Molecular Requirements for T Cell Recognition by a Major Histocompatibility Complex Class II–restricted T Cell Receptor: The Involvement of the Fourth Hypervariable Loop of the Vα Domain

The CD4⁻, TCR α⁻β⁻ cell line 58α⁻β⁻ (40) was provided by Dr. Stephen Hedrick (University of California at San Diego, CA), with permission from Dr. Bernard Malissen (INSERM-CNRS, Marseille-Luminy, France). The I-A^u expressing B cell line, PL-8 (derived by fusing LPS-activated splenocytes from H-2^u mice with the M-12.C3 lymphoblast line, [41]) was used as the APC line and was provided by Dr. David Wraith (University of Bristol, UK). The hybridoma producing the anti-Vβ8 mAb F23.1 (42) was a gift from Drs. John Kappler and Philippa Marrack (University of Colorado Health Science Center, Denver, CO). Hybridomas expressing the anti–I-A mAbs Y3P (HB-183), 10.2.16 (TIB-93), and the anti-CD4 mAb GK1.5 (TIB-207) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Anti-CD3ε mAb 145-2C11 (CRL-1975; ATCC) was purchased from PharMingen (San Diego, CA). The horseradish peroxidase- conjugated anti–mouse/rat IgG antibodies used as secondary antibodies for immunofluorescence studies were purchased from ICN Biomedicals, Inc. (Costa Mesa, CA). The NH₂-terminal peptide Ac1-11 of rat myelin basic protein (MBP) and an analog in which lysine at position 4 is substituted by tyrosine (Ac1-11[4Y]), were synthesized at the peptide synthesis unit of the Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, Texas.

The α and β shuttle vectors (43) used as TCR expression vectors in this study were a kind gift of Dr. Mark Davis (Stanford University, Palo Alto, CA). The construction of the 1934.4 α and β chain expression plasmids using the 1934.4 Vα and Vβ domain genes (isolated as in reference 44), was carried out using 1934.4 TCR-specific oligonucleotide primers and essentially the strategy of Patten and colleagues (43). Mutagenesis of HV4α residues K68 and E69 (numbering as in reference 37; in reference 4 these residues are numbers 67 and 68, respectively) together with E69 were carried out as described by Kunkel et al. (45). E69 was substituted by alanine, and K68 and E69 were both substituted by alanine to generate the mutants E69A and K68AE69A, respectively. The oligonucleotides used for mutagenesis were: 5′ AGG TGG CTG CTT TAT TG 3′ for E69A, and 5′ GAG GTG GCT GCT GCA TTG TAT GT 3′ for K68AE69A. Mutated bases are indicated by underlining. K68 was substituted by alanine to generate K68A using the splicing by overlap extension method (46) and the following complementary oligonucleotides (mutated bases underlined): 5′ GTG GCT TCT GCA TTG TAT GT 3′ and 5′ ACA TAC AAT GCA GAA GCC ACC 3′. The presence of the mutations, and the absence of second site mutations, was confirmed by nucleotide sequencing using the Thermo Sequenase ³³P radiolabeled terminator cycle sequencing system (Amersham, Arlington Heights, IL). All (sub)cloning steps and PCRs were carried out using standard methods. An expression plasmid encoding murine CD4 (14) was a generous gift of Dr. Syamal Datta (Northwestern University Medical School, Chicago, IL).

For transfections, the 1934.4 α (mutant and WT), 1934.4 β, and CD4 expression plasmids were purified using Qiagen (Chatsworth, CA) plasmid midi-prep kits. 5 μg of α shuttle vector and 10 μg of β shuttle vector were used per transfection. The plasmid encoding the murine CD4 molecule (14) was used together with α and β shuttle vectors at 10 μg per transfection. As transfection recipient, the TCR⁻, CD4⁻ cell line 58α⁻β⁻ (40) was used (1 × 10⁷ cells per transfection). Cells were washed once with ice-cold Hepes-buffered saline, resuspended in 0.5 ml phosphate-buffered saline (PBS) and placed on ice in Bio-Rad (Hercules, CA) electroporation cuvettes (0.4 cm gap). The relevant constructs were added and mixed with the cells by gentle pipetting. Cells were kept on ice for 10 min and pulsed in a Bio-Rad Gene Pulsar at 250V/cm and 960-μF capacitance. After pulsing, cells were immediately diluted into ice-cold non-selective medium (RPMI 1640 supplemented with 10% FCS, 5 × 10⁻⁵ M 2-ME, 100 U/ml of penicillin G, and 100 μg/ml streptomycin) and placed on ice for 10–15 min. Electroporated cells were plated into two 96-well plates at 100 μl/well. 2 d later, 100 μl of 2× selection medium (2 μg/ml mycophenolic acid, 400 μg/ml xanthine) was added per well. 4 d later, 80% of the medium was replaced with fresh selective medium. Mycophenolic acid–resistant transfectants (30–50 per transfection) were obtained and screened for surface expression of the transfected TCRs by flow cytometry.

For analysis of cell surface expression of TCR and CD4, transfectants (1 × 10⁵/well in a 200 μl volume) were incubated with 10 μg/ml of anti-Vβ8 mAb (F23.1) or with 10 μg/ml anti-CD4 mAb (GK1.5) at 4°C for 1 h. Cells were washed three times with PBS containing 1% BSA (Sigma Chemical Co., St. Louis, MO) and were incubated with anti–mouse IgG–FITC conjugate for TCR staining or anti–rat IgG–FITC conjugate for CD4 staining at 4°C for 1 h. Controls were incubated with secondary conjugates only. Cells were washed three times with 1% BSA containing PBS, resuspended in PBS, and analyzed in a flow cytometer (Becton Dickinson). Results were analyzed using the Cellquest program.

Transfectants (1 × 10⁵/well in 96-well plates) were stimulated either with 10 ng/ml PMA + 500 ng/ml ionomycin, plate-bound anti-Vβ8 antibody, F23.1, or plate bound anti-CD3ε antibody, 145-2C11. For the antibody stimulations, plates were incubated overnight with 10 μg/ml antibody and washed twice with PBS before adding the transfectants. Controls were run in parallel without any stimulation. Culture supernatants were harvested after 24 h, and after a single freeze-thaw, 80 μl aliquots were incubated with an IL-2–dependent cell line, CTLL-2 (TIB214; ATCC; 5 × 10³ cells/well). Cells were pulsed 24 h later with 1 μCi of ³H/well for 16–18 h and thymidine incorporation analyzed by liquid scintillation spectroscopy (LKB Pharmacia, Uppsala, Sweden). All assays were done in triplicates and data is expressed as mean values of triplicate measurements following background subtraction (in cpm) ± SD.

Transfectants    (1 × 10⁵/well in 96-well plates) were incubated with graded doses of the peptides Ac1-11 or Ac1-11[4Y] together with PL-8 cells (1 × 10⁵/well) as APCs. No peptide was added to the control wells. For anti-CD4 or anti–I-A^u inhibition, graded doses of anti-CD4 (GK1.5) or anti–I-A (Y3P, 10.2.16) mAbs were used. Appropriate isotype matched antibodies were used as controls. The responses of transfectants were measured by IL-2 production that was detected by the proliferative response of CTLL-2 cells as above. All stimulation assays were done in triplicates and data expressed as percent of response to the plate-bound anti-CD3ε antibody, 145-2C11.

Far-ultraviolet CD analyses were performed as previously described (44), using an AVIV model 60DS circular dichroism spectrophotometer at 25°C and a cell of 0.2 cm path length.

To analyze the effects of mutating HV4α residues 68 and 69 of the 1934.4 TCR on antigen responsiveness, WT and mutated α chain genes were transfected with the WT β (Vβ8.2-Jβ2.3) chain into a TCRα⁻β⁻ thymoma, 58α⁻β⁻ (40). This murine T cell thymoma line lacks an endogenous TCR, but has a functional CD3–TCRζ complex that can be expressed on the cell surface with the transfected TCR α and β chains. Fig. 1 shows the location of the residues that were targeted for mutagenesis on the X-ray crystallographic structure of the 1934.4 Vα domain (4).

Mycophenolic acid–resistant transfectants were analyzed for expression levels of surface TCR by flow cytometry using a Vβ8-specific mAb, F23.1. Since the expression levels varied between transfectants, transfectants with comparable levels of surface TCR were chosen for further analysis (Fig. 2). These transfectants were analyzed for responsiveness (assessed by quantitating IL-2 production) to PMA in addition to anti-Vβ8 (F23.1) and anti-CD3ε (145-2C11) antibody-mediated cross-linking. Responses to all three types of stimulation did not differ markedly, indicating that the signaling machinery of the transfectants was intact and that the Vα mutations do not affect signaling via antibody-mediated β chain cross-linking (Fig. 2 D). Differences observed upon 145-2C11 (Fig. 2 C) stimulation probably reflect minor differences in the surface TCR levels observed in Fig. 2 A. To control for variability, responses to cognate pMHC (below) have been normalized with respect to those obtained from stimulation with 145-2C11 and expressed as percentages of 145-2C11 responses. A similar approach was taken by Patten and colleagues for the analysis of anti-cytochrome c–I-E^k responses by transfectants expressing the WT 2B4 TCR and its mutated derivatives (43).

The 1934.4 TCR recognizes the NH₂-terminal 11-mer (or nonamer) of myelin basic protein (MBP) associated with I-A^u and the acetylation of the NH₂ terminus of this peptide (abbreviated to Ac1-11) is essential for T cell recognition (39). Position 4 analogs (position 4 substituted by alanine and tyrosine, designated Ac1-11(4A) and Ac1-11(4Y), respectively) of this peptide bind with higher affinity to I-A^u (47, 48), resulting in shifts of dose response curves of 1934.4 hybridoma cells (47). We tested the ability of the WT versus mutant transfectants to recognize the Ac1-11 peptide presented in the context of the MHC class II molecule I-A^u. When Ac1-11 was used for stimulation (Fig. 3 A), the WT transfectants respond, albeit poorly, whereas there is an almost undetectable response observed for the three types of mutant transfectants (K68A, E69A, and K68AE69A). This poor responsiveness is most likely due to the lack of CD4 expression by the transfectants, which in other systems is known to decrease antigen responsiveness (31, 49). Therefore, to make quantitative comparisons between the WT and mutant transfectants in the absence of CD4, we tested their responses to the higher affinity peptide Ac1-11(4Y). Fig. 3 B shows that the response of the WT transfectants to Ac1-11(4Y) is significantly better compared with their response to Ac1-11 (Fig. 3 A), and this is reminiscent of the data of others for the parent 1934.4 hybridoma (47, 48, 50). The mutant transfectants also show higher and detectable levels of responses to the higher affinity peptide. Replacement of glutamic acid at position 69 with alanine (E69A) results in a slight, but significant, reduction in response as compared with WT; however, the double mutants (K68AE69A) and the K68A mutants show substantial decreases in their responses relative to WT transfectants.

The data indicate that lysine at position 68 of HV4α plays a crucial role in the TCR–pMHC interaction, most likely by either directly contacting Ac1-11–I-A^u or indirectly by affecting the conformation of neighboring CDRs in the Vα domain. Importantly, several observations indicate that the mutations analyzed in this study do not have significant effects on the conformation of the TCR Vα domain. First, the WT and mutant transfectants express similar levels of surface TCR, and aberrant folding of the TCR α chain due to mutation would be expected to affect this. Second, CD spectroscopic analysis of the WT and mutant Vα domains, expressed as recombinant proteins in Escherichia coli, indicate that the Vα domains are folded into structures of high β-sheet content (data not shown). Unfortunately, the lack of an anti-Vα4.2 antibody that is conformationally dependent precludes analyses using such a reagent.

Since the differences in response to peptide were most striking between the WT and the K68A mutant, transfectants expressing these TCRs were analyzed further. The T cell coreceptor, CD4, has been shown to enhance the capability of thymocytes and mature T cells to recognize antigen (reviewed in references 19 and 51). To analyze whether CD4 can compensate for the effect of the K68A mutation, cotransfections of the 1934.4 WT or K68A mutant α chain together with WT β chain and CD4 expression constructs using the 58α⁻β⁻ thymoma cell line as recipient were carried out. The surface TCR and CD4 levels are comparable between the WT and K68A transfectants (Fig. 4 A), although the TCR levels for the transfectant K68A/CD4-32 are slightly lower. These CD4⁺ transfectants show similar responses to 145-2C11 and F23.1 stimulation (Fig. 4, B and C). In addition to shifting the dose response curve of the WT transfectants, cotransfection of CD4 almost completely (K68A/CD4-12 mutant) or partially (K68A/CD4-32 mutant) restores the K68A mutant responses to cognate pMHC to the levels seen with the WT transfectant, WT/CD4-43 (Fig. 5). The WT transfectant WT/CD4-33 is, however, still more responsive than the two mutant transfectants and this is particularly so for the Ac1-11 peptide (Fig. 5 A). This significant gain of function for the K68A mutants suggests that either the increased avidity and/or enhancing the efficiency of TCR signaling by CD4 coexpression can compensate, in part at least, for the suboptimal nature of the K68A TCR–pMHC interaction. In addition, the enhancing effect of CD4 is much greater for the K68A mutants than for the WT transfectants (Figs. 3 and 5).

To demonstrate that the increased IL-2 production by the K68A mutants is due to CD4 coexpression, the sensitivity of the WT and K68A transfectant responses following Ac1-11(4Y)–I-A^u exposure to blockade by the anti-CD4 mAb GK1.5 was investigated. K68A transfectants are significantly more sensitive to the effects of GK1.5 than the WT transfectants (Fig. 6 A). Importantly, this difference is observed for K68A/CD4-12 relative to WT/CD4-43, and these two transfectants show essentially the same dose response to Ac1-11(4Y)–I-A^u. The effects of GK1.5 are consistent with the observation that WT transfectants without CD4 coexpression are responsive to antigen, in contrast to the K68A transfectants (Fig. 3). Finally, the sensitivity of the WT and K68A transfectants to blockade by anti–I-A antibodies was also investigated, because this was expected to reveal differences in affinities of the corresponding TCR–pMHC complexes. Two anti–class II mAbs (10-2.16 and Y3P) that recognize I-A^u were used, and for both anti–MHC class II antibodies the K68A transfectants are more sensitive to inhibition than the WT transfectants (Fig. 6 B; data shown only for 10.2.16). As with the GK1.5 inhibition, this difference in sensitivity to anti–MHC class II inhibition is seen when the WT/CD4-43 and K68A/ CD4-12 transfectants are compared. Taken together with the similarity of the dose responses to cognate antigen in the absence of antibodies for these two transfectants (Figs. 5 and 6), this indicates that the affinity of the K68A TCR for cognate antigen is lower than that of the WT TCR.

To date, there are no high-resolution structural data available for a TCR–pMHC class II complex, but functional studies (14, 15) of class II–restricted TCRs indicate that the orientation of the TCR is similar to the diagonal configuration observed in the X-ray structures of several class I–restricted TCRs (10–13). The crystal stuctures indicate that HV4α residues sometimes (11), but not always (10, 12, 13), make contact with cognate ligand. In addition, binding studies using a recombinant class I–restricted TCR indicate a minor contribution for these residues to the binding energy of the interaction (38). However, the functional relevance of HV4α residues in T cell activation have not, to our knowledge, been investigated for either class I– or class II–restricted TCRs, and the current study addresses this issue for an autoreactive TCR that recognizes MBP Ac1-11 bound to I-A^u. In contrast to amino acids located in HV4α, the functional significance of HV4β residues have been more extensively analyzed, and this TCR region is known to play a role in contacting several bacterial and endogenous superantigens (52–54). In addition, recent modeling/X-ray structural studies for the interaction of the murine 2C TCR with allo-ligand have indicated that the HV4β residue R69 might contribute toward the binding energy through electrostatic effects (55).

Our findings identify a role for HV4α residues of the 1934.4 TCR in pMHC recognition. This is consistent with the X-ray crystallographic structure of the 1934.4 Vα domain (Vα4.2), in which HV4α forms part of a relatively flat continuous surface with CDR1, 2, and 3, leading to the earlier suggestion that it might be involved in interactions with cognate pMHC ligand (4). In the current study, the role of the two central exposed residues (K68, E69) of this loop in pMHC recognition have been investigated by expressing 1934.4-derived TCRs with mutations to alanine at these positions in T cell transfectants. K68 is highly conserved in murine and human Vα sequences, whereas the variability at position 69 is higher (37). Antigen responsiveness of the TCR transfectants has been analyzed in the presence and absence of CD4 coexpression and compared with that of 1934.4 WT transfectants. These studies have shown that K68 plays an important role in antigen responsiveness, as assessed by quantitating IL-2 secretion, whereas E69 has a lesser role. In addition, mutation of both K68 and E69 to alanine within the same TCR results in responsiveness to Ac1-11(4Y)–I-A^u complexes that is intermediate between that of the transfectants expressing K68A and E69A TCRs. However, transfectants expressing this double mutant show an almost undetectable response similar to that of the K68A mutants when presented with the lower affinity peptide Ac1-11 in the context of I-A^u. Thus, the effect of the double mutation appears to be analogous to that of the K68A mutation, reiterating the importance of K68 in the interaction.

The role of K68 in pMHC responsiveness of the 1934.4 TCR could be through several possible mechanisms that are not mutually exclusive. First, K68 may contact either peptide or I-A^u (or both) in the pMHC complex either directly or via ordered water, which has been shown to play a role in stabilizing antibody–antigen interactions (56, 57). Electrostatic interactions, which can occur over distances up to 20 Å (58), may also occur between K68 and cognate pMHC. Consistent with this possibility and assuming that the I-A^u structure is similar to that of the recent I-A^k/I-A^d structures (59, 60), several acidic I-Aβ^u residues (E84, E85; numbering as in reference 37) would contact/be in proximity to HV4α if the orientation of 1934.4 TCR binding resembles that in TCR–pMHC class I complexes (10–13). Alternatively, the effects of mutation of K68 may be indirect through destabilization of other CDR loops of Vα4.2 that are involved in pMHC binding. However, we favor a more direct effect of the mutation, since the X-ray crystallographic structure of Vα4.2 (4) indicates that K68 is exposed and does not make stabilizing H-bonds with CDR residues. Furthermore, it is unlikely that the mutations analyzed in this study have resulted in gross structural perturbations since the CD spectra of the corresponding recombinant Vα domains indicate that they are correctly folded, and the WT and mutant transfectants express similar levels of surface TCR.

Regardless of the mechanism by which the effect of mutation of K68 occurs, the almost total abrogation of responsiveness of the CD4⁻ mutant transfectants to Ac1-11–I-A^u complexes is unexpected since K68 would be predicted to be at the periphery of the interacting surfaces of TCR and pMHC. However, the absence of CD4 together with the low affinity of this peptide for I-A^u most likely contribute toward the unresponsiveness, and in this context CD4⁻ WT transfectants also respond relatively poorly to this ligand. In contrast, when the density of cognate pMHC is increased by using Ac1-11(4Y) which has a ∼1000-fold higher affinity for I-A^u (61), responsiveness of the K68A and K68AE69A transfectants is observed albeit at considerably lower levels than that of the WT transfectants. This supports the concept that the K68A mutant TCR binds to cognate antigen (Ac1-11–I-A^u complexes) with an affinity/ avidity which, in the absence of CD4, falls below the threshold needed for effective signaling (IL-2 production in the current study). Consistent with this, coexpression of CD4 partially compensates for the defective signaling of transfectants expressing K68A. The enhancing effect of CD4 could be through one or more mechanisms. First, CD4 may increase the affinity/avidity of the TCR for pMHC by stabilizing the trimeric complex via CD4–MHC interactions (19–21, 33), or by increasing the affinity of the TCR-pMHC interaction in an analogous way to that demonstrated for CD8 (24, 25). Second, CD4 is able to recruit the phosphotyrosine kinase p56^lck to the TCR–CD3 complex increasing the strength of the signal delivered postantigen recognition by the TCR (22, 23). In the current study, this could compensate for the loss in affinity of the K68A mutant. Consistent with this, several studies have shown a significant loss of CD4 effects on T cell activation in the absence of p56^lck-CD4 interaction (62–64). However, CD4 lacking its cytoplasmic lck-binding domain can still augment T cell reactivity (65), and in one study the extracellular domains of CD4 and not its cytoplasmic tail are responsible for restoration of IL-2 secretion (66).

Recent studies have shown that reduced CD4 availability can convert the functional and biochemical effects of agonist peptides into those characteristic of partial agonists (32) or partial agonists into antagonists (67, 68). Reciprocally, CD4 coexpression can convert partial agonists into agonists, but has no effect on antagonist activity (31). The lack of effect of CD4 on antagonists which form short-lived TCR–pMHC complexes (17) supports the hypothesis that CD4 engagement follows TCR–pMHC complex formation, and that the latter complex needs to be sufficiently long lived to allow CD4 recruitment (31, 32). By extension, in the current study, the enhancing effect of CD4 coexpression on the responsiveness of the K68A mutant suggests that the half life of the K68A TCR–pMHC interaction is long enough to allow CD4 recruitment to the ternary TCR–pMHC complex. However, without CD4 coexpression, the interaction of this TCR with Ac1-11–I-A^u complexes appears to be below the threshold needed for T cell activation. In contrast, the WT 1934.4 TCR–pMHC interaction appears to be sufficiently long lived in the absence of CD4 to result in signaling. This is also consistent with the relative resistance of this latter interaction to blockade by the anti-CD4 antibody, GK1.5. In addition, the enhancement in responsiveness by CD4 coexpression is much greater for the K68A mutant than the WT TCR. The effect of CD4 therefore appears to be maximal for suboptimal TCR–pMHC interactions that attain a threshold affinity, and this is consistent with observations using other antigen recognition systems (31, 69). However, in these other systems, the effects of CD4 on responses to agonists/partial agonists/antagonists were analyzed, and this is in contrast to the current study where the affinity of the TCR–pMHC (agonist) interaction has been affected by mutating the TCR. Taken together, our data provide further support for sequential engagement models of T cell signaling in which both the affinity (off-rate) of the TCR– pMHC complex and coreceptor involvement affect the outcome of T cell contact with APCs bearing cognate ligands (31, 32, 34).

In summary, a single amino acid substitution of the exposed, highly conserved residue K68, which is located outside the CDRs of the TCR, appears to have a significant impact on the outcome of the interaction between a TCR and its pMHC ligand. Given the indications that the diagonal orientation observed for the binding of class I–restricted TCRs to pMHC complexes is general (10–13), it is likely that the functional role of this HV4α amino acid that we have defined will be observed for other TCR–pMHC interactions.

We are indebted to Drs. Bertram Ober and Mark Mummert for providing the wild-type TCR transfection constructs and advice with CD analyses, respectively. We also thank Drs. Nicolai van Oers and Stephen Thompson for critical review of the manuscript and Dr. Mischa Machius for assistance with Fig. 1.

This work was supported by grants from the National Multiple Sclerosis Society (RG-2411), National Institutes of Health (AI/NS 42949) and Yellow Rose Gala. E.S.Ward is an Established Investigator of the American Heart Association (Grant 9640277N).