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The Journal of Immunology, 1999, 163: 4811-4818.
Copyright © 1999 by The American Association of Immunologists

Differential Requirements for CD4 in TCR-Ligand Interactions1

Karine Vidal2,*, Claude Daniel3,*, Mark Hill{dagger}, Dan R. Littman{dagger} and Paul M. Allen4,*

* Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110; and {dagger} Howard Hughes Medical Institute, The Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The coreceptor molecule, CD4, plays an integral part in T cell activation; it is involved in both extracellular Ag recognition and intracellular signaling. We wanted to examine the functional role of CD4 in the recognition of agonist and altered peptide ligands (APLs). We generated two CD4-deficient T cell lines expressing well-characterized TCRs specific for Hb(64–76)/I-Ek. Although the responsiveness of the T cell lines to the agonist peptide was differently affected by the loss of CD4 expression, the recognition of APLs was in both cases dramatically reduced. Nearly full responsiveness to the agonist peptide was achieved by expression of a CD4 variant that did not associate with p56lck; however, the stimulation by APLs was only partially restored. Importantly, the expression of a CD4 variant in which domains interacting with MHC class II molecules have been mutated failed to restore the reactivity to all ligands. CD4-deficient T cells were able to be antagonized by APLs, indicating that CD4 was not required for antagonism. Overall, these findings support the concepts that CD4 is an integral part of the initial formation of the immunological synapse, and that the requirement for different CD4 functions in T cell activation varies depending upon the potency of the ligand.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcell activation occurs as the result of a multimolecular interaction between T cells and APCs. The TCR/CD3 complex recognizes antigenic peptides bound to MHC molecules. T lymphocytes that recognize peptide/MHC class II complexes also express the coreceptor, CD4, which is involved in the interaction between the TCR and its ligand. CD4 binds to nonpolymorphic regions on the MHC class II molecules (1, 2, 3, 4), and its cytoplasmic tail interacts with the Src family tyrosine kinase p56lck, which plays a critical role in early events of T cell activation (5, 6, 7). CD4 enhances T cell activation in two ways: stabilization of the TCR/peptide/MHC complex and increased signal transduction (8). Thus, optimal T cell activation is thought to require binding of CD4 to the same MHC molecule as the TCR and activation of CD4-associated p56lck.

We and others have reported that T cell recognition of altered peptide ligands (APLs)5 can result in partial T cell activation and produce a wide range of biological effects (9, 10, 11). APLs are defined as analogues of antigenic peptides bearing substitutions at the TCR contact residues that alter TCR/ligand interaction without affecting peptide binding to MHC class II molecules. Agonist ligands induce full T cell activation, and weak agonists stimulate similarly, but require higher concentrations. In contrast, several distinct types of APLs have been described (9): partial agonists, which stimulate a subset of T cell effector functions (12, 13, 14, 15), and antagonist peptides, which engage the TCR but actively inhibit biological responses (16, 17, 18). A series of studies has revealed that depending on the ligand, distinct early TCR-mediated signaling events can be transduced into T cells (19, 20, 21, 22, 23). Several models have been proposed to explain how minor variations in the structures of the ligands can be translated into altered TCR signaling and aberrant T cell activation (9, 10, 11, 24). Significant insights were recently gained in this area when we showed that there is an ordered phosphorylation of the TCR{zeta} molecule and that is critically involved in maintaining the fidelity of T cell activation (25). Stimulation by APLs results in only a partial completion of the TCR{zeta} phosphorylation steps, thereby explaining the observed partial T cell activation.

CD4 expression has been shown to influence TCR specificity (26, 27, 28). We and others have demonstrated that blockade or reduction in coreceptor levels can convert a typical agonist ligand into an antagonist (29, 30, 31), resulting in the same biochemical and functional responses as those elicited by APLs (23). The role of CD4 in APL recognition has been extensively investigated in the MCC(88–103)/I-Ek Ag system. Davis and colleagues used 2B4 T cells to demonstrate that CD4 enhances the response to agonist ligands, but not to antagonists (32). In addition, they did not find any requirement for CD4 in antagonism assays, in contrast to the results reported by Madrenas et al. (23). Bottomly and co-workers (33) using AND T cells found that the early TCR-mediated biochemical events induced by a strong agonist did not require CD4, whereas the altered signaling events induced by APLs did. Therefore, from these studies, no clear role for CD4 in APL stimulation has been established.

We wanted to explore the role of CD4 in modulating the efficacy of the TCR to recognize agonist ligands and APLs using our well-established Hb(64–76)/I-Ek Ag system. To provide a comprehensive analysis of the role of CD4, we wanted to investigate the relative contributions of the two CD4 functions and extend our study to examine in parallel two different T cells. The 3.L2 and 2.102 T cells are both specific for Hb(64–76)/I-Ek complexes and respond to Hb(64–76) with similar sensitivities (EC50); however, they recognize different sets of APLs (12, 34, 35, 36). The TCR chains of 3.L2 and 2.102 were expressed in a TCR- CD4- T cell hybridoma, wild-type and mutant forms of CD4 were introduced, and the responses to a panel of agonists and APLs were determined. These two different T cells demonstrated different CD4 coreceptor requirements, with 3.L2 T cells only requiring the CD4 binding function, whereas 2.102 T cells required both binding and signaling functions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and Abs

PMA and ionomycin were purchased from Calbiochem (San Diego, CA), polybrene was obtained from Sigma (St. Louis, MO), and geneticin (G418) was purchased from Life Technologies (Grand Island, NY). mAbs GK1.5 (anti-murine CD4) and 500.A2 (anti-murine CD3{epsilon}) (37) were used as hybridoma culture supernatants. The anti-murine TCR{alpha}ß (H57–597 mAb) (38) was purchased from PharMingen (San Diego, CA). Purified unconjugated goat anti-hamster IgG, FITC-conjugated goat anti-hamster IgG(H+L), and FITC-conjugated goat anti-rat IgG(H+L) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Antigens

The Hb(64–76) peptide and the APLs were synthesized on either an Applied Biosystems model 432 (Foster City, CA) or a Rainin Symphony Multiplex synthesizer (Woburn, MA) and purified by HPLC using a C18 column (36). The composition, purity, and concentration of the peptides were determined by amino acid analysis using a Beckman model 6300 amino acid analyzer and by mass spectrometry at the Washington University Mass Spectrometry Facility. APLs of Hb(64–76) are referred to using the one-letter amino acid code for the substituted amino acid followed by its position. For example, V72 refers to Hb(64–76) peptide that has Val substituted for Asn at position 72. The nonnatural amino acid {alpha}-aminobutyric acid is abbreviated Abu. The sequences of the peptide used in these studies are: Hb(64–76), GKKVITAFNEGLK; K69, GKKVIKAFNEGLK; S69, GKKVISAFNEGLK; S70, GKKVITSFNEGLK; T72, GKKVITAFTEGLK; V72, GKKVITAFVEGLK; Q72, GKKVITAFQEGLK; Abu72, GKKVITAFAbuEGLK; D73, GKKVITAFNDGLK; N73, GKKVITAFNNGLK; M75, GKKVITAFNEGMK; and Q75, GKKVITAFNEGQK. All APLs bind to I-Ek in the same manner as Hb(64–76) (36).

Plasmids encoding the TCR {alpha}ß-chains

Complete cDNAs encoding the 3.L2 TCR{alpha} and TCRß (GenBank accession no. U46580 and U46841) chains were obtained from RT products derived from the 3.L2-12 hybrid of the Th1 clone 3.L2 (35). cDNAs encoding the 2.102 TCR{alpha} (U46581) and TCRß (U46842) chains were obtained from G2, a T cell hybrid of the Th2 clone 2.102 (35). cDNA encoding the 3.L2 TCR {alpha}-chain was cloned into pBSR{alpha}EN (3.L2-{alpha}EN), a eukaryotic expression vector containing the SV{alpha} promoter and a neomycin resistance cassette. The cDNA encoding the 3.L2 TCR ß-chain was cloned into pBSR{alpha}EH (3.L2-ßEH), which differs from pBSR{alpha}EN by having a hygromycin resistance cassette. The cDNA encoding 2.102 TCR{alpha} was cloned into pBSR{alpha}EN (2.102-{alpha}EN), and 2.102 TCRß was cloned into pSFFV-neo (2.102-pSFFV-neo).

T cell hybridomas and transfectants

The T cell hybridomas 3.L2-12 and 2.102 are both specific for Hb(64–76)/I-Ek as previously described (17). We attempted to isolate CD4 loss variants of 3.L2 and 2.102 T cell hybridomas, but were unsuccessful in two separate experiments. We then chose to generate the CD4-deficient T cell lines, 3.L2-{Delta}CD4 and 2.102-{Delta}CD4, by introducing the respective TCR {alpha}- and ß-chain genes into the 58{alpha}-ß- lymphoma, which expresses neither TCR nor CD4 (39). Plasmids encoding the 3.L2 TCR {alpha}- and ß-chains were cotransfected into 58{alpha}-ß- identically using 7.5 µg of 3.L2-{alpha}EN and 30 µg of 3.L2-ßEH followed by one pulse of 960 µF at 350 V using a Gene Pulser (Bio-Rad, Hercules, CA). Plasmids encoding the 2.102 TCR {alpha}- and ß-chains were cotransfected into 58{alpha}-ß- by electroporation using 6.25 µg of 2.102-{alpha}EN and 25 µg of 2.102ß-pSFFV-neo. Cells were seeded into flat-bottom 96-well microtiter plates, and stable transfectants were selected by the addition of G418 (0.5 mg/ml). Drug-resistant colonies were analyzed for the expression of cell surface TCR/CD3 complexes by FACS. Individual clones (seven for 3.L2 and six for 2.102) derived from either transfection were selected for their high TCR levels. A single clone of each that expressed the appropriate TCR at high levels and responded to anti-CD3 stimulation to a similar degree as the parental 3.L2.12 and 2.102 hybridomas were chosen for all subsequent studies. 3.L2-{Delta}CD4 and 2.102-{Delta}CD4 T cell lines were cultured at 37°C in 5% CO2, in RPMI 1640 (Life Technologies, Grand Island, NY) medium supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 2 mM Glutamax (Life Technologies), 50 µg/ml gentamicin (Life Technologies), 2 x 10-5 M 2-ME (Sigma), and 0.5 mg/ml G418.

Retrovirus-mediated CD4 gene transfer

Stable expression of wild-type and mutated forms of murine CD4 molecule by 3.L2-{Delta}CD4 and 2.102-{Delta}CD4 T cell hybridomas was obtained via retrovirus-mediated gene transfer. Packaging cells producing retroviruses encoding wild-type CD4 molecule or the MCA1/2 and mm4 mutants have been previously described (40, 41). The MCA1/2 variant has the Cys418 and Cys420 residues required for binding of p56lck, substituted by alanine residues (40). The mm4 variant has mutations in the D2-A strand (residues 101–107: KVTFSPG to GLTTTT) that disrupt binding of CD4 to MHC class II molecules (41). The packaging cell lines were maintained in DMEM supplemented with 10% heat-inactivated FBS (HyClone, Logan, UT), 2 mM Glutamax (Life Technologies), 100 U/ml penicillin G, 100 µg/ml streptomycin (Life Technologies), and 2 x 10-5 M 2-ME (Sigma). The infection of 3.L2-{Delta}CD4 and 2.102-{Delta}CD4 cells was performed as previously described (40). Briefly, 5 x 105 T cells were cultured in presence of polybrene (8 µg/ml) with 8 ml of culture supernatant derived from confluent culture of packaging cells. After 24 h, cells were washed and resuspended in fresh complete medium. After expansion, cells expressing cell surface CD4 molecules (5–10% of the overall population) were enriched using dextroferritin beads coated with anti-CD4 Abs, as described below. Cells (107/ml) in HBSS plus 1% FBS were mixed with 4 x 107 Dynabeads mouse CD4 (Dynal, Lake Success, NY) and incubated at 4°C for 45 min. Cells bound to beads were separated from the unbound cells using a magnet. The beads and cells were resuspended in 3 ml of HBSS/1% FBS and then reisolated. This process was repeated four times. Following the manufacturer’s recommended procedure, cells bound to beads were detached using DETACHabeads mouse CD4 (Dynal). The recovered cells were then expanded in culture. CD4-positive cells were sorted using a FACS-Vantage (Becton Dickinson, Mountain View, CA) and clonally expanded. Three to six clones of each line were analyzed, and one clone from each line was selected on the basis of equal and high levels of CD4 expression and wild-type levels of anti-CD3 or PMA and ionomycin stimulation.

Flow cytometric analysis

Cells (106/sample) were incubated on ice for 30 min with the primary mAb (neat culture supernatant), washed twice in PBS supplemented with 0.5% BSA and 0.1% sodium azide, and labeled for 30 min with FITC-conjugated goat anti-rat IgG(H+L) or goat anti-hamster IgG(H+L) (1 µg/106 cells). Controls were performed using FITC-conjugated goat anti-rat IgG alone. Cells were washed twice, resuspended in PBS containing 1% paraformaldehyde, and analyzed on a FACScan flow cytofluorometer using CellQuest software (Becton Dickinson).

T cell activation and IL-2 assays

Ag stimulation and antagonism experiments of the T cell hybridomas were performed following established protocols (17, 35). Stimulation by cross-linking of TCR/CD3 complexes was performed using immobilized anti-CD3 mAb (500A2). Briefly, 96-well flat-bottom microtiter plates were coated with goat anti-hamster IgG Ab (1 µg/well) for 90 min at 37°C, followed by three washes and incubation with 500A2 mAb (1/50 dilution of supernatant culture); after extensive washing, T cells were added at 5 x 104/well. In parallel, T cell hybridomas were stimulated with PMA (50 ng/ml) and ionomycin (1 µM). T cell activation was ascertained by measurement of IL-2 released in supernatants after 24-h culture using the IL-2-dependent cell line CTLL-2 (35).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Loss of CD4 expression differentially affects the responses of 3.L2 and 2.102 T cells to Hb(64–76)

CD4-deficient 3.L2 and 2.102 T cell lines, 3.L2-{Delta}CD4 and 2.102-{Delta}CD4, were generated by transfection of the TCR-/CD4- 58{alpha}-ß- T cell hybridoma with the TCR {alpha}- and ß-chain genes isolated from either 3.L2 or 2.102 T cells (see Materials and Methods). Flow cytometric analysis of stable transfectants 3.L2-{Delta}CD4 and 2.102-{Delta}CD4 showed that 3.L2 and 2.102 TCR {alpha}ß-chains were expressed on the cell surface in association with the endogenous CD3 polypeptides (Fig. 1Go). Both T cell lines remained CD4 negative (Fig. 1Go, A and B). To recapitulate the phenotype of the original 3.L2 and 2.102 Th cells, the wild-type murine CD4 molecule was expressed in the 3.L2 and 2.102 CD4-deficient T cell lines. FACS analysis showed that following retrovirally mediated CD4 gene transfer, both 3.L2 and 2.102 T cells gained high surface expression of the CD4 molecule (Fig. 1Go, C and D). The CD4-reconstituted 3.L2 and 2.102 T cell lines are referred to as 3.L2-CD4 and 2.102-CD4, respectively. The CD4-deficient and CD4-positive T cell lines produced the same level of IL-2 upon cross-linking of TCR/CD3 complexes or treatment with PMA and ionomycin (data not shown).



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  		FIGURE 1. FACS analysis of the cell surface expression of TCR/CD3 complexes and CD4 molecules on 3.L2 and 2.102 T cell lines. The top panels show cell surface expression of the CD3 molecule on the CD4-deficient T cell lines, 3.L2-{Delta}CD4 (A) and 2.102-{Delta}CD4 (B), generated as described in Materials and Methods. The bottom panels show the expression of CD3 and CD4 molecules by the two CD4-positive T cell lines, 3.L2-CD4 (C) and 2.102-CD4 (D). The CD4-positive lines were obtained by retrovirus-mediated gene transfer of the full-length cDNA encoding for the murine wild-type CD4 molecule. T cells (106/sample) were incubated with either 500A2 mAb specific for the CD3{epsilon} molecule (broken line) or the anti-CD4 mAb GK1.5 (solid line), followed by incubation with an FITC-conjugated goat anti-hamster or goat anti-rat Ab, respectively. As a negative control, cells were stained with the secondary Ab alone (dotted lines). Mean fluorescence intensity is plotted on log scale (x-axis) against the relative number of cells (y-axis).

 
We next analyzed the capacity of these T cells to respond to the agonist Hb(64–76) peptide. Both reconstituted 3.L2-CD4 and 2.102-CD4 T cells produced IL-2 in response to Hb(64–76)/I-Ek complexes in a similar dose-dependent manner (Fig. 2Go). Interestingly, in the absence of CD4 expression, 3.L2 and 2.102 T cells manifested very different patterns of reactivity to this ligand. The 3.L2-{Delta}CD4 T cells responded vigorously to Hb(64–76) (Fig. 2GoA), whereas the ability of 2.102 T cells to respond to the same agonist ligand was dramatically reduced upon loss of CD4 (Fig. 2GoB). The 3.L2-{Delta}CD4 T cells had a 10-fold shift in responsiveness, whereas the 2.102-{Delta}CD4 T cells weakly responded even at the highest Ag concentrations. To confirm that these observed responses correlated with the loss of CD4 surface expression, we performed activation assays of CD4-positive T cells, 3.L2-CD4 and 2.102-CD4, in the presence of the GK1.5 anti-CD4 mAb and observed that with nonsaturating concentrations of GK1.5, IL-2 production by both CD4-positive T cell lines in response to Hb(64–76) peptide can be reduced to the level secreted by CD4-deficient T cells (data not shown). Overall, these studies revealed that the two Hb(64–76)-specific T cells had significantly different requirements for CD4, with 3.L2 T cells not requiring CD4 to respond fully to Hb(64–76), whereas 2.102 T cells did.



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  		FIGURE 2. Ag-specific activation of 2.102 T cells is more dependent on CD4 expression than activation of 3.L2 T cells. CD4-deficient ({square}) and CD4-positive ({blacksquare}) 3.L2 (A) and 2.102 (B) T cells (5 x 104/well) were incubated for 24 h with the indicated concentrations of Hb(64–76) peptide presented by the B cell lymphoma CH27 (5 x 104/well). T cell activation was measured by IL-2 production using the CTLL-2 cell line. The values represent the mean of triplicate determinations of [3H]TdR incorporation by the IL-2-dependent CTLL-2 cells. The SD were <20% of the mean, and the data are representative of seven independent experiments.

 
Differential CD4 requirements for APL function

We previously identified a series of APLs for the 3.L2 TCR (34, 35, 36) and the 2.102 TCR (12). These APLs have been ranked according to their relative activities and included agonists, weak agonists, and antagonists. We first verified that the APLs identified as weak agonists for the 3.L2 TCR or the 2.102 TCR were also able to activate our reconstituted 3.L2-CD4 and 2.102-CD4 T cells. As shown in Fig. 3GoA, Abu72, T72, and V72, representatives of weak agonists for the 3.L2 TCR, were able to stimulate IL-2 production by 3.L2-CD4 T cells. These APLs required a 10-fold (Abu72) to 100-fold (T72 and V72) higher concentration to achieve the same level of T cell activation as that obtained by Hb(64–76). The same pattern of responsiveness was observed in previous studies using the 3.L2 T cell hybridoma, Th1 T cell clones, and TCR transgenic T cells (36, 42). For the 2.102 TCR, APLs containing substitutions at positions 69, 70, and 75 have been previously identified, with K69 being a strong agonist, and S70, M75, and Q75 being weak agonists for the 2.102 T cell hybridoma, Th2 clones, or TCR transgenic T cells (12, 35, 43). The same pattern of reactivity was observed for the reconstituted 2.102-CD4 T cells (Fig. 3GoC). Thus, expression of the 3.L2 or 2.102 TCR and CD4 in the 58{alpha}-ß- T cell hybridoma reproduced the phenotypes of the original Th cells.



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  		FIGURE 3. CD4 expression is required for the activation of 3.L2 and 2.102 T cells by APLs. 3.L2 T cells (5 x 104/well) expressing wild-type CD4 molecules (A), deficient for CD4 expression (B), 2.102 CD4 positive (C), or CD4 deficient (D) were incubated with increasing concentrations of the indicated peptides presented by CH27 cells (5 x 104/well). After 24 h of culture, supernatants were assayed for IL-2 production as described in Fig. 2Go. The SD were <15% of the mean. The results are representative of seven experiments.

 
The capacity of the CD4-deficient 3.L2 and 2.102 T cell lines to be activated upon recognition of their respective APLs was then investigated. Activation of 3.L2 T cells by Abu72, V72, and T72 peptides was dramatically affected by the loss of CD4 expression (Fig. 3Go). The response to Abu72 was almost entirely eliminated, and those to V72 and T72 were completely eliminated. The responses of 2.102 T cells to APLs were also clearly dependent on the expression of CD4 molecules, with only K69 weakly stimulating (Fig. 3GoD). These data reveal that recognition of APLs by 3.L2 TCR is much more dependent on CD4 expression than the response to Hb(64–76), while the responses of 2.102 to both Hb(64–76) and the APLs definitely require CD4. Therefore, CD4 increases the response to a strong agonist less than that to a weak agonist or antagonist.

The function of CD4 varies depending upon the ligand being recognized

To elucidate what functions of the CD4 molecule were involved in the responses of 3.L2 and 2.102 to the different ligands, we used two well-characterized mutant forms of CD4. The mm4 mutation eliminated the interaction of the CD4 with MHC class II molecule (41), and the MCA1/2 mutations abolished the ability of CD4 to associate with p56lck (40). These mutant forms of CD4 were introduced into both CD4-deficient 3.L2 and 2.102 cells by retrovirus-mediated gene transfer (see Materials and Methods), and lines were obtained that expressed high levels of CD4 and TCR/CD3 levels, comparable to the parental CD4-deficient T cells.

We first addressed what function of CD4 was involved in the 10-fold enhancement by CD4 in 3.L2 T cells stimulated by the Hb(64–76) ligand. The MCA1/2 mutant was only slightly less active than the wild-type CD4, whereas the mm4 mutant was essentially the same as the CD4-deficient cells (Fig. 4GoA). Thus, for the strong agonist, Hb(64–76), the role of CD4 only involves its interaction with class II. The response to the APLs was also enhanced by the MCA1/2 mutant, but not by the mm4 mutant (Fig. 4Go, B–D). The stimulation of both CD4-deficient T cell lines was not increased when the agonist ligand was presented by ICAM-1-positive APCs compared with ICAM-1-negative APCs (data not shown), indicating that an increase in cell-cell adhesion via ICAM-1/LFA-1 was insufficient to restore full responsiveness. Thus, the role of CD4 in the response of 3.L2 T cells to Hb(64–76) and APLs largely involves interaction with class II molecules and not p56lck.



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  		FIGURE 4. Effect of the expression of mutated forms of the CD4 molecule on 3.L2 T cell responsiveness. The reactivities of the 3.L2 T cell lines expressing the wild-type CD4 molecule (3.L2-CD4), the mm4 variant (3.L2-mm4), or the MCA1/2 variant (3.L2-MCA1/2) were compared with that of CD4-deficient 3.L2 T cells (3.L2-{Delta}CD4). T cells (5 x 104/well) were incubated with increased concentrations of either the Hb(64–76) peptide (A) or the indicated APLs (B–D) presented by CH27 cells (5 x 104/well). 3.L2 T cells were chosen to express comparable levels of CD3/TCR and CD4. The levels of expression (mean fluorescent intensity) of the CD4 mutants were: 3.L2-CD4, 18.7; 3.L2-MCA1/2, 14.6; and 3.L2-MM4, 15.7. T cell activation was measured as described in Fig. 2Go. The results are representative of four experiments.

 
The response of 2.102 T cells expressing the mutant CD4 molecules was markedly different from that of 3.L2 (Fig. 5Go). The responses to Hb(64–76) and K69 were fully reconstituted with the MCA1/2 mutant, but not the mm4 (Fig. 5Go, A and B). Thus, for stimulation with these ligands, CD4 binding to class II molecules was sufficient. In contrast, full activation in response to S70 and Q75 required the wild-type CD4 molecule, with neither mutant fully reconstituting (Fig. 5Go, C and D). Thus, for these weaker ligands, the role of CD4 involved its ability to bind to both class II molecules and p56lck. Taken together, these data indicate in two different T cell lines that the MHC class II binding function of CD4 is sufficient for responses to strongly agonistic peptides and APLs, but that the additional binding of p56lck is required for responses to weak APLs.



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  		FIGURE 5. Effect of the expression of mutated forms of the CD4 molecule on 2.102 T cell responsiveness. The reactivities of 2.102 T cells expressing the wild-type CD4 molecule (2.102-CD4), the variant mm4 (2.102-mm4), or the variant MCA1/2 (2.102-MCA1/2) were compared with the activation of CD4-deficient T cells (2.102-{Delta}CD4). T cells (5 x 104/well) were incubated with increased concentrations of the Hb(64–76) peptide (A) or the indicated APLs (B–D) presented by CH27 cells (5 x 104/well). 2.102 T cells were chosen to express comparable levels of CD3/TCR and CD4. The levels of expression of the CD4 mutants were: 2.102-CD4, 58.2; CD4-MCA1/2, 59.2; and 2.102-mm4, 42.6. T cell activation was measured as described above. The results are representative of four experiments.

 
CD4 is not required for antagonism by APLs

We next examined the role of CD4 in T cell antagonism using 3.L2 and 2.102 T cells lacking CD4 expression. The D73 peptide was able to antagonize both 3.L2-CD4 and 3.L2-{Delta}CD4 cells with similar IC50 values (17, 36) (Fig. 6GoA). The null peptide Q72 did not affect the T cell responsiveness of either T cell. We additionally tested whether APLs that required CD4 for full agonist activity could act as antagonists in the absence of CD4. The T72 and V72 APLs were as potent antagonists of the CD4-deficient 3.L2 T cells to Hb(64–76) as D73 (Fig. 6GoB).



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  		FIGURE 6. TCR antagonism of the 3.L2 T cells in the absence of CD4 expression. 3.L2-CD4 (A) and 3.L2-{Delta}CD4 T cells (B; 5 x 104/well) were incubated with increased concentrations of the indicated APLs presented by Hb(64–76)-prepulsed CH27 cells (5 x 104/well). CH27 cells were prepulsed for 2 h at 37°C with 50 µM of the agonist Hb(64–76) peptide. Activation of 3.L2-CD4 and 3.L2-{Delta}CD4 T cells in the absence of added APLs is depicted as a dashed line. T cell responsiveness was measured as described in Fig. 2Go. The results are representative of at least four independent experiments.

 
The 2.102 antagonists, S69, N73, and D73 (17), were assessed for their ability to inhibit the different 2.102 T cell lines. The activation of CD4-deficient 2.102 T cells in response to Hb(64–76) peptide was reduced in a dose-dependent manner by the presence of all three antagonists (Fig. 7GoA). The 2.102-mm4 and 2.102-MCA T cell lines also were antagonized to a similar degree. There was no observable change in the activity (IC50) of these different antagonists in any of the different 2.102 cell lines, even when the level of agonist stimulation differed. These results confirm and extend our observations on the effect of CD4 blockade in converting a weak agonist into an antagonist ligand (31). Taken together, these data clearly demonstrate that TCR antagonism does not require CD4 expression.



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  		FIGURE 7. TCR antagonism of 2.102 T cells in the absence of CD4 expression. TCR antagonism assays were performed on the four 2.102 T cell lines generated: 2.102 T cells expressing the wild-type CD4 molecule (2.102-CD4; A), 2.102 T cells deficient of CD4 expression (2.102-{Delta}CD4; B), expressing the MCA1/2 variant (2.102-MCA1/2; C), or the mm4 variant of the CD4 molecule (2.102-mm4; D) were incubated with increasing concentrations of the indicated APLs presented by Hb(64–76)-prepulsed CH27 cells (5 x 104/well). CH27 were prepulsed for 2 h at 37°C with 10 µM (A and C) or 100 µM (B and D) of the agonist Hb(64–76) peptide. Activation of T cells in the absence of added APLs is depicted as a dashed line. T cell activation was measured as described in Fig. 2Go. The results are representative of four independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In these studies we used the well-defined Hb(64–76)/I-Ek-specific TCR system to assess the role of the CD4 molecule in TCR recognition of a series of different potency ligands. We examined the T cell responsiveness to ligands that were previously identified as strong agonist, partial agonist, or antagonist ligands. The 3.L2 and 2.102 TCRs derived from two different Th cells recognize the same Hb(64–76)/I-Ek ligand, but have distinct fine specificity. By generating CD4-deficient 3.L2 and 2.102 T cell lines, we showed that these two T cells demonstrate a differential dependency on CD4 expression for an optimal T cell response to the agonist Hb(64–76) peptide. However, the responsiveness of both T cell lines to their respective suboptimal ligands was strongly CD4 dependent. In our system the ability of CD4 to interact with class II molecules was critically involved, whereas its intracellular signaling role was most evident with the weaker ligands. In addition, we showed that TCR antagonism can similarly be obtained in both CD4-deficient and CD4-positive T cell lines, providing evidence that there is no direct involvement of the CD4 molecule and CD4-associated p56lck kinase in TCR antagonism.

By examining two different T cells in parallel, we are also able to provide some perspective on the extensive literature involving CD4. No clear consensus on the function of CD4 emerged from these studies because each used a different Ag system. For example, the HEL-specific T cell, 171, absolutely required CD4 association with p56lck for activation (40). For another HEL-specific T cell, 3A9, CD4 was not required when the naturally processed peptides, HEL(48–62) and HEL(42–63), were used. However, it was absolutely required when the HEL(48–61) peptide was used (28). 3A9 stimulation by HEL(48–61) was restored by either the mm4 or cytoplasmic tail-less mutant of CD4. Our studies using two T cells and a series of different potency ligands for each essentially recapitulate these collective findings. Thus, our studies reveal that the role of CD4 in T cell activation is flexible and varies depending upon the nature of the ligand being recognized by the TCR.

Successful signaling through the TCR requires oligomeric clustering of the TCR and the creation of an immunological synapse characterized by the clustering of key molecules and the exclusion of others (44, 45, 46, 47). Our recent studies, using a real-time quantitative imaging analysis system, revealed that immunological synapse formation is a multistage process (48). We examined the role of CD4 in the immunological synapse formation by exploiting the ability of 3.L2 transgenic T cells to develop in the absence of CD4. CD4 T cells were much less efficient at stopping and forming synapses. Thus, CD4 plays a critical role in very early stages of immunological synapse formation. We have previously determined the binding kinetics and affinity of the 3.L2 TCR for five different ligands, including many of those studied here (49). Each of these ligands was examined using the image analysis system along with an equally defined set of ligands in the 2B4/MCC/I-Ek system. We found that the amount of MHC/peptide accumulated in the synapses correlated strongly in both Ag systems to the t1/2 of the complexes and not with the Kd or kon. A model for the role of CD4 then emerges from these studies that supports the findings in our present study. In this kinetic model, the t1/2 of the TCR-ligand interaction is the critical parameter for the role of CD4. The time required to recruit CD4 to the engaged TCR would provide a kinetic window that determines the ability of ligands to initiate early signaling events. This model would predict that there would be at least two different thresholds for full T cell stimulation. The t1/2 of the TCR:ligand interaction must be above a first threshold, which would allow the time for CD4 to be recruited into the complex. TCR-ligand interactions with a t1/2 less than this first threshold would not result in T cell activation. These types of interactions still may play a functional role, especially in T cell development in the thymus. The second threshold would be for TCR-ligand interactions with longer t1/2, such as 3.L2:Hb(64–76). This interaction is above a threshold, for which CD4 is not essential for the subsequent steps to occur. For T cells in this category, CD4 can be eliminated experimentally; however, physiologically, CD4 is still an integral part of T cell synapse formation and subsequent activation. We would predict that T cells with a t1/2 between these two thresholds would represent the majority of class II-restricted T cells, and that they would require CD4.

An apparent paradox of our findings on the role of CD4 involves the phenomenon of antagonism. Our findings and those of Davis and colleagues clearly demonstrate that CD4 is not needed for antagonism (32). If CD4 augments the activity of weak ligands, then why is it not needed for antagonism by weak ligands? The antagonism assay involves stimulation with APCs pulsed with suboptimal concentrations of an agonist and the continual presence of high concentrations of an antagonist. It is thus possible that, under these limited conditions, CD4 does not make any appreciable contribution. The mechanism of antagonism is still unknown, and its identification should help to address this issue.

Overall, our studies highlight the multifunctional role that CD4 plays in T cell activation. The CD4 molecule can interact with the TCR, class II molecules, and p56lck. The requirement for each of these interactions in T cell activation is therefore defined by the nature of the recognition of the peptide/MHC complex by the TCR.


   Acknowledgments
 
We thank Dave Donermeyer for assistance in cloning the TCR genes, Parveen Chand for assistance in cell sorting, and Gil Kersh for help with the initial 3.L2 antagonism studies. We are also grateful to Steve Horvath for synthesis, purification, and amino acid analysis of the peptides. We thank the Washington University Mass Spectrometry facility for mass spectrometry analysis. We thank Jerri Smith for her assistance with the preparation of this manuscript. Special thanks to Andrey Shaw and Mike Dustin for fruitful discussions.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health. Back

2 Current address: Centre de Recherche Nestle, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland. Back

3 Current address: Human Health Research Center, Institut Armand-Frappier, 531 boulevard des Prairies, Laval, Quebec, Canada H7N 4Z3. Back

4 Address correspondence and reprint requests to Dr. Paul M. Allen, Department of Pathology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8118, St. Louis, MO 63110. E-mail address: Back

5 Abbreviations used in this paper: APL, altered peptide ligand; Hb, hemoglobin. Back

Received for publication April 16, 1999. Accepted for publication August 19, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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