The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.

T cells recognize MHC/peptide complexes by means of their clonally distributed Ag receptors, or TCRs. TCR recognition of ligand involves specific interactions with amino acid residues of the peptide as well as of the α-helices of the MHC molecule (1). Interaction of the TCR with MHC residues has been shown to be important for allele-specific recognition (2); however, CTL clones specific for the same MHC molecule can differ in certain MHC residues that they contact (3, 4, 5, 6). Because each MHC allele can bind a unique repertoire of peptides, the precise set of peptides bound to each MHC molecule also contributes to allele-specific recognition. Yet, it remains unclear whether this functional recognition by TCRs varies in terms of the relative dependence upon interaction with peptide vs MHC residues.

Recent evidence suggests that alloreactive T cells, like self-restricted T cells, recognize a complex of both MHC and endogenous peptide ligand, and that alloreactive T cells are capable of the same degree of specificity as self MHC-restricted T cells (2, 7, 8, 9, 10, 11). However, there has been speculation that alloreactive TCRs are more dependent on interactions with MHC, rather than peptide residues, as compared with self MHC-restricted TCRs (12, 13, 14). Whereas T cells with high affinity for self MHC molecules will be deleted during development, a T cell with a high inherent affinity for an allo-MHC molecule does not undergo selection on this allo-MHC molecule, and thus can mature. As a consequence, alloreactive TCRs might be more biased toward interactions with MHC residues than syngeneic TCRs. Recent studies have established that TCRs do have an inherent reactivity toward MHC molecules (15, 16), and studies examining the effects of changes in peptide residues on recognition by a given T cell have reported a greater tolerance for changes in peptide residues with allo-, rather than self MHC ligands (13, 14). This has been used to argue that the recognition of an allogeneic ligand must be more dependent upon interaction with MHC residues than peptide residues. However, there have been no studies that have directly addressed the relative MHC dependence of allogeneic vs syngeneic TCRs.

Examination of how TCRs interact with their MHC/peptide ligands has led to the proposal that there is a common pattern for TCR recognition of a given MHC molecule and that TCRs may interact with class I and class II MHC proteins in this same orientation (5, 17). The solution of the structures of four individual TCR-MHC/peptide complexes, in addition to a functional study of TCR interaction with Kb, have led to the suggestion that this common orientation is parallel to the β-pleated sheets of the MHC molecule (5, 18, 19, 20, 21). However, subtle differences in the angle of this interaction could occur if the residues contacted by TCRs differ (20, 21), and patterns of TCR-MHC engagement distinct from this proposed common orientation have been identified (22, 23). Thus, it is not clear whether all TCRs engage all MHC molecules in this same common orientation, or whether allele-specific recognition of MHC molecules is in part a result of distinct orientations of TCR engagement.

In this study, using an extensive panel of site-directed mutants of Ld together with a large panel of CTL clones, we determine which MHC class I residues are important for TCR recognition by Ld-restricted and Ld-alloreactive CTL clones that are specific for the same and different peptides. Our approach is different from earlier studies that compared self-restricted vs allorecognition of different MHC alleles by a single T cell clone. In contrast to these earlier studies, we find that alloreactive CTL clones are no more cross-reactive with the Ld mutants than Ld-restricted clones, indicating that allogeneic and syngeneic TCRs are equally dependent on interaction with MHC residues. Importantly, the same primary MHC contact residues are used by self-restricted and alloreactive T cells. A high affinity clone, 2C, interacts with the same primary contact residues, but is less affected by mutation of Ld, suggesting that dependence on interaction with MHC residues can be overcome by high affinity. This analysis reveals an allele-specific recognition pattern of Ld with greater dependence upon interaction with residues in the center of both α-helices of Ld than what has been observed with other class I molecules, yet is consistent with a common diagonal orientation of TCR-MHC/peptide engagement.

BALB/c (H-2d), BALB/c-H-2dm2 (dm2, Ld loss mutant), and DBA/2 (H-2d) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) or Charles River Laboratories (Wilmington, MA) and were housed and bred in the barrier animal facility at Washington University School of Medicine (St. Louis, MO).

The amino acid sequence of the murine CMV (MCMV)4 peptide corresponds to residues 168–176 (YPHFMPTNL) of the MCMV immediate early protein pp89 (24). The amino acid sequence of the tum peptide corresponds to residues 14–22 (TQNHRALDL) of the mutant protein P91A (exon 4) from the tum P815 variant (25). The p2Ca and QL9 peptides are both derived from the endogenous mitochondrial protein α-ketoglutarate dehydrogenase, and the sequences are LSPFPFDL and QLSPFPFDL, respectively (8, 12, 26). Peptides were synthesized using Merrifield’s solid-phase method (27) on a peptide synthesizer (model 431A; Applied Biosystems, Foster City, CA). Peptides were purified (>95%) by reverse-phase HPLC, and purity was assessed, as described (28).

28-14-8S is a mAb of the mouse IgG2a isotype specific for the α3 domain of Ld and was used for detection of Ld and the Ld mutants (29). The following mAbs were used in flow-cytometric studies to establish the TCR Vβ usage of our CTL clones. F23.1 is a mouse IgG2a mAb that is specific for the mouse Vβ8.1, Vβ8.2, and Vβ8.3 regions (30). MR9-4, a mouse mAb specific for the Vβ5.1 and Vβ5.2 regions; RR4-7, a rat mAb specific for the Vβ6 region; and MR12-3, a mouse mAb specific for the Vβ13 region were generous gifts from Osami Kanagawa (Washington University). 1B2 is a clonotypic mouse mAb specific for the 2C TCR (31).

DAP-3 is the murine Ltk fibroblast cell line (H-2k), and L-Ld was generated by introducing the Ld gene into DAP-3 cells (32). LM1.8, a kind gift from Phillipe Kourilsky (INSERM, Institut Pasteur, Paris, France), is the L cell (H-2k) fibroblast cell line transfected with ICAM-1 under HAT selection. LM1.8-Ld is the LM1.8 cell line transfected with the Ld cDNA under G418 resistance. Cell lines were maintained at 37°C, 5% CO2 in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 2 mM l-glutamine, 10% (v/v) bovine calf serum (HyClone Laboratories, Logan, UT), 0.1 mM nonessential amino acids, 1.25 mM HEPES, 1 mM sodium pyruvate, and 100 U/ml penicillin/streptomycin. Transfected cell lines were grown in medium containing 0.6 mg/ml G418 (Geneticin; Life Technologies, Grand Island, NY).

The Ld-alloreactive CTL clone 2C, generated from a BALB.B (H-2b) mouse that had been immunized with H-2d cells, and shown to specifically recognize Ld (33), was a generous gift from Herman Eisen (MIT, Cambridge, MA). The Ld-alloreactive clone, L3 (34), was provided by Matt Thomas (Washington University). The clone 42F3 was derived in vitro from a dm2 anti-BALB/c response and was shown to be specific for Ld+ p2Ca, and Vβ8+, but 1B2 (35). The Ld-alloreactive, MCMV-specific CTL clone 2.11.2 and the Ld-restricted, tum-specific CTL clones P15 and P24 were previously described (2, 36, 37). All clones were maintained in 24-well plates in sensitization medium (RPMI 1640 medium supplemented with l-glutamine, sodium pyruvate, nonessential amino acids, 100 U/ml penicillin/streptomycin, 50 μM 2-ME, and 10% (v/v) FCS (HyClone Laboratories)) and stimulated weekly with 5 × 106 irradiated (2000 rad) BALB/c splenocytes/ml and 10 U/ml murine rIL-2 (Biosource, Camarillo, CA). Peptide-specific clones were supplemented with 10−5 M peptide.

The MCMV-specific, Ld-alloreactive clone 2.3.3 was generated in vitro in the presence of the MCMV peptide, as described (2). Clones 1C2, 1A1F7, and 1A1G9 were generated using dm2 mice that had been primed with BALB/c splenocytes in vivo. Two weeks after priming, spleens from the mice were removed and the splenocytes were isolated. A total of 7.5 × 106 responding dm2 splenocytes/well was cocultured with 3.5 × 106 BALB/c splenocytes/well (irradiated at 2000 rad) in 24-well Linbro plates (Flow Laboratories, ICN, Horsham, PA) containing 2 ml sensitization medium. After incubation for 5 days at 37°C and 5% CO2, effector cells were analyzed for Ld specificity. For the generation of CTL clones, effector cells were resuspended in fresh sensitization medium and cloned by limiting dilution into 96-well, round-bottom plates in the presence of 2.5 × 106 irradiated BALB/c splenocytes/ml and 10 U/ml rIL-2. The clones were restimulated weekly by replacing 100 μl of the medium with fresh medium containing 5 × 106 irradiated BALB/c splenocytes/ml and 10 U/ml rIL-2. Clones were selected for recognition of Ld-expressing targets and maintained by weekly restimulation in 24-well plates with 0.5–1 × 106 T cells/well, 5 × 106 irradiated splenocytes/well, and 10 U/ml rIL-2.

The generation of the tum- and MCMV-specific CTL clones was performed as previously described (38). The MCMV-specific clones 8, C5, D7, and A8 were generated from BALB/c splenocytes, while the clones IF5, 1C6, and 2C4 were generated from DBA/2 splenocytes. The tum-specific clones ID3, IC10, and IG10 were generated from DBA/2 splenocytes.

The Ld mutants were made by PCR from an Ld cDNA template, and sequenced to confirm the presence of the mutation and the fidelity of the polymerase reaction. Each Ld mutant was cloned into the expression vector RSV.5.neo (39), and the constructs were transfected, following the instructions provided by the manufacturer, into LM1.8 cells using Lipofectin (Life Technologies, Gaithersburg, MD).

Cells were washed and incubated on ice in PBS containing 0.2% BSA in the presence of a saturating concentration of mAb, or PBS + 0.2% BSA alone, for 30 min, washed twice, and incubated with a saturating concentration of fluorescein-conjugated, Fc-specific, affinity-purified F(ab′)2 fragment of goat anti-mouse, or goat anti-rat, IgG (Organon-Teknika Cappel, West Chester, PA) for 30 min on ice. Cells were washed twice and resuspended, and the viable cells, gated by forward and side light scatter, were analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Mean fluorescence values were converted from logarithmic amplification by linear regression analysis using the CellQuest 30 software (Becton Dickinson).

For peptide inductions, cells were cultured overnight at 37°C in the presence or absence of peptide in RPMI medium (Life Technologies, Grand Island, NY) supplemented with 10% BCS (HyClone, Logan, UT), 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 1.25 mM HEPES, and 100 U/ml penicillin/streptomycin. After incubation, the cells were harvested, labeled, and analyzed, as described above.

A total of 1 × 106 target cells was labeled for 1 h with 150–200 μCi of 51Cr (Na51CrO4, NEN, Boston, MA; 1 Ci = 37 GBq) in 200 μl of RPMI 1640 medium + 10% bovine calf serum at 37°C in 5% CO2. Effector cells were plated at various concentrations into 96-well microtiter plates, and 2.5 × 103 or 5 × 103 washed target cells per well were added. In some experiments, the targets were resuspended in medium containing peptide before plating. The plates were centrifuged at 50 × g for 1 min and incubated for 4 h at 37°C in 5% CO2. Radioactivity in 100 μl of supernatant was measured in an Isomedic gamma counter (ICN Biomedicals, Huntsville, AL). The mean of triplicate samples was calculated, and percentage 51Cr release was determined according to the following equation: percentage 51Cr release = 100 × ((experimental 51Cr release − control 51Cr release)/(maximum 51Cr release − control 51Cr release)), where experimental 51Cr release represents counts from target cells mixed with effector cells; control 51Cr release represents counts from target cells incubated in medium alone (spontaneous release); and maximum 51Cr release represents counts from target cells lysed with 5% Triton X-100. Spontaneous release ranged from 8–20% of maximum release.

CTL assays were performed on day 4 after clone restimulation to determine the lowest E:T ratio that gave maximal lysis on LM1.8 cells expressing wild-type Ld. This was done to ensure strong lysis by all of the clones and to avoid the effect of a mutation being masked by too high of an E:T ratio. On day 5 after clone restimulation, this E:T ratio was used for CTL assays with the entire panel of target cell lines. All of the clones gave strong lysis of wild-type LM1.8-Ld (mean specific lysis 48 ± 2% (SEM)). For the data in Fig. 2, for a given clone, the lysis of each Ld mutant was calculated relative to the lysis of wild-type Ld. The lysis of mutant Ld by any one clone was divided by the lysis of wild-type Ld by the same clone (× 100) to obtain the relative lysis of that clone on the mutant.

FIGURE 2.

Recognition of H-2Ld mutants by allogeneic and syngeneic CTL clones. The peptide specificities and expression of Vβ8, where known, of the clones are indicated. The lysis of each Ld mutant is relative to lysis of wild-type Ld. Relative lysis was calculated as the percent specific lysis of LM1.8 cells transfected with mutant Ld divided by the percent specific lysis of LM1.8 cells transfected with wild-type Ld. Black boxes indicate CTL lysis of the mutant at levels less than 25% of wild-type Ld; grey boxes indicate lysis between 25 and 50% of wild-type Ld; and white boxes indicate lysis comparable with that of wild-type Ld (greater than 50% of wild-type Ld). Assays were run in continuous peptide at 10−5 M. For each clone, the lowest E:T ratio that gave peak lysis on LM1.8-Ld was used to test for recognition of the mutants. All data represent means of two to five independent experiments. The mean specific lysis of LM1.8 cells expressing wild-type Ld was 48 ± 2% (SEM).

FIGURE 2.

Recognition of H-2Ld mutants by allogeneic and syngeneic CTL clones. The peptide specificities and expression of Vβ8, where known, of the clones are indicated. The lysis of each Ld mutant is relative to lysis of wild-type Ld. Relative lysis was calculated as the percent specific lysis of LM1.8 cells transfected with mutant Ld divided by the percent specific lysis of LM1.8 cells transfected with wild-type Ld. Black boxes indicate CTL lysis of the mutant at levels less than 25% of wild-type Ld; grey boxes indicate lysis between 25 and 50% of wild-type Ld; and white boxes indicate lysis comparable with that of wild-type Ld (greater than 50% of wild-type Ld). Assays were run in continuous peptide at 10−5 M. For each clone, the lowest E:T ratio that gave peak lysis on LM1.8-Ld was used to test for recognition of the mutants. All data represent means of two to five independent experiments. The mean specific lysis of LM1.8 cells expressing wild-type Ld was 48 ± 2% (SEM).

Close modal

A model of the Ld/MCMV complex was built using the refined coordinates of the Ld/P29 complex (40), where residues P3(N), P4(V), P5(N), P6(I), P7(H), and P9(F) of the P29 peptide (YPNVNIHNF) were replaced with those of the MCMV peptide (YPHFMPTNL). The residues of the Ld heavy and light chains were not tampered with during the modeling. The atomic interactions between the heavy chain atoms and the MCMV peptide atoms were manually checked for close contacts. Molecular dynamics calculations and a 200-step conjugate gradient energy minimization were performed using DISCOVER module in INSIGHT (Molecular Simulation, San Diego, CA). Fig. 5 was generated using the energy-minimized model of Ld/MCMV and the program SETOR (41). The model was stable during the molecular dynamics simulations and reached equilibrium on the basis of the leveling with time of the root-mean-square deviation (1.11 angstrom) of the C α atom positions. The proline at P6 of the MCMV peptide in the energy-minimized conformation adopts the same orientation as the proline at P6 of QL9 in the energy-minimized Ld/QL9 model of Speir et al. (42).

FIGURE 5.

TCR interaction sites on Ld/MCMV. A ribbon diagram of the H-2Ld crystal structure modeled with the MCMV peptide, in yellow, viewed from ∼30o away from the α1 α2 pseudo-dyad axis. The side chains, and for Gly the main chain carbonyl oxygen, of the five primary TCR contact residues on Ld (Gly69, Gln72, Val76, Tyr155, and Arg157), based on data from Fig. 2, and the side chain of the primary TCR contact site on the MCMV peptide (Pro at P6) are shown as ball-and-stick models (grey, carbons; blue, nitrogens; red, oxygens).

FIGURE 5.

TCR interaction sites on Ld/MCMV. A ribbon diagram of the H-2Ld crystal structure modeled with the MCMV peptide, in yellow, viewed from ∼30o away from the α1 α2 pseudo-dyad axis. The side chains, and for Gly the main chain carbonyl oxygen, of the five primary TCR contact residues on Ld (Gly69, Gln72, Val76, Tyr155, and Arg157), based on data from Fig. 2, and the side chain of the primary TCR contact site on the MCMV peptide (Pro at P6) are shown as ball-and-stick models (grey, carbons; blue, nitrogens; red, oxygens).

Close modal

To determine which residues of the MHC class I molecule H-2Ld are important for functional interaction with TCRs, amino acid residues of both the α1 and α2 domains of Ld, predicted to interact with TCRs, were mutated. Of the 18 Ld residues indicated in Fig. 1, 14 were mutated independently, and residues 144, 145 and 155, 157 were mutated together. These residues, with the exception of 107 on a loop on the α2 domain, span much of the α-helices, and point up and, thus, are predicted to interact directly with TCRs (41, 43). Where possible, to minimize gross alterations in the class I structure, the amino acid residue found in Ld was changed to an amino acid residue found in other class I molecules at that position. The nonconservative amino acid substitutions made at each of these Ld residues are shown in Table I. Flow-cytometric analysis demonstrates that the cell lines transfected with mutant Ld molecules express normal levels of Ld, within a 4-fold range of the transfectant expressing wild-type Ld, LM1.8-Ld (Table II).

FIGURE 1.

Location of the mutated H-2Ld amino acid residues and the recognition patterns of H-2Ld and H-2Kb. The indicated residues were mutated, and 20 Ld-alloreactive and Ld-restricted clones were tested for recognition of these Ld mutants. The solid line outlines the functional TCR-interaction area on Ld and includes positions 69, 72, 76, and 155/157. The dashed line outlines the functional TCR-interaction area on H-2Kb as found by Sun et al. (5 ), and includes positions 80, 82, 158, 166, 167, and 174.

FIGURE 1.

Location of the mutated H-2Ld amino acid residues and the recognition patterns of H-2Ld and H-2Kb. The indicated residues were mutated, and 20 Ld-alloreactive and Ld-restricted clones were tested for recognition of these Ld mutants. The solid line outlines the functional TCR-interaction area on Ld and includes positions 69, 72, 76, and 155/157. The dashed line outlines the functional TCR-interaction area on H-2Kb as found by Sun et al. (5 ), and includes positions 80, 82, 158, 166, 167, and 174.

Close modal
Table I.

Amino acid substitutions at mutated Ld residues

Residue MutatedAmino Acid SubstitutionNew Amino Acid Found ina
58 E → K Conserved 
65 Q → R b,c 
69 G → D Kq, Dq, Dp 
72 Q → A d 
76 V → E b,c 
82 L → R c 
107 G → W Kd, Kk, Lq, Dk, Db, Dp, —c 
144 R → K Kq, Kk, Kb, Dk 
145 R → H Kq, Kk, Kb, Dk, —b,c 
149 Q → A c 
154 E → A d 
155 Y → H Dq, Lq, Db 
157 R → K Dq, Lq, Db, —b 
158 A → T b 
162 G → D d 
166 E → K b 
169 H → R Kd, Kk, Dd, Dk, Dp, —b,c 
173 K → E Kd, Dk, Dp, —bc 
Residue MutatedAmino Acid SubstitutionNew Amino Acid Found ina
58 E → K Conserved 
65 Q → R b,c 
69 G → D Kq, Dq, Dp 
72 Q → A d 
76 V → E b,c 
82 L → R c 
107 G → W Kd, Kk, Lq, Dk, Db, Dp, —c 
144 R → K Kq, Kk, Kb, Dk 
145 R → H Kq, Kk, Kb, Dk, —b,c 
149 Q → A c 
154 E → A d 
155 Y → H Dq, Lq, Db 
157 R → K Dq, Lq, Db, —b 
158 A → T b 
162 G → D d 
166 E → K b 
169 H → R Kd, Kk, Dd, Dk, Dp, —b,c 
173 K → E Kd, Dk, Dp, —bc 
a

Determined from Refs. 43 and 58.

b

Found in some class Ib molecules.

c

Found in some HLA alleles.

d

Not found in other class I molecules.

Table II.

Surface expression and peptide induction of the Ld mutants

CellsMean Fluorescence IntensityaFold Increase in Ld Surface Expressionb
Peptide
MCMVtump2Ca
LM1.8 5.4    
LM1.8-Ld 137.3 6.8 5.5 6.0 
LM1.8-Ld-E58K 59.8 5.3 5.1 4.8 
LM1.8-Ld-Q65R 105.7 5.5 4.5 4.8 
LM1.8-Ld-G69D 36.3 4.5 3.5 3.9 
LM1.8-Ld-Q72A 32.6 6.6 5.3 5.6 
LM1.8-Ld-V76E 78.1 4.0 3.6 4.1 
LM1.8-Ld-L82R 25.4 8.5 6.5 7.2 
LM1.8-Ld-G107W 39.1 6.2 5.1 4.6 
LM1.8-Ld-R144K/R145H 153.4 6.4 4.9 6.4 
LM1.8-Ld-Q149A 148.6 6.7 6.7 6.6 
LM1.8-Ld-E154A 94.2 5.4 5.2 5.6 
LM1.8-Ld-Y155H/R157K 51.8 7.0 6.6 6.8 
LM1.8-Ld-A158T 161.1 5.3 5.7 5.4 
LM1.8-Ld-G162D 104.9 6.2 5.5 5.4 
LM1.8-Ld-E166K 43.9 3.0 3.1 2.7 
LM1.8-Ld-H169R 205.1 6.3 4.8 5.5 
LM1.8-Ld-K173E 153.0 6.7 5.1 6.6 
CellsMean Fluorescence IntensityaFold Increase in Ld Surface Expressionb
Peptide
MCMVtump2Ca
LM1.8 5.4    
LM1.8-Ld 137.3 6.8 5.5 6.0 
LM1.8-Ld-E58K 59.8 5.3 5.1 4.8 
LM1.8-Ld-Q65R 105.7 5.5 4.5 4.8 
LM1.8-Ld-G69D 36.3 4.5 3.5 3.9 
LM1.8-Ld-Q72A 32.6 6.6 5.3 5.6 
LM1.8-Ld-V76E 78.1 4.0 3.6 4.1 
LM1.8-Ld-L82R 25.4 8.5 6.5 7.2 
LM1.8-Ld-G107W 39.1 6.2 5.1 4.6 
LM1.8-Ld-R144K/R145H 153.4 6.4 4.9 6.4 
LM1.8-Ld-Q149A 148.6 6.7 6.7 6.6 
LM1.8-Ld-E154A 94.2 5.4 5.2 5.6 
LM1.8-Ld-Y155H/R157K 51.8 7.0 6.6 6.8 
LM1.8-Ld-A158T 161.1 5.3 5.7 5.4 
LM1.8-Ld-G162D 104.9 6.2 5.5 5.4 
LM1.8-Ld-E166K 43.9 3.0 3.1 2.7 
LM1.8-Ld-H169R 205.1 6.3 4.8 5.5 
LM1.8-Ld-K173E 153.0 6.7 5.1 6.6 
a

Data shown are the mean fluorescence intensity of Ld surface expression as measured by flow cytometry as described in Materials and Methods using the mAb 28-14-8, which recognizes the α3 domain of Ld.

b

Data shown represent the fold increase in Ld surface expression as measured by flow cytometry as described in Materials and Methods using the mAb 28-14-8 after overnight incubation of the cell lines with 250 or 500 μM of the indicated peptides.

To verify that the Ld mutants can stably bind peptides, Ld mutant-expressing cell lines were incubated overnight with p2Ca, MCMV, and tum peptide, and surface Ld expression was examined by flow cytometry. Surface Ld expression was found to increase a minimum of 2.7-fold (average 5.5-fold) for the mutants (Table II). This increase in expression of Ld has been shown to result from peptide binding to and stabilization of surface Ld molecules, and thus a high proportion (≥80%) of surface Ld molecules is occupied with the exogenously added peptide ligand (44, 45). This indicates that the mutant Ld molecules clearly retain their ability to bind these three different peptide ligands and that much of the surface Ld is occupied with the added peptide. The ability of all the mutant Ld molecules to bind peptides together with their substantial surface expression validates their use in functional analyses using peptide-specific CTL clones reactive with Ld.

An extensive panel of CTL clones was used to determine whether similar residues of Ld are involved in recognition by self-Ld-restricted clones specific for different peptides, as well as in recognition by alloreactive and self-restricted clones specific for the same or different peptides. The panel of independently derived CTL clones tested for recognition of the Ld mutants includes eight Ld-alloreactive clones, four of which have known peptide specificity, and twelve Ld-restricted clones (Table III). In addition, nine of the clones are Vβ8+, including alloreactive and self-restricted clones with different peptide specificities. This permits a comparison of the Ld residues recognized by Vβ8+ vs Vβ8 TCRs.

Table III.

CTL clones

ClonePeptide SpecificityTCRaStrain CombinationRef.
Ld-alloreactive     
2.3.3 MCMV NT dm2 α BALB/c Present study 
2.11.2 MCMV NT dm2 α BALB/c 
2C p2Ca Vβ8+ BALB/B α H-2d 8, 31 
42F3 p2Ca Vβ8+ dm2 α BALB/c 35 
L3 Unknown NT C57BL/6 α DBA/2 34 
1C2 Unknown NT dm2 α BALB/c Present study 
1A1F7 Unknown NT dm2 α BALB/c Present study 
1A1G9 Unknown Vβ13+ dm2 α BALB/c Present study 
Ld-restricted     
MCMV Vβ8 BALB/c Present study 
C5 MCMV Vβ8 BALB/c Present study 
D7 MCMV Vβ8+ BALB/c Present study 
P15 tum Vβ8+ BALB/c 37 
P24 tum Vβ8+ BALB/c 36 
ID3 tum Vβ8 DBA/2 Present study 
IC10 tum Vβ8 DBA/2 Present study 
IG10 tum Vβ8+ DBA/2 Present study 
IF5 MCMV Vβ8+ DBA/2 Present study 
A8 MCMV Vβ8 BALB/c Present study 
1C6 MCMV Vβ8+ DBA/2 Present study 
2C4 MCMV Vβ8+ DBA/2 Present study 
ClonePeptide SpecificityTCRaStrain CombinationRef.
Ld-alloreactive     
2.3.3 MCMV NT dm2 α BALB/c Present study 
2.11.2 MCMV NT dm2 α BALB/c 
2C p2Ca Vβ8+ BALB/B α H-2d 8, 31 
42F3 p2Ca Vβ8+ dm2 α BALB/c 35 
L3 Unknown NT C57BL/6 α DBA/2 34 
1C2 Unknown NT dm2 α BALB/c Present study 
1A1F7 Unknown NT dm2 α BALB/c Present study 
1A1G9 Unknown Vβ13+ dm2 α BALB/c Present study 
Ld-restricted     
MCMV Vβ8 BALB/c Present study 
C5 MCMV Vβ8 BALB/c Present study 
D7 MCMV Vβ8+ BALB/c Present study 
P15 tum Vβ8+ BALB/c 37 
P24 tum Vβ8+ BALB/c 36 
ID3 tum Vβ8 DBA/2 Present study 
IC10 tum Vβ8 DBA/2 Present study 
IG10 tum Vβ8+ DBA/2 Present study 
IF5 MCMV Vβ8+ DBA/2 Present study 
A8 MCMV Vβ8 BALB/c Present study 
1C6 MCMV Vβ8+ DBA/2 Present study 
2C4 MCMV Vβ8+ DBA/2 Present study 
a

TCR Vβ usage was determined as described in Materials and Methods. NT indicates clones that were not tested.

Recognition of the Ld mutants by these CTL clones was assessed in 51Cr release assays in the presence of 10−5 M continuous peptide to maximize surface expression of Ld/peptide complexes and to minimize minor differences in peptide binding. In previous analyses, this concentration gave maximal recognition regardless of the affinity of the clone (Ref. 38 and unpublished observations). This same rationale extends to the alloreactive clones specific for an endogenous peptide, p2Ca (2C and 42F3). Each combination was tested in two to five independent experiments, and the results of all the experiments are summarized in Fig. 2. The black boxes indicate a dramatic reduction in T cell recognition, whereas the grey boxes indicate an intermediate effect and white boxes indicate little or no effect.

We find that mutations of the amino acid residues at positions 69, 72, 76, or 155/157 of Ld result in the most dramatic effect on CTL recognition. Each of these mutations reduces CTL recognition to less than 50% of wild-type Ld for 75% or more of the CTL clones. In fact, for many of the clones, recognition is reduced to less than 25% of wild-type Ld. Even though peptide is present at a high concentration, and thus peptide occupancy is high, mutation of these residues has a noticeable effect on recognition, suggesting that these are major TCR contact points. In addition, for Ld molecules mutated at residues 58, 82, 158, 162, or 166, recognition is reduced to less than 50% of wild-type Ld for 50% or more of the clones. The importance of TCR interaction with each of these amino acid residues, although clearly significant, appears to vary depending upon the particular CTL clone. It is important to note that a unique aspect of this analysis is that it includes both Ld-restricted and Ld-alloreactive CTL specific for the same peptide, MCMV. Thus, a direct comparison can be made between allogeneic and syngeneic CTL that recognize the same MHC/peptide complex. These data reveal a recognition pattern of Ld by both alloreactive and self-restricted T cells, which spans much of the α1 α-helix and the middle of the α2 α-helix toward the C-terminal end, with the Ld residues most important for recognition clustered in the middle of both α-helices (Fig. 1). Thus, the majority of clones analyzed show a common recognition pattern of Ld that involves both relatively conserved (72, 76) and polymorphic (69, 155) amino acid residues.

Next we wanted to determine whether, in addition to common residues, there are differences in other residues that are involved in recognition of a specific MHC/peptide ligand. Given that the nature of the peptide bound to Ld can influence the conformation of the MHC/peptide complex (46), it might be expected that different MHC residues would be involved in TCR interaction depending on the sequence of the Ld-bound peptide. However, comparison of recognition of the Ld mutants by CTL clones specific for the MCMV peptide vs those specific for the tum peptide shows a remarkable similarity. The most dramatic effect on CTL recognition is seen for mutation of the same key positions noted earlier. MHC residues 69, 72, 76, and 155/157 are important for recognition by both tum-specific and MCMV-specific clones, with 60% or more of each type affected by these mutations. There are some differences, namely the amino acids at positions 82 and 169 are more important for MCMV-specific clones than tum-specific clones. This suggests, although a larger sample size would need to be examined to verify this, that in addition to common MHC residues important for recognition of Ld (69, 72, 76, 155/157), the importance of other MHC residues for recognition may vary depending on the sequence of the peptide bound.

The preponderance of Vβ8+ TCRs among Ld-alloreactive and Ld-restricted clones suggests that these TCRs may contact similar residues of Ld (35, 36, 47). However, our data show that the Ld residues important for interaction with TCRs are similar regardless of whether the CTL is Vβ8+ or Vβ8. Differences between TCR-MHC contacts might be expected for TCRs with different Vβ se- quences. Instead, comparing the nine clones that are known to be Vβ8+ (2C, 42F3, D7, P15, P24, IG10, IF5, 1C6, and 2C4) with the six that are known to be Vβ8 (1A1G9, 8, C5, ID3, IC10, and A8), there are only a few differences. Actually, our data demonstrate that clones that share usage of Vβ8 show some differences in the Ld residues contacted, indicating similar TCRs can interact with distinct MHC residues. Because the TCR β-chain contacts both peptide and MHC residues, as shown in crystal structure analyses (18, 20, 48), the precise MHC residues contacted by a given TCR most likely depend upon the specificity of the peptide bound to the MHC molecule.

To address whether allogeneic clones are biased toward interaction with MHC residues compared with syngeneic clones, we assessed the effect of the mutations on recognition by each type of clone. If alloreactive clones are more MHC dependent, then they should recognize fewer Ld mutants than the syngeneic clones. However, both the Ld-alloreactive clones as well as the Ld-restricted clones, on average, recognize a similar number of the MHC mutants. The Ld-alloreactive clones recognized 53% of the mutants and the Ld-restricted clones recognized 48% of the mutants (Fig. 3). Thus, there does not appear to be an increased requirement for interaction with MHC amino acid residues by the Ld-alloreactive CTL clones.

FIGURE 3.

Ld-alloreactive and Ld-restricted CTL clones show similar levels of cross-reactivity with the Ld mutants. Each point represents the number of Ld mutants recognized at levels between 50 and 100% of recognition of wild-type Ld by each clone, with the 8 alloreactive clones on the left and the 12 Ld-restricted clones on the right. Horizontal lines indicate the group average.

FIGURE 3.

Ld-alloreactive and Ld-restricted CTL clones show similar levels of cross-reactivity with the Ld mutants. Each point represents the number of Ld mutants recognized at levels between 50 and 100% of recognition of wild-type Ld by each clone, with the 8 alloreactive clones on the left and the 12 Ld-restricted clones on the right. Horizontal lines indicate the group average.

Close modal

In addition, the specific residues important for recognition of Ld vary little between allogeneic and syngeneic CTL clones. The same residues that comprise the common recognition pattern of Ld (positions 69, 72, 76, and 155/157) are important for both types of clones, although mutation of the amino acid residue at position 72 affects somewhat more alloreactive clones (88%) than syngeneic clones (67%). There are differences at three other sites on the Ld α-helices; the amino acid residue at position 82 is more important for Ld recognition by alloreactive clones, while the residues at positions 144/145 and 162 are more important for the Ld-restricted clones. For the remainder of the Ld residues, a similar number of allogeneic and syngeneic clones is affected. Together these data suggest that Ld-restricted clones are as dependent on interaction with MHC residues as Ld-alloreactive clones.

The alloreactive clone, 2C, is not affected by any of the Ld mutations at the high peptide concentration used for analysis, whereas all of the other clones were affected by at least some of the Ld mutations (Fig. 2). This suggests that, unlike what has been reported (13), 2C shows a decreased dependence upon interaction with MHC residues. Another p2Ca-specific, Ld-alloreactive clone (42F3) and two other peptide-specific, Ld-alloreactive clones (2.3.3 and 2.11.2) are affected by several of the Ld mutations. Thus, the insensitivity of 2C to the Ld mutations does not appear to be a common feature of peptide-specific, Ld-alloreactive clones.

2C’s unique recognition pattern of Ld could reflect a reduced dependence upon interaction with MHC residues due to the high affinity interaction of the 2C TCR with ligand (12), or 2C’s recognition pattern of Ld may not include the mutated residues. To distinguish between these two possibilities, we tested the ability of 2C to recognize those Ld mutants shown to have the most dramatic effect on recognition by the other T cell clones, over a wide range of peptide concentrations. Decreasing the peptide concentration effectively lowers the ligand density and reduces the avidity of the interaction. We observed that 2C recognizes wild-type Ld and Q149A, which does not affect recognition by most of the clones, in the absence of exogenous peptide (Fig. 4). However, 2C recognizes Q72A and Y155H/R157K at high, but not lower peptide concentrations (Fig. 4). This indicates that the 2C TCR recognizes similar MHC determinants as other Ld-restricted and alloreactive clones, and thus the common recognition pattern of Ld applies also to 2C.

FIGURE 4.

Amino acid residues of Ld important for interaction with 2C can be unmasked at lower peptide concentrations. Assays were performed at an E:T ratio of 2:1 in the continuous presence of the QL9 peptide.

FIGURE 4.

Amino acid residues of Ld important for interaction with 2C can be unmasked at lower peptide concentrations. Assays were performed at an E:T ratio of 2:1 in the continuous presence of the QL9 peptide.

Close modal

Although 2C shares at least some of the same key contact residues with the majority of Ld-alloreactive and Ld-restricted clones tested, the ability of 2C to interact with several Ld mutants at a peptide concentration that reduces recognition by the other clones suggests that 2C is less dependent upon interaction with MHC residues. Thus, because of the overall high affinity of 2C for its ligand, mutation at any one site does not interfere with functional engagement at high ligand density. This analysis demonstrates that although 2C displays unique cross-reactivities that may reflect its high affinity interaction with ligand, it still interacts with the same key Ld residues, and thus shares the same orientation as other Ld-reactive TCRs.

The mechanistic basis of the strength of alloreactive T cell responses compared with self MHC-restricted responses is unknown, but may reflect inherent properties of the T cell repertoire and how it is selected. Indeed, the preselected repertoire appears to be biased toward MHC reactivity (15, 16), and because T cells with high affinity for alloantigens would not be specifically deleted during thymic development, it has been speculated that alloreactive TCRs are more MHC dependent and less peptide dependent than self-restricted TCRs. To rigorously assess the MHC dependency of self-restricted vs alloreactive TCRs, we compared the activity of several CTL on an extensive panel of targets expressing Ld molecules with mutations at positions that potentially engage TCRs. Whereas other studies compared recognition of different MHC alleles by the same T cell, our study is the first to compare the MHC dependency of T cells that are either self restricted or alloreactive to the same class I molecule. In contrast to the results of these other studies, we show in this work that Ld-restricted and Ld-alloreactive T cells are comparably dependent on interaction with MHC alleles and in fact contact the same primary residues. Furthermore, the MHC recognition pattern is also independent of peptide specificity.

The most dramatic effects on T cell recognition of Ld include the mutation of residues at 155/157. Other studies, both structural and functional, have also implicated the importance of the class I MHC residue 155 for TCR recognition. These include recognition of Kb/dEV8 by 2C, and recognition of HLA-A2, HLA-B7, H-2Kd, and H-2Ld by different TCRs (2, 4, 6, 18, 20, 48, 49, 50, 51). Thus, position 155 serves as a principal anchor on most class I molecules, and may be required for docking the TCR on its MHC/peptide ligand. In addition to 155/157, mutation of residues at positions 69, 72, and 76 had the most dramatic effect on recognition of Ld, and residues at positions 58, 82, 158, 162, and 166 were also influential. It is important to note that the dramatic reduction in recognition of the Ld molecules mutated at these positions occurred at high peptide concentration, and thus could not be rescued at high ligand density. This underscores the importance of these residues for Ld recognition. Thus, these are the primary contact residues for Ld-reactive T cells.

The residues implicated in Ld interaction with TCRs in our study are generally consistent with the diagonal orientation of TCRs with MHC, first proposed in a study examining TCR recognition of Kb (5). Based on the effect of mutations at positions 80, 82, 158, 166, 167, and 174, it was proposed that the TCR engages Kb/peptide parallel to the MHC β-pleated sheets and diagonal to the MHC α-helices. This model has more recently been supported by class II mutagenesis and TCR-class I MHC crystallographic studies (17, 18, 19, 20, 21). Such a common orientation for TCR-MHC engagement implies that TCR specificity is achieved by TCR engagement of a unique combination of amino acid residues, including polymorphic MHC residues and distinct peptide residues.

Although our results are consistent with the diagonal model, there are important differences that most likely dictate the allele specificity. For example, the primary TCR contact residues at positions 69, 72, 76, and 155/157 of Ld were not among the mutants that inhibited recognition of Kb (5). As shown in Fig. 1, the location of these residues on Ld is distinct from the location of residues on Kb implicated in TCR interaction, such that the important Kb residues are skewed more to the C-terminal regions of both α-helices. Also, mutation of Kb residues at positions 167 and 174 affected recognition, while we did not see a dramatic effect on TCR recognition of Ld by mutations at positions in this range (positions 169 and 173). A difference between TCR interaction with Ld vs Kb is also supported by the crystal structure of the 2C TCR-Kb/dEV8 complex (48). In that study, the Kb residue at position 69 is not contacted by the TCR, whereas in our study, position 69 is a primary TCR contact residue for recognition of Ld. Collectively, these findings suggest that TCRs may engage Ld in a distinct orientation compared with Kb, such that with Ld the TCR is slightly more perpendicular to the α-helices (Fig. 1). However, independent of predicting orientation, the reciprocal differences critical for TCR interaction with Kb vs Ld most likely define the structural basis of allotypic T cell recognition of these two class I molecules.

Comparison of our study with the aforementioned Kb mutagenesis study reveals significant differences in the location of residues on Ld vs Kb that are important for functional interaction with their respective TCRs. However, it is difficult to adapt our data to the recently published model predicting how the 2C TCR interacts with Ld/QL9 (42). This model was predicted from their structural resolution of Ld loaded with heterogeneous peptides, and was based on resolution of the structure of the 2C TCR with Kb/dEV8. It was also assumed that most of the TCR interactions with MHC are through conserved α-helical residues shared by Kb and Ld. However, our data indicate that both conserved and polymorphic MHC residues are important for recognition of Ld. In addition, we demonstrate that the primary functional MHC interaction sites are the same for all TCRs reactive with Ld, but differ between Ld and Kb. Solution of a crystal structure of Ld/peptide with TCR will help to resolve this discrepancy.

To visualize the topology of the critical residues on the surface of Ld/peptide complexes as seen by TCRs, we modeled the Ld/MCMV structure based on our crystal structure of Ld/P29 (41). We chose to model the MCMV peptide into the Ld groove because Ld-restricted and Ld-alloreactive, MCMV-specific CTL were characterized in this study and earlier experiments using alanine-substituted peptides identified the P6 proline as the most critical for CTL recognition (data not shown). Fig. 5 shows the orientation of the side chains (the main chain carbonyl oxygen in the case of Gly) of the residues we determined to be most critical for interaction of Ld with TCRs, namely Gly69, Gln72, Val76, Tyr155, and Arg157. The close proximity of the critical peptide side chain of the P6 proline between the critical Ld side chains highlights the importance of the central region of the MHC/peptide complex for TCR interaction. The side chain of the P6 proline of MCMV points out of the cleft, in part due to the hydrophobic ridge that transverses the center of the Ld Ag-binding groove (41, 42). Importantly, the p2Ca and QL9 peptides have a proline in the same position (P5 of the p2Ca octamer, P6 of the QL9 nonamer) that is critical for CTL recognition (52, 53, 54). The side chains of the P6 proline of QL9 and MCMV have a similar orientation when modeled bound to Ld (42 and Fig. 5). The fact that proline is the critical residue for TCR interaction with both MCMV and QL9 is a unique feature of Ld/peptide interactions with TCRs. Most structure/function studies have identified a central charged residue in the peptide that forms a salt bridge with the complementary charged residue in complementarity-determining region 3 of the TCR (as first shown by Jorgensen et al., in Ref. 22). Because proline cannot form a salt bridge, its function as the most critical peptide residue may be more a matter of shape complementarity.

As a corollary to the proposed difference in MHC dependency, it has also been speculated that self MHC-restricted TCRs are more peptide dependent than alloreactive TCRs (12, 13, 14). Recent studies have used peptide libraries to compare the peptide dependencies of individual T cell clones that display both self-restricted and alloreactive responses. For example, Brock et al. (13) reported that 2C recognition of self-Kb/SIYR-8 was highly peptide specific, whereas the allo-Ld/p2Ca response was very peptide degenerate. However, one recent study has shown that the recognition of self-Kb molecules by 2C T cells is peptide degenerate (55), and other studies have shown that 2C recognition of its allo-ligand is highly peptide specific (52, 53, 56). These approaches are limited by the use of a single T cell clone that detects different MHC molecules bound by different peptides with different anchor motifs. Thus, it is difficult to generalize from these studies in regard to the structural basis of allorecognition, and therefore the relative peptide dependency of 2C, and other alloreactive T cells, remains controversial. In this study, we have taken a reciprocal approach by comparing the MHC degeneracy of alloreactive vs self-restricted T cells. Another unique feature of our study is that all T cells compared were reactive with the same MHC molecule and several were specific for the same peptide.

The affinity of a TCR for MHC/peptide ligand can also influence whether mutations in peptide and/or MHC residues affect T cell recognition. Based on the higher affinity of the 2C TCR for its allo-ligand, Ld/QL9, compared with the clone 4G3 for its self ligand, Kb/SIINFEKL, it was speculated that alloreactive TCRs may be of a higher affinity than self MHC-restricted TCRs (12). A recent model of how Ld/QL9 interacts with the 2C TCR predicts that peptide, relative to class I, contributes 33% of the binding energy and 46% of the contacts with TCR (42, 57). This contribution of peptide to the binding energy could explain why in our study 2C T cells were strikingly tolerant to changes in Ld α-helical residues at high peptide concentrations in comparison with other Ld-reactive T cells. However, when peptide was limiting, 2C T cells displayed a similar pattern of recognition on the panel of Ld mutants. This result is consistent with 2C having a higher affinity for Ld than other T cells, but sharing a common orientation when engaging Ld. Given that 2C is atypical, at least among the T cells we compared, generalizations regarding affinity of alloreactive vs self-restricted T cells need to be confirmed using a wider panel of CTL clones. However, regardless of whether or not there is an affinity difference between alloreactive and self MHC-restricted TCRs, our study definitively shows that alloreactive T cells are not more dependent on MHC residues, but rather both types of T cells are comparably dependent upon interaction with the same MHC α-helical residues.

We thank Linda A. Walp for technical assistance, and Drs. Osami Kanagawa, Wayne Yokoyama, Yuri Sykulev, and Paul Allen for critical review of the manuscript.

1

This work was supported by National Institutes of Health Grants AI27568 and AI19687. T.M.C.H. is supported by the National Institutes of Health Training Grant AI07163.

4

Abbreviation used in this paper: MCMV, murine CMV.

1
Bjorkman, P. J..
1997
. MHC restriction in three dimensions: a view of T cell receptor/ligand interactions.
Cell
89
:
167
2
Alexander-Miller, M. A., K. Burke, U. H. Koszinowski, T. H. Hansen, J. M. Connolly.
1993
. Alloreactive cytotoxic T lymphocytes generated in the presence of viral-derived peptides show exquisite peptide and MHC specificity.
J. Immunol.
151
:
1
3
Ajitkumar, P., S. S. Geier, K. V. Kesari, F. Borriello, M. Nakagawa, J. A. Bluestone, M. A. Saper, D. C. Wiley, S. G. Nathenson.
1988
. Evidence that multiple residues on both the α-helices of the class I MHC molecule are simultaneously recognized by the T cell receptor.
Cell
54
:
47
4
Jaulin, C., J.-L. Casanova, P. Romero, I. Luescher, A.-S. Cordey, J. L. Maryanski, P. Kourilsky.
1992
. Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.
J. Immunol.
149
:
3990
5
Sun, R., S. A. Shepherd, S. S. Geier, C. T. Thomson, J. M. Sheil, S. G. Nathenson.
1995
. Evidence that the antigen receptors of cytotoxic T lymphoctyes interact with a common recognition pattern on the H-2Kb molecule.
Immunity
3
:
573
6
Smith, K. D., C. T. Lutz.
1997
. Alloreactive T cell recognition of MHC class I molecules: the T cell receptor interacts with limited regions of the MHC class I long α helices.
J. Immunol.
158
:
2805
7
Heath, W. R., K. P. Kane, M. F. Mescher, L. A. Sherman.
1991
. Alloreactive T cells discriminate among a diverse set of endogenous peptides.
Proc. Natl. Acad. Sci. USA
88
:
5101
8
Udaka, K., T. J. Tsomides, H. N. Eisen.
1992
. A naturally occurring peptide recognized by alloreactive CD8+ cytotoxic T lymphocytes in association with a class I MHC protein.
Cell
69
:
989
9
Malarkannan, S., F. Gonzalez, V. Nguyen, G. Adair, N. Shastri.
1996
. Alloreactive CD8+ T cells can recognize unusual, rare, and unique processed peptide/MHC complexes.
J. Immunol.
157
:
4464
10
Shi, Y., K. D. Smith, C. T. Lutz.
1998
. TAP-independent MHC class I peptide antigen presentation to alloreactive CTL is enhanced by target cell incubation at subphysiologic temperatures.
J. Immunol.
160
:
4305
11
Wang, W., S. Man, P. H. Gulden, D. F. Hunt, V. H. Engelhard.
1998
. Class I-restricted alloreactive cytotoxic T lymphocytes recognize a complex array of specific MHC-associated peptides.
J. Immunol.
160
:
1091
12
Sykulev, Y., A. Brunmark, T. J. Tsomides, S. Kageyama, M. Jackson, P. A. Peterson, H. N. Eisen.
1994
. High-affinity reactions between antigen-specific T-cell receptors and peptides associated with allogeneic and syngeneic major histocompatibility complex class I proteins.
Proc. Natl. Acad. Sci. USA
91
:
11487
13
Brock, R., K.-H. Wiesmüller, G. Jung, P. Walden.
1996
. Molecular basis for the recognition of two structurally different major histocompatibility complex/peptide complexes by a single T-cell receptor.
Proc. Natl. Acad. Sci. USA
93
:
13108
14
Daniel, C., S. Horvath, P. M. Allen.
1998
. A basis for alloreactivity: MHC helical residues broaden peptide recognition by the TCR.
Immunity
8
:
543
15
Ignatowicz, L., J. Kappler, P. Marrack.
1996
. The repertoire of T cells shaped by a single MHC/peptide ligand.
Cell
84
:
521
16
Zerrahn, J., W. Held, D. H. Raulet.
1997
. The MHC reactivity of the T cell repertoire prior to positive and negative selection.
Cell
88
:
627
17
Sant’Angelo, D. B., G. Waterbury, P. Preston-Hurlburt, S. T. Yoon, R. Medzhitov, S.-C. Hong, C. A. Janeway, Jr.
1996
. The specificity and orientation of a TCR to its peptide-MHC class II ligand.
Immunity
4
:
367
18
Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, D. C. Wiley.
1996
. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.
Nature
384
:
134
19
Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson, P. A. Peterson, L. Teyton, I. A. Wilson.
1996
. An αβ T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex.
Science
274
:
209
20
Ding, Y.-H., K. J. Smith, D. N. Garboczi, U. Utz, W. E. Biddison, D. C. Wiley.
1998
. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids.
Immunity
8
:
403
21
Teng, M.-K., A. Smolyar, A. G. D. Tse, J.-H. Liu, J. Liu, R. E. Hussey, S. G. Nathenson, H.-C. Chang, E. L. Reinherz, J.-H. Wang.
1998
. Identification of a common docking topology with substantial variation among different TCR-peptide-MHC complexes.
Curr. Biol.
8
:
409
22
Jorgensen, J. L., U. Esser, B. F. de St. Groth, P. A. Reay, M. M. Davis.
1992
. Mapping T-cell receptor-peptide contacts by variant peptide immunization of single-chain transgenics.
Nature
355
:
224
23
Sim, B.-C., P. J. Travers, N. R. J. Gascoigne.
1997
. Vα3.2 selection in MHC class I mutant mice: evidence for an alternate orientation of TCR-MHC class I interaction.
J. Immunol.
159
:
3322
24
Reddehase, M. J., J. B. Rothbard, U. H. Koszinowski.
1989
. A pentapeptide as minimal antigenic determinant for MHC class I-restricted T lymphocytes.
Nature
337
:
61
25
Lurquin, C., A. Van Pel, B. Mariamé, E. DePlaen, J.-P. Szikora, C. Janssens, M. J. Reddehase, J. Lejeune, T. Boon.
1989
. Structure of the gene of tum transplantation antigen P91A: the mutated exon encodes a peptide recognized with Ld by cytolytic T cells.
Cell
58
:
293
26
Udaka, K., T. J. Tsomides, P. Walden, N. Fukusen, H. N. Eisen.
1993
. A ubiquitous protein is the source of naturally occurring peptides that are recognized by a CD8+ T-cell clone.
Proc. Natl. Acad. Sci. USA
90
:
11272
27
Merrifield, R. B. 1963. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149.
28
Gorka, J., D. W. McCourt, B. D. Schwartz.
1989
. Automated synthesis of a C-terminal photoprobe using combined Fmoc and t-Boc synthesis strategies on a single automated peptide synthesizer.
Pept. Res.
2
:
376
29
Evans, G. A., D. H. Margulies, B. Skykind, J. G. Seidman, K. Ozato.
1982
. Exon shuffling, mapping polymorphic determinants on hybrid mouse transplantation antigens.
Nature
300
:
755
30
Staerz, U. D., H. G. Rammensee, J. D. Benedetto, M. J. Bevan.
1985
. Characterization of a novel monoclonal antibody specific for an allotypic determinant of T cell antigen receptor.
J. Immunol.
134
:
3994
31
Kranz, D. M., S. Tonegawa, H. N. Eisen.
1984
. Attachment of an anti-receptor antibody to non-target cells renders them susceptible to lysis by a clone of cytotoxic T lymphocytes.
Proc. Natl. Acad. Sci. USA
81
:
7922
32
Lie, W.-R., N. B. Myers, J. M. Connolly, J. Gorka, D. R. Lee, T. H. Hansen.
1991
. The specific binding of peptide ligand to Ld class I major histocompatibility complex molecules determines their antigenic structure.
J. Exp. Med.
173
:
449
33
Kranz, D. M., D. H. Sherman, M. V. Sitkovsky, M. S. Pasternack, H. N. Eisen.
1984
. Immunoprecipitation of cell surface structures of cloned cytotoxic T lymphoctyes by clone-specific antisera.
Proc. Natl. Acad. Sci. USA
81
:
573
34
Glasebrook, A. L., F. W. Fitch.
1979
. T-cell lines which cooperate in generation of specific cytolytic activity.
Nature
278
:
171
35
Connolly, J. M..
1994
. The peptide p2Ca is immunodominant in allorecognition of Ld by β chain variable region Vβ8+ but not Vβ8 strains.
Proc. Natl. Acad. Sci. USA
91
:
11482
36
Solheim, J. C., M. A. Alexander-Miller, J. M. Martinko, J. M. Connolly.
1993
. Biased T cell receptor usage by Ld-restricted, tum peptide-specific cytotoxic T lymphocyte clones.
J. Immunol.
150
:
800
37
Alexander-Miller, M., R. A. Robinson, J. D. Smith, W. E. Gillanders, L. G. Harrison, T. H. Hansen, J. M. Connolly, D. R. Lee.
1994
. Definition of TCR recognition sites on Ld-tum complexes.
Int. Immunol.
6
:
1699
38
Alexander, M. A., C. A. Damico, K. M. Wieties, T. H. Hansen, J. M. Connolly.
1991
. Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes.
J. Exp. Med.
173
:
849
39
Long, E. O., S. Rosen-Bronson, D. R. Darp, M. Malnati, R. P. Sekaly, D. Jaraquemada.
1991
. Efficient cDNA expression vectors for stable and transient expression of HLA-DR in transfected fibroblast and lymphoid cells.
Hum. Immunol.
31
:
229
40
Balendiran, G. K., J. C. Solheim, A. C. M. Young, T. H. Hansen, S. G. Nathenson, J. C. Sacchettini.
1997
. The three-dimensional structure of an H-2Ld-peptide complex explains the unique interaction of Ld with β2 microglobulin and peptide.
Proc. Natl. Acad. Sci. USA
94
:
6880
41
Evans, S. V..
1993
. SETOR: hardware lighted three-dimensional solid model representations of macromolecules.
J. Mol. Graph.
11
:
134
42
Speir, J. A., K. C. Garcia, A. Brunmark, M. Degano, P. A. Peterson, L. Teyton, I. A. Wilson.
1998
. Structural basis of 2C TCR allorecognition of H-2Ld peptide complexes.
Immunity
8
:
553
43
Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, D. C. Wiley.
1987
. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens.
Nature
329
:
512
44
Lie, W.-R., N. B. Myers, J. Gorka, R. J. Rubocki, J. M. Connolly, T. H. Hansen.
1990
. Peptide ligand-induced conformation and surface expression of the Ld class I MHC molecule.
Nature
344
:
439
45
Smith, J. D., W.-R. Lie, J. Gorka, C. S. Kindle, N. B. Myers, T. H. Hansen.
1992
. Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface.
J. Exp. Med.
175
:
191
46
Solheim, J. C., B. M. Carreno, J. D. Smith, J. Gorka, N. B. Myers, Z. Wen, J. M. Martinko, D. R. Lee, T. H. Hansen.
1993
. Binding of peptides lacking consensus anchor residue alters H-2Ld serologic recognition.
J. Immunol.
151
:
5387
47
Tjoa, B. A., D. M. Kranz.
1994
. Sequence restriction in T cell receptor β-chains that have specificity for a self-peptide/Ld complex.
Mol. Immunol.
31
:
705
48
Garcia, K. C., M. Degano, L. R. Pease, M. Huang, P. A. Peterson, L. Teyton, I. A. Wilson.
1998
. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen.
Science
279
:
1166
49
Moots, R. J., M. Matsui, L. Pazmany, A. J. McMichael, J. A. Frelinger.
1991
. A cluster of mutations in HLA-A2 α2 helix abolishes peptide recognition by T cells.
Immunogenetics
34
:
141
50
Louie, K. A., J. Ochoa-Garay, P.-J. Chen, D. McKinney, S. Groshen, M. McMillan.
1996
. H-2Ld-alloreactive T cell hybridomas utilize diverse Vα and Vβ T cell receptor chains.
Mol. Immunol.
9
:
747
51
Noun, G., M. Reboul, J.-P. Abastado, P. Kourilsky, F. Sigaux, M. Pla.
1998
. Strong alloantigenicity of the α-helices residues of the MHC class I molecule.
J. Immunol.
161
:
148
52
Sykulev, Y., A. Brunmark, M. Jackson, R. J. Cohen, P. A. Peterson, H. N. Eisen.
1994
. Kinetics and affinity of reactions between an antigen-specific T cell receptor and peptide-MHC complexes.
Immunity
1
:
15
53
Al-Ramadi, B. K., M. T. Jelonek, L. F. Boyd, D. H. Margulies, A. L. M. Bothwell.
1995
. Lack of strict correlation of functional sensitization with the apparent affinity of MHC/peptide complexes for the TCR.
J. Immunol.
155
:
662
54
Gillanders, W. E., H. L. Hanson, R. J. Rubocki, T. H. Hansen, J. M. Connolly.
1997
. Class I-restricted cytotoxic T cell recognition of split peptide ligands.
Int. Immunol.
9
:
81
55
Tallquist, M. D., A. J. Weaver, L. R. Pease.
1998
. Degenerate recognition of alloantigenic peptides on a positive-selecting class I molecule.
J. Immunol.
160
:
802
56
Schlueter, C. J., T. C. Manning, B. A. Schodin, D. M. Kranz.
1996
. A residue in the center of peptide QL9 affects binding to both Ld and the T cell receptor.
J. Immunol.
157
:
4478
57
Manning, T. C., C. J. Schlueter, T. C. Brodnicki, E. A. Parke, J. A. Speir, K. C. Garcia, L. Teyton, I. A. Wilson, D. M. Kranz.
1998
. Alanine scanning mutagenesis of an αβ T cell receptor: mapping the energy of antigen recognition.
Immunity
8
:
413
58
Pullen, J. K., R. M. Horton, Z. Cai, L. R. Pease.
1992
. Structural diversity of the classical H-2 genes: K, D, and L.
J. Immunol.
148
:
953