothesis being supported by the relative absence of DP precursors in the H-2k/bm8 thymus. To address the first possibility, we tested whether exposure of mature SP CD8hightgTCR+ T cells from H-2k/k mice to H-2Kbm8 would induce a CD8low phenotype. Upon injection in irradiated (CBAxbm8)F1 mice, tgTCR+ cells expanded and remained as CD8high (median CD8 fluorescence = 144) as when injected in irradiated CBA mice (median CD8 fluorescence = 170) in which they did not expand. As a control, injected CD8lowtgTCR+ cells maintained a CD8low phenotype (median CD8 fluorescence, ~40; see below). Thus no evidence for the acquisition of the CD8low phenotype was found for mature SP T cells. To formally prove the second proposed scheme, we performed RTOC using thymocytes recovered from H-2k/k tgTCR+ newborn offspring and thymic stromal cells isolated from day 15 (CBAxB6)F1, (CBAxbm8)F1 or CBA embryos. First, we verified that our in vitro conditions allowed the differentiation of mature SP CD8+ thymocytes by putting whole newborn thymic lobes in culture for several days and measuring by immunostaining the percentage of SP CD8+ cells at different times: this number rose from 6 to 7, 11, 17, and 31% at day 0, 1, 2, 4 and 8, respectively (unpublished data). Next, we asked whether H-2Kbm8 presentation by thymic stromal cells was able to induce the deletion at the DP stage. As shown in Figure 4GoA, coculture of tgTCR+ thymocytes and H-2Kb-expressing stromal cells induced the complete and rapid (already seen after 24 h) loss of the DP population. In contrast, the reaggregation with H-2k/bm8 stroma triggered only a small decrease in the percentage of DP thymocytes, which may correspond to the ongoing differentiation as suggested by the appearance of mature SP CD8high cells. As a control, the differentiation observed in the presence of CBA (H-2k/k) stromal cells was similar to that seen with H-2k/bm8 stromal cells: in the former situation, a more pronounced decrease of the DP subset was seen, which correlated with a more efficient positive selection (11 vs 7% of CD8+ SP thymocytes) and may be due to the higher expression of the positively selecting MHC products (H-2k/k). Additionally, a very weak activation of DP thymocytes cultured in the presence of H-2k/bm8 stromal cells was revealed by analyzing CD69 expression (respectively, 6 and 22% of CD69+ cells after H-2k/bm8 and H-2k/b in vitro stimulation (unpublished data)). This was also accompanied by a slight down-modulation of both CD4 and CD8 on the remaining DP population, possibly corresponding to the "dulling effect" observed upon feeble TCR engagement at the DP stage (36). Engagement of the tgTCR by H-2Kbm8 at the DP stage thus appeared inappropriate for the efficient induction of either positive or negative selection.



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  		FIGURE 4. Phenotype of thymocytes which differentiated in RTOC. A, Total thymocytes from tgTCR H-2k/k newborn mice were stained with anti-CD8 and anti- CD4 mAb as they were harvested (left) and than cultured for 24 h with either H-2k/b (CBA/B6), H-2k/bm8 (CBA/bm8), or H-2k/k (CBA) stromal cells; CD4/CD8 staining of tgTCR+ cells is shown. B, The DN population was enriched from thymi of either tgTCR or tgTCR Rag-1-/- newborn mice and cultured for 4 days as described in A; CD4/CD8 staining of either total or tgTCR+ cells is shown.

 
We then asked whether the CD8lowtgTCR+ thymocytes could directly differentiate from the DN precursors. For this determination, we enriched DN thymocytes from newborn tgTCR+ H-2k mice by negative magnetic sorting. Upon reaggregation with H-2k/b stromal cells, we observed the differentiation of some DN into DP and CD4+ SP cells after 4 days (Fig. 4GoB). However, none of these cells expressed the tgTCR, as they were excluded if we gated on cells positive for the anticlonotypic mAb. In contrast, H-2k/bm8 stromal cells were able to potentiate a massive differentiation of CD8+ cells, all tgTCR+, without any maturation of the DP population (Fig. 4GoB). The same result was obtained when DN thymocytes from newborn tgTCR+Rag-1-/- H-2k/k mice were exposed to H-2k/bm8 stromal cells (Fig. 4GoB), indicating that the tgTCR alone was responsible for this differentiation. As a control, reaggregation with H-2k/k stromal cells was shown to allow the differentiation from DN to DP TCRtg+ thymocytes, and further, to CD4+ SP for cells expressing a TCR{alpha}e/TCRßtg combination. Mature SP CD8+ T cells appeared only on day 10 in this system (unpublished data).

Previous reports, using altered peptides delivered during thymocyte differentiation in fetal thymic organ culture, have suggested that positive and negative selections are influenced not only by quantitative parameters (threshold of TCR engagement by the same MHC + peptide complex (37, 38)), but also by qualitative parameters (different signaling resulting from the recognition of an agonist as compared with a partial agonist (39, 40)). In our model, we have previously shown that the early TCR expression in tgTCR mice leads, in the case of H-2Kb expression, to the arrest of tgTCR+ cells already at the DN stage (15). Here, we showed that both in vivo and in RTOC (Fig. 4GoB), only the thymocytes that expressed a TCR{alpha}e/TCRßtg combination were allowed to mature in the presence of the full agonist, H-2Kb. These cells probably developed from thymocytes that escaped because they did not express the TCR{alpha}tg but rather the pT{alpha}/TCRß (41) and followed their maturation until the stage in which endogenous TCR{alpha} genes rearrange. Then, reaching the DP stage, these thymocytes could be positively selected on an endogenously encoded MHC class II-restricted TCR, giving rise to the CD4+ population (14). In the case of H-2Kbm8 expression, the decreased avidity of the interaction between the tgTCR and (MHC + peptide) complexes allowed the persistence of DN tgTCR+ precursors (Fig. 3Go). Based on the recent observation that a tgTCR{alpha}ß can replace the pT{alpha}/ß complexes in pT{alpha}-/- mice (41) for selection at the DN CD44-CD25+ stage, we can hypothesize that most of the DN tgTCR+ precursors may continue their maturation based on a tgTCR rather than on a pT{alpha}/ß selection. However, at the next stage (ISP), when the CD8 coreceptor is up-regulated, the overall avidity resulting from the interaction between both the tgTCR and CD8, and the H-2Kbm8 ligand induces a signal that appears to select these CD8low cells. At the same time, this signal may down-regulate Rag gene expression, preventing TCR{alpha}e gene rearrangement from occurring and tgTCR-CD4+ cells from developing. This model is supported by the fact that Rag-1 and Rag-2 genes are transcriptionally down-regulated in rapidly cycling late DN and ISP thymocytes (42) and is also in agreement with a previous study indicating that engagement of the TCR at the CD8low ISP stage prevents further differentiation (43). Moreover, this scheme is consistent with the observation that the higher cellularity in k/bm8 as compared with k/b mice thymi is not associated with a higher absolute number of either DP or CD4 SP thymocytes (Table IGo).

Emerging CD8lowtgTCR+ cells are cycling

Intrathymic maturation of T cell precursors not only involves a differentiation process but also a proliferation step, which takes place between the late DN (CD44-CD25-) and the early DP stages (blastic) encompassing the ISP precursors (12). We thus evaluated the proliferative status of thymocytes in tgTCR H-2k/bm8 vs H-2k/k mice after in vivo pulse labeling with the thymidine analogue, BrdU. As shown in Figure 5GoA, 90 min after BrdU injection, the number of thymocytes that have incorporated BrdU was slightly higher in H-2k/bm8 than in H-2k/k tgTCR mice. However, this difference was markedly increased 24 h later, as 62.4 vs 19.8% of thymocytes were BrdU+, although the level of immunofluorescence decreased as a result of cell division. Moreover, the forward cell scatter indicated that H-2k/bm8 thymocytes were largely blastic as compared with those recovered from H-2k/k mice (unpublished data). To define more precisely the thymic subsets that were proliferating, we analyzed the level of BrdU incorporated in three populations defined by their CD8 expression (CD8-, CD8low and CD8high cells) (Fig. 5GoB). In thymocytes from H-2k/k tgTCR mice, 18% of the CD8low and 36% of the CD8high cells were labeled (the latter population corresponded to the immature CD4+8+ as assessed by triple staining with an anti-CD4 mAb; unpublished data). In H-2k/bm8 tgTCR mice, CD8high thymocytes were absent due to a lack of DP thymocytes. Both the DN and the CD8low populations were BrdU+ (respectively, 45 and 80% of labeled cells at 24 h). Thus, engagement of the TCR by a partial agonist selected the ISP CD8low to exit the normal differentiation pathway at a stage at which proliferation occurs.



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  		FIGURE 5. Thymic cell proliferation assessed by BrdU incorporation in thymocytes from tgTCR H-2k/k and tgTCR H-2k/bm8 mice. A, BrdU staining measured on total thymocytes before, 1.5 h after, or 24 h after the first BrdU injection. Percentage of positive cells is indicated. For the 24-h point, two percentage values are given; the value under the bar additionally takes into account BrdU+ cells with low intensity due to dilution of BrdU in proliferating cells (see text, Results). B, CD8 staining is shown, allowing the definition of CD8-, CD8low, and CD8high populations (see bars on histograms); for each of these cell subsets, the percentage of BrdU-positive cells is reported.

 
CD8lowtgTCR+ ISP precursors migrate to the periphery and express CD8{alpha}ß heterodimers

As the CD8lowtgTCR+ cells were also tgTCRhigh, normally a hallmark for positive selection, we wondered whether such cells would also acquire the capacity to exit the thymus and migrate to the periphery. Indeed, CD8low cells were found in lymph nodes of H-2k/bm8 mice, and in a manner similar to CD8high T cells in H-2k/k mice, these cells expressed a high level of tgTCR (Fig. 6Go). Consistent with the thymic phenotype, we noted the absence of CD4+ T cells in peripheral lymphoid organs of tgTCR H-2k/bm8 mice.



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  		FIGURE 6. Analysis of lymph node cells from tgTCR H-2k/k and tgTCR H-2k/bm8 mice; CD8low tgTCRhigh cells are present in the latter. Total CD8 staining is shown as well as the level of tgTCR on CD8+ cells. Percentage of positive cells and mean fluorescence (in parentheses) are reported.

 
A heterogeneity in the composition of surface CD8 molecules exists. Most CD8+ T lymphocytes express CD8{alpha} molecules associated through disulfide bonds with the CD8 ß-chain, whereas NK cells, TCR{gamma}{delta} T cells, and extrathymically differentiated TCR{alpha}ß T cells have been described as expressing CD8{alpha} homodimers (44, 45). Furthermore, CD8 {alpha}-chains may exist in two forms produced by alternative splicing: CD8{alpha} and CD8{alpha}'. The latter does not contain a cytoplasmic tail and therefore cannot bind p56lck, being devoid of signaling capacity (46). We verified that the peripheral CD8lowtgTCR+ cells expressed CD8{alpha}ß heterodimers (by immunofluorescence staining using mAb specific for CD8{alpha}- and CD8 ß-chains; unpublished data). Additionally, by immunoprecipitation of CD8 molecules after cell surface labeling, we observed that both CD8{alpha} and CD8ß forms were present, CD8{alpha}' being undetectable (unpublished data). Except for their low expression level, the characteristics of CD8 molecules on CD8lowtgTCR+ cells are thus similar to those on thymus-dependent mainstream TCR{alpha}ß lineage cells.

In the thymus of the tgTCR H-2k/bm8 mice, the CD8lowtgTCR+ population was slightly expanded as compared with CD8low populations in tgTCR H-2k/k mice or in normal mice (Table IGo). In the periphery, they constituted ~30% of the total cells in lymph nodes, a proportion equivalent to that of CD8high tgTCR+ cells in H-2k/k mice (Fig. 6Go), suggesting that they were rapidly exported from the thymus and accumulated in the periphery.

A CD8low (CD8{alpha}ß) population, together with a major DN population, has been found in male mice coexpressing an H-Y-reactive tgTCR and the corresponding nominal Ag (47); these cells were recently suggested to belong to the TCR{gamma}{delta} lineage (48). The arguments presented above, which include the cycling status of these cells in the thymus, indicate that the CD8lowtgTCR+ thymocytes, which we find only in the presence of the partial agonist H-2Kbm8 but not in the presence of the full agonist H-2Kb, appear to exit the thymus after a selection at the ISP stage that is concomitant with a blockade of the main TCR{alpha}ß lineage at that stage. Since on the mainstream TCR{alpha}ß lineage, the step of commitment to the CD4 vs CD8 lineage takes place at the DP stage, which follows the ISP stage, it was interesting to characterize the functional potential of these CD8lowtgTCR+ cells.

CD8lowtgTCR+ cells selected in the presence of H-2Kbm8 express a partial functional program in response to H-2Kb

Cells with a CD8low tgTCRhigh phenotype have previously been shown to differentiate in the presence of low concentrations of a full agonist (39), and it was concluded that such cells lacked biologic function, as they failed to proliferate in response to Ag. We thus determined the functional phenotype of the CD8lowtgTCR+ cells in vitro and in vivo. Effectors from H-2k/bm8 mice were able to lyse H-2Kb-expressing target cells after a 3-day in vitro stimulation with B6 splenocytes. In these conditions, IFN-{gamma} secretion was also detected (Fig. 2GoB). For these two functions, CD8lowtgTCR+ cells from H-2k/bm8 mice stimulated by H-2Kb were as efficient as CD8hightgTCR+ cells from H-2k/k controls (Fig. 2GoA). However, no IL-2 was detected in supernatants of CD8lowtgTCR+ cells stimulated by H-2Kb, indicating that in these cells cytotoxicity and IFN-{gamma} production but not IL-2 synthesis were induced in response to B6. Finally, effectors from H-2k/bm8 mice were fully tolerant of bm8 for all the tested functions (Fig. 2GoB), and the CD8lowtgTCR+ phenotype remained stable in all the stimulating conditions (unpublished data). Upon adoptive transfer into sublethally irradiated mice, the CD8lowtgTCR+ cells maintained a CD8low phenotype (median CD8 fluorescence = 42), whether transferred into H-2k/k, H-2k/bm8, or H-2k/b mice. Only in the latter strain did the CD8low cells acquire an activated phenotype (CD69highCD44high) and H-2Kb-specific CTL function (on day 5 after transfer, CTL activity on RMA target cells was 8.0, 1.7, and 64.0%, respectively, in H-2k/k, H-2k/bm8, and H-2k/b mice, at 60:1 E:T ratio).

These data led us to define two situations of elicitation of a partial activation program for T cells expressing the same TCR: 1) CD8hightgTCR+ cells stimulated by a TCR partial agonist; 2) CD8lowtgTCR+ cells stimulated by a TCR full agonist. The reason for the latter could either be related to differences in the intrinsic acquisition of a functional program at the ISP as compared with the DP thymocyte stage or from the signaling resulting from engagement of the TCR/CD3 and coreceptors.

Molecular mechanisms involved in partial activation of CD8lowtgTCR+ T cells: selective deficiency of AP-1 transactivation

To understand how the triggering of one TCR can lead to the synthesis of one type of cytokine (IFN-{gamma}) but not of another (IL-2), both being transcriptionally controlled, we analyzed the activation of transcription factors such as NF-{kappa}B and AP-1 known to act on the IL-2 gene promoter (49). For this purpose, we crossed the tgTCR mice with mice transgenic for a reporter gene, the luciferase, controlled by either two AP-1 (17) or two NF-{kappa}B (R.J.P. and S.G., unpublished observations) DNA binding sites. As shown in Figure 7Go, A and B, CD8hightgTCR+ cells isolated from H-2k/k tgTCR mice expressed both NF-{kappa}B and AP-1 transactivation, whereas for CD8lowtgTCR+ cells isolated from H-2k/bm8 tgTCR, only NF-{kappa}B, but not AP-1 activity was detected in response to H-2Kb stimulation (Fig. 7Go, C and D). The partial activation by H-2Kbm8 of CD8hightgTCR+ cells from H-2k/k mice also appeared to be associated with defective AP-1 transactivation (Auphan et al., manuscript in preparation).



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  		FIGURE 7. Partial reactivity correlated with a lack of AP-1-mediated transactivation. CD8+ purified and lymph node T cells (106) from double tg (TCR x AP-1/luciferase) or (TCR x NF-{kappa}B/luciferase), respectively, H-2k/k (A and B) and H-2k/bm8 (C and D) mice, were cultured in the presence of either H-2-matched (B10.BR or CBA) or B6-stimulating cells. Luciferase activities measured at each time point are reported as the mean of three independently analyzed mice.

 
Thus, for two types of CD8+ T cells, TCR triggering in conditions of partial activation did not induce either active AP-1 or IL-2 production, whereas it succeeded in activating NF-{kappa}B and IFN-{gamma} secretion. It is not clear, however, whether the defects in signaling leading to inefficient AP-1 transactivation are similar in both instances. Further biochemical analyses will be performed in the two cases of partial activation of CD8+ T cells described here to identify the point of divergence of signals generated through the TCR after recognition of a partial or full agonist and responsible for the efficient NF-{kappa}B vs the inefficient AP-1 activations, two factors known to share numerous stimuli.

Conclusion

Two main issues were addressed in this study relating to the consequences of tgTCR engagement by a partial as compared with a full agonist. The first deals with the distinct stages of thymocyte development at which tgTCR engagement by these ligands leads to a block in the mainstream TCR{alpha}ß thymocyte differentiation and to the "selection" of alternative thymocyte subsets. The second addresses the question of the molecular basis for elicitation of partial functional programs from T cells.

Concerning the first point, we previously showed that intrathymic engagement of this H-2Kb-reactive "CD8--independent" tgTCR with the full agonist led to a block in the development of tgTCR+ DP thymocytes and to the selection of tgTCR+/CD3low DN NK1.1+ thymocytes (15, 35), which were not selected in the presence of the partial agonist H-2Kbm8. We have shown in the present study that thymic expression of the partial agonist leads to the enrichment of CD8low tgTCR+/CD3high thymocytes at the ISP stage. These results are compatible with the existence of discrete windows of selection in response to TCR/CD3 and CD8 engagement in developing thymocytes that can be revealed by the expression of a tgTCR. Furthermore, the tgTCR model described here indicates that engagement of the TCR at the CD8low ISP stage can lead to the selection of these cells and to their migration to the periphery as functional T cells. Selection at the DP stage is therefore not an absolute requirement for T cells to acquire a functional program, as suggested in a previous study (50).

Concerning the second issue, we report two distinct cases of partial reactivity of naive T cells using the same tgTCR. The first instance is that of CD8+ T cells reacting to a partial agonist as compared with a full agonist; the second is that of T cells developing in the context of the in vivo expression of the partial agonist, which now react with a partial activation program to the full agonist. In both cases, IL-2 secretion was affected whereas cytotoxic function and IFN-{gamma} production were maintained. At the molecular level, our study establishes for the first time a correlation between partial T cell activation and deficient AP-1 transactivation, despite efficient NF-{kappa}B transactivation. This is reminiscent of the defect observed in in vitro-anergized CD4+ T cell clones (51, 52). Indeed, a block in the Ras pathway leading to failure to transactivate AP-1 following TCR stimulation (53, 54), as well as a lack of induction of AP-1 components such as c-Fos, FosB, and JunB (55) have been reported. As for the maintenance of IFN-{gamma} gene activation, although AP-1 sites have been identified in the IFN-{gamma} gene promoter, their sequence seems to differ from the AP-1 binding sequences identified within the IL-2 promoter leading to the fixation of distinct transcription complexes (56), and their role in transcriptional activity remains to be established.

The approach of combining tgTCR and tg reporter genes controlled by defined transactivation elements should permit the identification of the pathways that are essential for elicitation of particular cytokine gene expression programs in response to Ag. This approach may also indicate which pathways have to be affected specifically by drugs designed as selective immunosuppressors or immunostimulators. Relevant to this point, mimics of a melanoma-specific peptide derived from nontumor sources have been described (57). If such altered ligands are presented in the thymus, our results predict that specific T cells may escape deletion but will become partially reactive against the tumor Ag, with possible functional consequences on the antitumor response.


   Acknowledgments
 
We thank Sylvie Guerder for help in establishing the luciferase assay and for criticism on the manuscript, for which we also thank He Hai-Tao, Bernard Malissen, Marie Malissen, Claude Boyer, and Annick Guimezanes. We thank M. Pontier and G. Warcollier for animal care.


   Footnotes
 
1 This work was supported by institutional grants from the Institut National de la Santé et de la Recherche Médicale and Centre National de la Recherche Scientifique and by grants from the Association pour la Recherche sur le Cancer, the Ligue Nationale Française contre le Cancer (LNFCC), the LNFCC-Comité des Bouches du Rhône, and the Groupement des Entreprises Françaises dans la Lutte contre le Cancer. A.K.S. and H.A. were, respectively, the recipients of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft and a predoctoral fellowship from the LNFCC. R.A.F. is an Investigator and S.G. an Assistant Investigator of the Howard Hughes Medical Institute. Back

2 Address correspondence and reprint requests to Dr. Nathalie Auphan, Centre d’Immunologie INSERM-CNRS de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, 13288 Marseille, Cedex 9, France. E-mail address: Back

3 Present address: Department of Medicine, University of Vermont, Burlington, VT 05405. Back

4 Abbreviations used in this paper: DN, double negative (for CD4 and CD8); BrdU, bromodeoxyuridine; DP, double positive (for CD4 and CD8); SP, single positive (CD4 or CD8); ISP, immature SP; RTOC, reaggregated thymic organ culture; TCRe, endogenous TCR; tg, transgenic; pT{alpha}, pre-TCR{alpha}. Back

Received for publication October 31, 1997. Accepted for publication January 22, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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