Characterization of the interaction of a TCR
chain variable domain with MHC II I-A molecules
Ayub Qadri1,2,
Jayant Thatte1,3,
Caius G. Radu1,
Bertram Ober1,4 and
E. Sally Ward1
1 Center for Immunology and Cancer Immunobiology Center, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235-8576, USA
Correspondence to:
E. S. Ward
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Abstract
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The
ß TCR recognizes peptides bound to MHC molecules. In the present study, we analyzed the interaction of a soluble TCR
chain variable domain (V
4.2J
40; abbreviated to V
4.2) with the MHC class II molecule I-Au. V
4.2 bound specifically to I-Au expressed on the surface of a transfected thymoma cell line. Modifications in the amino acid residues located within the three complementarity-determining regions (CDRs) of the V
domain did not markedly affect this interaction. However, mutation of glutamic acid to alanine at position 69 of the fourth hypervariable region (HV4
) significantly increased the binding. Antibody inhibition studies suggested that the binding site was partly contributed by a region of the ß chain of I-Au. Furthermore, the binding of V
4.2 to the MHC molecule was dependent on the nature of the peptide bound in the groove. Soluble V
4.2 specifically inhibited the activation of TCR transfectants by I-Au-expressing cells pulsed with an N-terminal peptide of myelin basic protein. V
4.2 also bound to MHC class II-expressing spleen cell populations from mice of the H-2u and H-2d haplotypes. The binding of V
4.2 to I-A molecules might explain the immunoregulatory effects reported previously for TCR
chains. This V
4.2 interaction may also be relevant to models of antigen presentation involving the binding of intact proteins to MHC class II molecules followed by their processing to generate epitopes suitable for T cell recognition.
Keywords: fourth hypervariable region, immunosuppression, MHC class II, TCR, V
domain
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Introduction
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The structure of a TCR resembles that of an Ig (1). However, unlike Ig, the TCR recognizes its ligand as a small peptide fragment bound to the groove of an MHC molecule (2,3). The interaction between antibodies and cognate antigens has been extensively studied by X-ray crystallography (4,5). These studies have demonstrated that the recognition of an antigen by an antibody molecule is primarily mediated by the hypervariable regions referred to as complementarity-determining regions (CDRs). In contrast to antibodies, the structural basis of peptideMHC recognition by the TCR has not been analyzed in such extensive detail, primarily because until recently it was not possible to produce soluble and functional TCR molecules in sufficient amounts for crystallographic studies. The crystal structures of MHC class I and class II with peptides bound to the groove have provided structural information concerning the TCR ligand (612). More recently, crystal structures of a TCR V
domain (13), a TCR ß chain (14), a TCRßstaphylococcal enterotoxin B (SEB) complex (15), several TCR
ßpeptideMHC class I complexes (1619) and TCR
ß heterodimers (20,21) have been reported. The 3-D structures of the ternary complexes confirmed the involvement of Ig-like CDRs in ligand binding. Consistent with the structures, the CDR1 and CDR2 of the TCR
chain have also been shown to control MHC restriction (22). The high-resolution crystal structure of a human TCRMHC class Ipeptide complex demonstrated, unexpectedly, that ß chain CDR 1 and 2 have limited, if any, contact with peptideMHC ligand (17), but this appears not to be the case for other tripartite complexes (18,19). Although the 3-D structures of these trimolecular complexes have provided a view of the TCRpeptideMHC class I interaction which is most likely general for class I-restricted TCRs, whether this configuration also holds for class II-restricted TCRs awaits the solution of structure(s) of TCRpeptideMHC class II complex(es). However, experimental support for a similar orientation for the latter has been obtained by the detailed analysis of antigen recognition by an I-Ak-restricted TCR and arguments for the generality of this configuration have been presented (23,24).
In this communication we describe the interaction between a soluble TCR V
domain (V
4.2J
40; abbreviated to V
4.2) and the MHC class II molecules I-Au and I-Ad. V
4.2 was isolated from the 1934.4 T cell hybridoma (25) that recognizes the N-terminal nonapeptide (or 11mer) of myelin basic protein (MBP) complexed with I-Au and the X-ray structure of this V
domain is known (13). The data demonstrate specific binding of V
4.2 to I-Au and I-Ad molecules, but not to I-Aq. Unexpectedly, CDR1, 2 and 3 residues of V
4.2 do not play a significant role in binding to I-Au, in contrast to a central residue of the fourth hypervariable loop. Thus, the mode of binding is distinct from that expected for a V
domain in the context of a TCR
ß heterodimer. The binding of soluble V
4.2 to I-Au may have relevance to the reported immunosuppressive functions of TCR
chains (2628). This interaction may also be representative of an antigen-presenting pathway in which intact, folded protein antigens act as ligands for the initial interaction with recycling, cell surface MHC class II molecules (29,30).
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Methods
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Cell lines, antibodies and peptides
The I-Au-expressing B cell line, PL-8 (31), and I-Au -restricted T cell hybridoma, 1934.4 (25), were generously provided by Dr David Wraith (University of Bristol, UK). The TCR thymoma cell line BW5147 and its I-Au-transfected derivatives, Utm6.15, Utm34a.1 and U400 (32,33), were kindly made available by Dr Harden McConnell (Stanford University, Palo Alto, CA). T cell lines expressing the 1934.4 TCR were made by transfecting the TCR genes into a CD4, TCR cell line 58
ß (34), kindly provided by Dr Stephen Hedrick (San Diego, CA) with permission from Dr Bernard Malissen (INSERM-CNRS, Marseille-Luminy, France), using the
and ß shuttle vectors (35) kindly provided by Dr Mark Davis (Stanford University School of Medicine, Palo Alto, CA). T cell transfectants were made as described elsewhere (36). TCR expression was analyzed by indirect immunofluorescence using the anti-Vß8 mAb F23.1 (37), a generous gift from Drs John Kappler and Philippa Marrack (University of Colorado Health Science Center, Denver, CO).
Hybridomas HB183 and TIB93 secreting anti-I-A mAb Y3P (38) and 10.2.16 (39), respectively were obtained from ATCC (Rockville, MD). mAb 10.3.6 (39) was kindly provided by Dr David Wraith. Anti-polyhistidine mAb was obtained from Sigma (St Louis, MO). FITC-labeled anti-mouse Ig and FITCstreptavidin were from Cappel. The N-terminal peptide (Ac1-11; acetylated at position 1) of rat MBP and an analog in which the wild-type 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, TX.
Expression of TCR V
4.2 and its mutants
TCR V
4.2 was produced in a bacterial expression system as described previously (40) and V
4.2 purified by affinity chromatography on Ni2+-NTA agarose (Qiagen, Germany) as described (40). V
4.2 produced using this approach was used previously for X-ray crystallography (13) and is therefore correctly folded.
Mutagenesis of residues encompassing CDR1, 2 and 3, and the fourth hypervariable region (HV4) were carried out as described by Kunkel (41). Four different derivatives of V
4.2 were produced: one in which CDR1 residues were mutated, the second in which CDR2 residues were mutated and the third with amino acid residues in all three CDRs mutated. A fourth derivative with glutamic acid at position 69 of HV4 mutated to alanine was also produced by the same strategy. The oligonucleotides (synthesized on an Applied Biosystems DNA synthesizer) used were: 5'-CCA GAA CAG AGC TCC GGC CGC TCC GGC TGA GTA GTT GCA-3' for mutating CDR1 residues at positions 27, 28, 29 and 30 [Kabat numbering (42)]; 5'-GCT GCT TCC TTT CGA TGC GCC CGC TGA GGC TCT AAA-3' for mutating CDR2 residues at positions 51, 52, 53 and 54; 5'-AGT TAT TTT CTC AGA TGC ACC GGC TTC ACT CAG AGC-3' for mutating CDR3 residues at positions 98, 99, 100 and 101; and 5'-AGG TGG CTG CTT TAT TG-3' for altering HV4 residue 69. The mutations were confirmed by determining the nucleotide sequence using Sequenase version 2.0 kits (USB, Cleveland, Ohio). The mutated derivatives of V
4.2 were expressed and purified as described above for the wild-type V
4.2.
Flow cytometric analysis of V
4.2 binding
The interaction of V
4.2 with I-A-expressing cells (in vitro cell lines, splenocytes or thymocytes) was analyzed by flow cytometry. Briefly, cells (3x105 cells/well in a 200 µl volume) were incubated with varying concentrations of V
4.2 at 37°C for 1 h. After washing with PBS containing 1% BSA, cells were incubated for 1 h at 4°C with anti-polyhistidine mAb (to detect the V
domain with a C-terminal polyhistidine tag) diluted in PBS/BSA. Subsequently cells were washed and incubated with FITC-labeled anti-mouse Ig for 1 h at 4°C. Controls were incubated only with anti-polyhistidine antibody and FITCanti-mouse Ig. Cells were washed with PBS/BSA, suspended in PBS/BSA and analyzed in a flow cytometer (Becton Dickinson, Mountain View, CA). In some assays, biotinylated V
4.2 was employed for binding studies [V
was biotinylated as described by Meier et al. (43)] and FITC-labeled streptavidin was used to detect binding. Binding studies were also carried out in the presence of anti-I-A antibodies or I-Au-binding peptides. For these experiments, V
was co-incubated with the cells and the antibodies or the peptides. The remainder of the procedure was as described above.
In vitro T cell stimulation assays
The effect of V
4.2 binding to I-Au on peptideMHC recognition by specific T cells was analyzed in vitro. T cell transfectants expressing the 1934.4 TCR were stimulated in vitro with MBP peptide Ac1-11 in the presence of varying concentrations of V
4.2 or, as a control, a V
domain (V
11J
17; abbreviated to V
11) derived from an irrelevant TCR designated qcII85.33 (44). Utm6.15, an I-Au-transfected BW5147 thymoma cell line (32,33), or PL-8, a B cell lymphoblastoid line expressing I-Au (31), were employed as antigen-presenting cells (APC). At 24 h post-stimulation, culture supernatants were collected and analyzed for IL-2 levels using an IL-2-dependent cell line CTLL-2 as described (45). Data shown for T cell stimulation assays represents the means of triplicate measurements with errors ranging from 5 to 10%.
Surface plasmon resonance (SPR) experiments
SPR experiments were carried out using a BIAcore 2000. Flow cells of CM5 chips were coupled with either V
4.2 or, as a reference cell, D1.3 antibody (46) using amine coupling chemistry. Densities of coupling were 1112 and 1773 RU (two flow cells for V
4.2) and 2752 RU (D1.3 antibody). Ac1-11 or its analog, Ac1-11[4Y], were injected over the flow cells using programmed methods and the BIAcore control software. Flow rates of 5 or 80 µl/min were used at a temperature of 25°C. Ac1-11 or Ac1-11[4Y] were injected at a concentration of 200 µg/ml in PBS, pH 7.2, plus 0.01% Tween 20. All injections were carried out in triplicates to ensure reproducibility. For data processing, the BIAevaluation 2.1 software was used. Data was zero adjusted and reference (D1.3 antibody coupled) flow cell data subtracted from V
4.2 flow cell data. Similar results were obtained when a `blank' flow cell (i.e. treated with coupling buffer only during coupling cycle) was used. In addition, the presence of immobilized V
4.2 was verified by injecting a 1:1000 dilution of anti-polyhistidine antibody (Sigma) over the sensor chip surface.
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Results and discussion
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The TCR
chain variable domain (V
4.2) binds to cell surface MHC class II molecules
In the present study, we analyzed the interaction of a TCR
chain with MHC class II by examining the binding of a soluble TCR
chain variable domain (V
4.2) to I-Au. TCR V
4.2 is one of the predominant
chains expressed by the TCR of encephalitogenic T cells isolated from PL/J mice (H-2u) following induction of experimental autoimmune encephalomyelitis (47). These autoreactive T cells recognize the immunodominant epitope Ac1-11 of MBP (48). To study the binding of V
4.2 to I-Au, two different types of I-Au -expressing cell lines were employed: PL-8 [a B cell line, derived by fusing LPS-activated splenocytes from H-2u mice with the M-12.C3 B lymphoblast line (31)] and BW5147 thymoma derived cell lines transfected with I-Au (Utm6.15, Utm34a.1 and U400). Utm6.15 and Utm34a.1 express membrane-bound I-Au, and U400 has GPI-linked I-Au (33). V
4.2 was expressed in Escherichia coli in a form previously used for X-ray crystallography (13) and the binding analyzed by indirect immunofluorescence using flow cytometry. In the absence of an antibody against TCR V
4.2, a mAb directed against the polyhistidine tag present at the C-terminus of the V
domain was employed to detect binding. V
4.2 showed a dose-dependent binding at 37°C to Utm6.15 which expresses high levels of I-Au (Fig. 1a
). Untransfected BW5147 cells did not show any significant binding, demonstrating the specificity of interaction with I-Au expressed by the transfectants (Fig. 1b
). Moreover, BW5147 transfectants Utm34a.1 and U400, expressing lower levels of surface I-Au (assessed by anti-I-A antibody, Y3P, binding), showed reduced binding of V
4.2 (Fig. 1c and d
). The specificity of this interaction was also demonstrated by the almost undetectable binding of soluble V
11J
17 [V
11; derived from the qcII85.33 hybridoma (44)] to Utm6.15 (Fig. 1e
). The secondary structure of V
4.2 after incubation at 37°C was similar to the native molecule (40) as revealed by CD spectroscopy (data not shown). The V
4.2I-Au interaction therefore appears to be distinct from the association of partially folded polypeptides with MHC class II molecules that has been reported for both invariant chain -positive (4953) and -negative cell lines (54). However, it cannot be excluded that following initial binding of V
4.2 to I-Au, some unfolding that is favored by V
4.2I-Au interaction occurs.
The strict correlation between the levels of surface I-Au and the extent of binding seen with BW5147 transfectants did not, however, hold true for the B cell line PL-8. V
4.2 bound at much lower levels to PL-8 relative to Utm6.15 although both cell lines expressed comparable levels of surface I-Au (Fig. 2a and b
). This was not due to increased internalization of I-Au by PL-8 cells as similar results were obtained with paraformaldehyde-fixed cells (data not shown). The levels of surface I-Au were determined by flow cytometry with three different anti-I-A mAb, Y3P, 10.2.16 and 10.3.6. There was no significant difference between PL-8 and Utm6.15 in binding to the three mAb (data not shown), suggesting that there were no major conformational differences between I-Au molecules present on the two cell lines although this does not exclude the presence of subtle differences. Utm6.15 was also more effective at activating peptide (MBP-derived N-terminal peptide, Ac1-11)-specific 1934.4 hybridoma cells (Fig. 2c
) although it did not differ significantly from PL-8 in its ability to bind a biotinylated high-affinity analog of Ac1-11 (Ac1-11[4Y]; data not shown), which due to replacement of the position 4 lysine of wild-type Ac1-11 by tyrosine has higher affinity for binding to I-Au (55). However, for these peptide binding studies with the PL-8 line the degree of MHC class II-specific binding of Ac1-11[4Y] cannot be determined with certainty since I-Au -negative PL-8 cells are not available. This is clearly not the case for Utm6.15 as untransfected BW5147 cells serve as a negative control.
The differences in binding of PL-8 and BW5147 transfectants to V
4.2 and in activating specific T cells are possibly due to differences in the peptide repertoires loaded on the I-Au molecules from the two cell types. A difference in the peptide repertoires of the two cell lines was also suggested by the inability of Utm6.15 to stimulate an alloreactive T cell hybridoma [qcII85.33 (44)] which PL-8 activated very efficiently (data not shown). Furthermore, Utm6.15 cells were less potent than PL-8 in mediating SEB-induced T cell activation (Fig. 2d
) and antigenic peptides have been shown to affect the efficiency of presentation of bacterial superantigens (5658).
The binding of V
4.2 to I-Au on the surface of splenocytes from PL/J mice was also investigated. Significant levels of binding to I-Au-positive splenocytes relative to H-2u thymocytes was observed (Fig. 3a
). This demonstrates that the interaction on PL-8 and Utm6.15 is not an artifact of using in vitro cell lines. Binding was also observed with ex vivo splenocytes but not thymocytes derived from Swiss or BALB/c mice (both H-2d haplotype; data shown only for Swiss mice, Fig. 3b
). V
4.2 did not, however, interact with a B cell-derived transfectant expressing I-Aq (data not shown) nor did it show significant MHC class II-specific binding to splenocytes from DBA1/Lac.J mice (H-2q haplotype; Fig. 3c
). The interaction therefore shows specificity for certain I-A haplotypes.
The putative V
4.2 interaction site involves the ß chain of I-Au
The demonstration of V
4.2 binding to I-Au offers a system to investigate which MHC class II chain the V
domain interacts with. This was carried out by analyzing binding of V
4.2 to I-Au in the presence of antibodies to MHC class II. Two of these, 10.2.16 and 10.3.6, are directed against the ß chain while the third (Y3P) recognizes an
chain-dependent determinant on the MHC class II heterodimer (59). 10.2.16 partially inhibited the binding of V
to I-Au (Fig. 4a
) suggesting that the interaction was at least partly mediated through residues on the ßu chain. The other two mAb had no significant effect (data shown only for Y3P, binding in presence of 10.3.6 was overlapping with V
4.2-binding in the absence of any antibody). The three antibodies showed comparable binding to I-Au on Utm6.15 (data not shown). Our results suggest that this inhibition may be a result of blockade of the interaction between the ß chain of I-Au and V
4.2. The data therefore indicate a role for V
4.2I-Au ß chain contacts in the interaction and this is supported by our observation that the binding was not affected by another MHC class II ligand, SEB (data not shown), which is believed to interact with the
chain of I-A molecules (60). However, these data do not exclude the possibility of an interaction of V
4.2 with other regions of the I-Au molecule distinct from the recognition sites for 10.3.6, Y3P or SEB. Interestingly, the importance of the 10.2.16-binding site in the 1934.4 TCR
ßpeptideMHC interaction was also indicated by more efficient blockade of 1934.4 T cell recognition of Ac1-11I-Au (on both PL-8 and Utm6.15 cells) by this antibody relative to Y3P (Fig. 4b
), although both antibodies bound equally well to I-Au on PL-8 and Utm6.15 cells. 10.2.16 does not recognize I-Aq and this might be one of the reasons for the lack of V
4.2 binding to this MHC class II haplotype.
Effect of antigenic peptide on the V
4.2I-Au interaction
The interaction site for V
on I-Au was further probed by analyzing binding in the presence of a known I-Au -binding peptide. The N-terminal peptide Ac1-11 of MBP was chosen for this purpose because it is an encephalitogenic immunodominant peptide in H-2u mice recognized in association with I-Au by T cells which predominantly express V
4 or V
2 (47). Perhaps unexpectedly, there was a dose-dependent reduction in the binding of V
4.2 to I-Au in the presence of Ac1-11 (Fig. 5a
). The effect was more pronounced when a high affinity derivative of this peptide, Ac1-11[4Y], was co-incubated with V
4.2 (Fig. 5b
). An unrelated peptide (derived from bovine type II collagen) that does not bind to I-Au did not have any effect (Fig. 5c
). To exclude the possibility that the inhibitory effects of Ac1-11 or Ac1-11[4Y] were due to binding of V
4.2 to these peptides, SPR experiments were carried out. These analyses demonstrated that binding of Ac1-11 or Ac1-11[4Y] at a concentration of 200 µg/ml to immobilized V
4.2 was not detectable under the conditions of the SPR experiments (Fig. 6
).
The peptide inhibition data suggest that the I-Au residues involved in binding to soluble V
4.2 are located close to or overlapping with the peptide-binding groove of I-Au. However, rather than competition between V
4.2 and the peptides for binding to the empty groove, we speculate that it is the differential ability of V
4.2 to displace peptides of different affinities from the groove that results in the affinity-dependent modulation of V
4.2 binding to I-Au by the MBP peptides. Studies carried out previously with I-Au molecules purified from the same BW5147 transfectants used in the current analyses (32,33) suggest that the large majority of these molecules are occupied by peptide. Therefore, the interaction reported here is unlikely to be due to binding of V
4.2 to empty I-Au molecules on Utm6.15. A major role for empty molecules in the V
4.2I-A interaction is also excluded by the ability of this V
domain to bind to ex vivo spleen cells from H-2d and H-2u mice (Fig. 3
). In our model for V
I-A interaction, the initial low-affinity binding might resemble the V
component of the TCR
ßpeptideMHC interaction and subsequently this V
would displace peptide from the groove to form a high-affinity, stable complex that is long lived enough to be detectable by flow cytometry. By analogy with the affinities of TCR
ßpeptideMHC interactions reported (6165) the affinity of the initial V
I-A complex would be expected to be very low and would not be sufficiently stable to be detected under the conditions used in our binding assays. Irrespective of the mechanism of binding, it is clear that the interaction between V
4.2 and I-Au/I-Ad is specific and the binding site on I-Au overlaps with the 10.2.16 interaction site. The interaction is also of sufficiently high affinity to be detected by flow cytometry and V
4.2 competes with antigenic peptide for binding to the I-Au groove. These unexpected features prompted us to probe further the molecular nature of the interaction by carrying out mutagenesis of the hypervariable loops of the V
4.2 domain.
The interaction is not significantly modulated by mutations in the CDR residues; mutation of HV4 residue 69 enhances binding
The role of CDR residues which are known in a number of systems to be involved in the recognition of peptideMHC complexes by TCR (1619,24,6668) was analyzed for the V
4.2I-Au interaction. For this analysis, the central amino acid residues of CDR1, 2 and 3, identified from the X-ray structure (13), were mutated. Three different derivatives of V
4.2 were produced: a CDR1 mutant in which serine, glycine, tyrosine and proline at positions 27, 28, 29 and 30 [Kabat numbering (42)] were mutated to glycine, alanine, alanine and glycine respectively; a CDR2 mutant in which arginine, aspartic acid, lysine and glutamic acid at positions 51, 52, 53 and 54 were mutated to alanine, glycine, alanine and serine respectively; and a mutant in which residues in all three CDRs were altered, with CDR3 residues asparagine, tyrosine, glycine and asparagine at positions 98, 99, 100 and 101 modified to alanine, glycine, alanine and serine respectively. A fourth analog was also produced in which glutamic acid at position 69 [Kabat numbering (42)] in HV4 of V
4.2 (HV4
) was mutated to alanine. When analyzed for binding to I-Au by flow cytometry, the three V
4.2 mutants with alterations of CDR residues did not differ markedly from the wild-type V
4.2 in the extent of interaction with I-Au. CDR2 and CDR1, 2 and 3 mutants showed slight reductions in binding, indicating a minor role for CDR2 residues in interacting with I-Au (Fig. 7a
). Interestingly, however, modification of glutamic acid to alanine at position 69 (designated E69A) in HV4
significantly increased the binding to I-Au (Fig. 7b
), suggesting an important role for this region of the soluble V
4.2 domain in the V
4.2I-Au interaction.
The data for the V
4.2 mutants indicate that the mode of binding observed here is unconventional insofar as a critical role for CDR residues is not observed. The nature of the interaction also appears to be distinct from that described for peptides/proteins which interact with MHC molecules at sites that do not encompass the peptide-binding groove (69,70). Analysis of the HV4
mutant E69A indicates that, in contrast to CDR residues, glutamic acid at position 69 plays a significant role in the binding of soluble V
4.2 to I-Au. Interestingly, in a recent study in which V
4.2 with the E69A mutation was expressed as a TCR
ß heterodimer in transfectants, a reduction in antigen responsiveness was observed (36). Thus, whilst both these analyses indicate involvement of residue 69 of HV4 in recognition of I-Au, alteration of glutamic acid to alanine appears to result in different consequences depending upon whether the V
is expressed as a single domain or TCR
ß heterodimer, with the former resulting in enhancement in the binding of soluble V
to I-Au and the latter resulting in a TCR with reduced affinity for cognate peptideI-Au complexes. This in turn underscores the differences between the two interactions. In addition, V
4.2 exists as a homodimer in solution (13) and it is currently an open question as to whether both monomers in a dimer are involved in binding to a single I-Au molecule.
V
4.2 binding to I-Au modulates recognition of the peptideMHC complex by the 1934.4 TCR
The TCR
chainMHC class II interaction was further studied by analyzing the effect of V
4.2 binding to I-Au on Ac1-11I-Au recognition by a specific TCR expressed by T cell transfectants (TCR 58
ß cells transfected with the 1934.4
and ß chain genes containing V
4.2J
40 and Vß8.2Jß2.3). Activation of peptide-specific T cells (1934.4 TCR transfectants) was investigated in the presence of V
4.2. This V
inhibited the recognition of the Ac1-11I-Au complex by the 1934.4 TCR transfectants in a dose-dependent fashion (Fig. 8a
). The inhibition was seen only for the wild-type peptide and not for the higher affinity derivative, Ac1-11[4Y] (data not shown). The mutated derivative of V
4.2, E69A, was more effective in blocking Ac1-11I-Au recognition by the T cells (Fig. 8b
), consistent with the higher affinity of this mutant for I-Au. In contrast to V
4.2 and the E69A mutant, addition of soluble V
11J
17 [derived from the qcII85.33 hybridoma (44)] at a concentration of 100 µg/ml did not have a significant effect on the stimulation of 1934.4 T cell transfectants (data not shown), demonstrating the specificity of the inhibitory effects. The data suggest that V
4.2 can specifically modulate the recognition of peptideMHC ligand by the TCR, the effect being dependent on the affinity of the peptide for MHC. Whether the V
mediates the inhibitory effects through displacement of Ac1-11 from I-Au or blockade of the interaction of Ac1-11I-Au by the TCR, or a combination of both, cannot be determined by the current experiments. The modulation of T cell recognition was seen only with Utm6.15 and not with PL-8 as the APC, which is consistent with the direct binding data (Fig. 1
). It is, however, possible that at higher concentrations V
4.2 may also modulate recognition of PL-8-mediated peptide presentation.
The affinity-dependent modulation of T cell recognition by soluble V
demonstrated in the present study might explain the immunoregulatory effects reported for soluble TCR
chains in a number of systems in vitro and in vivo (2628). The influence of the nature/affinity of the peptide bound in the groove of the MHC molecule on V
binding suggests that the phenomenon will not lead to generalized non-specific inhibition of peptideMHC recognition by T cells. The affinity dependence would impart some kind of specificity similar to that observed previously (27). The efficiency with which a soluble V
can bring about modulation of the T cell response would also depend on the relative affinity of a particular
chain for a given MHC haplotype and therefore different V
s might have differential effects. The present study does not, however, exclude other mechanisms which have been proposed for immunoregulation by soluble TCR
chains (28).
In summary, we have demonstrated specific binding of a correctly folded TCR V
domain to MHC class II I-A molecules on the surface of both in vitro cell lines and ex vivo splenocytes. In addition, V
4.2 can bind to I-Au in a manner that is influenced by the affinity of the peptide present in the groove. Thus, the interaction appears to be unconventional and in support of this, CDR mutations do not have significant effects whereas mutation of E69 in HV4 enhances binding. Further studies will be required to evaluate the generality of this type of interaction and assess the role of peptide repertoire/affinity in V
binding. The binding of soluble TCR
chain to MHC class II may be of relevance in certain physiological situations such as in mediating suppressor activity of
chains (2628) for which a mechanistic basis is currently unknown. This hypothesis is supported by the observation that V
4.2 binds to ex vivo splenocytes of the I-Au/I-Ad haplotypes and blocks T cell stimulation by peptideI-Au complexes in vitro. Finally, V
binding to I-A molecules may be an initial step in an antigen-presentation pathway reminiscent of that described by others, whereby intact proteins bind to surface MHC class II molecules prior to endocytic uptake and recycling (29,30,50). In this respect, it will be of interest to investigate whether the region of V
4.2 encompassing HV4
is immunodominant, consistent with determinant capture models of epitope generation (52).
 |
Acknowledgments
|
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We are indebted to Dr Mark Mummert for providing valuable advice concerning CD analyses. We would also like to thank Drs Harden McConnell, John Kappler, Philippa Marrack, Mark Davis, Stephen Hedrick and David Wraith for providing essential reagents. This study was supported by grants from the National Institutes of Health (AI31592, AI/NS42949), National Multiple Sclerosis Society (RG-2411) and the Yellow Rose Gala. E. S. W holds an Established Investigator Award from the American Heart Association (9640277N).
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Abbreviations
|
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APC | antigen-presenting cell |
CDR | complementarity-determining region |
HV4 | fourth hypervariable region |
MBP | myelin basic protein |
SEB | staphylococcal enterotoxin B |
SPR | surface plasmon resonance |
 |
Notes
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2 Present address: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India 
3 Present address: Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA 
4 Present address: Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, 924 East 57th Street, 4th Floor, Chicago, IL 60637-5420, USA 
Transmitting editor: C. Martinez-A
Received 13 December 1998,
accepted 2 March 1999.
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