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J. Biol. Chem., Vol. 277, Issue 23, 20840-20846, June 7, 2002

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Novel CD8⁺ T Cell Antagonists Based on ₂-Microglobulin^*

Meir Glick, David A. Price^§¶, Anne-Lise Vuidepot, Torben B. Andersen, Sarah L. Hutchinson^§, Bruno Laugel^§, Andrew K. Sewell^§, Jonathan M. Boulter, P. Rod Dunbar^§, Vincenzo Cerundolo^§, Annette Oxenius^§, John I. Bell^§, W. Graham Richards, and Bent K. Jakobsen^**

From the  Department of Chemistry, Central Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QH, the ^§ Nuffield Department of Clinical Medicine, Level 7, John Radcliffe Hospital, Headington, Oxford OX3 9DU, and  Avidex Ltd., 57 Milton Park, Abingdon, Oxon OX14 4RX, United Kingdom

Received for publication, February 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The CD8 coreceptor of cytotoxic T lymphocytes binds to a conserved region of major histocompatibility complex class I molecules during recognition of peptide-major histocompatibility complex (MHC) class I antigens on the surface of target cells. This event is central to the activation of cytotoxic T lymphocyte (CTL) effector functions. The contribution of the MHC complex class I light chain, ₂-microglobulin, to CD8 binding is relatively small and is mediated mainly through the lysine residue at position 58. Despite this, using molecular modeling, we predict that its mutation should have a dramatic effect on CD8 binding. The predictions are confirmed using surface plasmon resonance binding studies and human CTL activation assays. Surprisingly, the charge-reversing mutation, Lys⁵⁸  Glu, enhances ₂m-MHC class I heavy chain interactions. This mutation also significantly reduces CD8 binding and is a potent antagonist of CTL activation. These results suggest a novel approach to CTL-specific therapeutic immunosuppression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

The peptide-MHC¹ class I complex (pMHC) on a target cell (antigen presenting cell) is recognized by a specific T cell receptor on the surface of CD8⁺ cytotoxic T lymphocytes (CTL). The pMHC consists of a heavy chain, which is attached to the cell membrane and contains the peptide binding site, and a light chain, ₂-microglobulin (₂m). The CD8 molecule is a cell-surface glycoprotein present on CTL, which acts as a "coreceptor"; it is not peptide-specific, but binds to a conserved site on the pMHC molecule, which comprises several regions on the heavy chain and the small DE loop of ₂m consisting of residues 58-60 (Lys⁵⁸-Asp⁵⁹-Trp⁶⁰) (1).

After CTL engage pMHC, the earliest intracellular events induce specific phosphorylation of tyrosine residues in the immunoreceptor tyrosine activation motifs within the cytoplasmic tails of the TCR-associated CD3 complex. The cytoplasmic tail of the CD8 -chain is associated with the protein tyrosine kinase p56^lck. Active p56^lck initiates TCR signal transduction by phosphorylating the immunoreceptor tyrosine activation motifs within the CD3 complex. Inhibition of CD8 binding to pMHC therefore inhibits T cell activation (2).

Exogenous soluble ₂m can exchange with cell surface-associated ₂m complexed to pMHC (3). Therefore, by mutating the CD8 contact site on ₂m, and exchanging the mutant ₂m into the native MHC, it should be possible to inhibit CTL activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Molecular Dynamics and Free Energy Perturbations-- Initial coordinates were taken from the crystal structure of the complex between human MHC class I HLA-A2 and the T cell coreceptor CD8 solved at 2.65-Å resolution and deposited in the Protein Data Bank (4) under the name 1akj (1). Molecular dynamics and free energy perturbations were performed using CHARMM (version 27) (5) and the standard all-atom parameter set (6). Hydrogens were added using the HBUILD module in CHARMM. Water molecules were added to the complex by superimposing a 16-Å sphere of TIP3P water molecules centered at the ₂m Lys⁵⁸ N atom.

The solvent atoms were minimized by 500 steps of steepest descents followed by 1000 steps of conjugate gradient. At the next step, the entire system was relaxed with 500 steps of steepest descents that were switched to conjugate gradient until convergence criteria of r.m.s. gradient of the potential energy lower than 0.3 Kcal/mol·Å has been achieved. A 14-Å nonbonded cutoff was employed. The dielectric constant was unity. The system was simulated using a stochastic boundary molecular dynamics (7). The reference point for partitioning the system was the ₂m Lys⁵⁸ N atom. The system was divided into a 12-Å reaction region, a 4-Å buffer region, and a reservoir. The frictional coefficients for water oxygen and heavy atoms in the protein were 62 and 200 ps¹, respectively (8). The relaxed system was equilibrated at 300 K for 150 ps with a time step of 1 fs followed by 1 ns performed for data collection with coordinates and energies saved to a disc every 1 ps. The ₂m Lys⁵⁸ was mutated using the Biopolymer and Homology modules in the MSI software package. For each mutation the procedure described above has been repeated. Relative binding Helmholtz free energies were calculated by the perturbation method (9) as follows: G₃ = HLA-A2/₂m_(native) complex  HLA-A2/₂m_(LysGlu) complex and G₄ = HLA-A2/₂m_(native)/CD8 complex  HLA-A2/₂m_(LysGlu)/CD8 complex.

Since free energy is a state function, it is path-independent, and the free energy difference: G₄-G₃ is equal to the difference G₂-G₁ (see "Results"). Each perturbation was performed in two steps using a total number of 26 windows. At the first 16 windows the lysine H1, H1, H2, N, H1, H2, and H3 atoms (including their charges) were deleted. C atom type was modified to sp² carbonyl carbon. H2 and C atom types were modified to carboxylate oxygens. At the last 10 windows, the charges of the remaining side chain were adjusted to asp side chain. Trajectories were produced by MD simulations at the same conditions to these described above with 150 ps of equilibration and 100 ps of data collection at every window.

Soluble CD8 Preparation-- The extracellular fragment of soluble CD8 (residues 1-120) was expressed in Escherichia coli, refolded and purified as described previously (1). The CD8 concentration was determined from the extinction coefficient (32,480 M¹ × cm¹, determined by amino acid analysis), assuming 100% activity.

Soluble TCR Preparation-- The TCR used for the SPR experiments derives from the JM22 T cell clone (10, 11). It is specific for an HLA-A2-restricted peptide (GILGFVFTL) from the influenza matrix protein (58-66) and uses gene segments TCRAV10S2J9S11C1 and TCRBV17S1J2S7C2. The two fragments of soluble JM22-TCR (residues 1-204 for the -chain and 1-245 for the -chain) were expressed in E. coli, refolded and purified as described previously (12). The TCR concentration was determined from the extinction coefficient (105,500 M¹ x cm¹, determined by amino acid analysis), assuming 100% activity.

Soluble HLA-A2/₂m Complex Preparation-- Soluble influenza peptide-HLA-A2/₂m complexes were prepared by refolding HLA-A2 heavy chain carrying the biotin tag with ₂m wildtype or mutant (both expressed in E. coli) and the synthetic peptide corresponding to influenza matrix protein 58-66 GILGFVFTL (Genosys, Woodlands, TX) as described in Garboczi et al. (13). The refolded complexes were purified by both anion exchange and gel filtration before being used in SPR experiments. HLA-A2 heavy chain was enzymatically biotinylated as described (14) using N-hydroxysuccinimidobiotin (Sigma) and BirA enzyme. Tetrameric pMHC I complexes were produced by conjugation with phycoerythrin-labeled extravidin as described previously (14). DNA constructs encoding the ₂m mutants (Lys⁵⁸ to Arg, Asp, Glu, Ser, Val, Tyr, Cys, SES, Trp, and GRG) were produced using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) and checked by DNA sequencing of the entire coding fragment. The mutant proteins were expressed and the inclusion bodies purified as for the wild type.

Surface Plasmon Resonance Experiments-- SPR binding studies were performed at 25 °C using a BIAcore^{TM 3000} (BIAcore AB, St. Albans, UK) in HBS (BIAcore AB). HBS contains 10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% Surfactant P20. Peptide-HLA class I complex was bound to the BIAcore chip by producing recombinant soluble pMHC fused to a biotinylation tag, which was specifically biotinylated in vitro (14) and flowed over a streptavidin-coated chip surface. Streptavidin (Sigma) was covalently coupled to Research Grade CM5 sensor chip (BIAcore) via primary amines using the Amine Coupling kit (BIAcore). For coupling, the streptavidin was dissolved in 10 mM sodium acetate, pH 5.5, and injected at 0.2 mg ml¹. Immobilization level was 6000 response units on average. Biotinylated HLA-A2/₂m (wild type and mutants) complexes were immobilized at 12,000-12,500 Response units by injection of 5-35 µl at 40-100 µg ml¹, at a flow rate of 5 µl min¹. The injections of the different CD8 (sCD8) and JM22-TCR (sTCR) solutions were performed at a flow rate of 5 µl min¹. K_d values were obtained either by Scatchard plots or by nonlinear fitting of the Langmuir binding isotherm (A + B  AB) equation [AB = B × AB_max/(K_d + B)] (where B is sCD8 (or sTCR) concentration and AB_max is maximum sCD8 (or sTCR) binding) to the data using the Levenberg-Marquardt algorithm as implemented in the Windows 98 application Origin (version 6.1; Microcal Software, Northampton, MA).

Cell Culture and CTL Activation Assays-- pBMC were isolated from fresh blood by Ficoll-Hypaque density gradient centrifugation. CD8⁺ CTL clones were generated and maintained as described previously (15). Target cells in cytotoxicity assays were HLA-matched immortalized T cell hybridomas or Epstein-Barr virus transformed B-lymphoblastoid cell lines (B-LCL) incubated with peptide as shown, or infected with recombinant vaccinia virus (rVV), and labeled with ⁵¹Cr (Amersham Biosciences, Amersham, UK). Peptides were synthesized using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and were >90% pure as determined by high performance liquid chromatography (Research Genetics, Huntsville, AL). Vaccinia infections were effected at 3-5 plaque-forming units/cell for 1 h and followed by a 6-h incubation period to allow expression prior to ⁵¹Cr labeling. The rVV expressing HIV-1 Nef was constructed from a full-length proviral clone isolated from donor SC1 according to standard protocols; wild type WR rVV was used to infect control target cells (16). Lysis assays were performed in low percentage fetal calf or human serum using standard ⁵¹Cr release methodology (17). Tetrameric pMHC I complex activation assays were performed by measurement of extracellular RANTES release after 30-min exposure as described previously (18). All data points for both lysis and RANTES release assays represent the mean of triplicate readings; y axis error bars show the S.D. in each case.

Flow Cytometric Analysis-- Cells were stained with phycoerythrin-labeled tetrameric pMHC I complexes containing wildtype or Lys⁵⁸  Glu forms of ₂m as described previously (19). Stained cells were analyzed using a Becton Dickinson Calibur flow cytometer with CellQuest software.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

We employed 1-ns MD simulations to study the interactions between CD8, HLA-A2, and ₂m molecules. We focused on the ₂m Lys⁵⁸ residue, which forms two key hydrogen bonds with CD8-1 Asp⁷⁵ O2 and Val²⁴ O (1), to design and predict the effects of novel ₂m mutants on the interaction between CD8 and pMHC. Five classes of Lys⁵⁸ mutations were studied: a mutation that preserves the positive charge (Lys⁵⁸  Arg), mutations to short polar side chains (Lys⁵⁸  Ser, Lys⁵⁸  C), mutations introducing steric hindrances via bulky side chains (Lys⁵⁸  Tyr, Lys⁵⁸  Trp) or insertions (Lys⁵⁸  SES, Lys⁵⁸  GRG), a mutation to a medium length hydrophobic side chain (Lys⁵⁸  Val), mutations to negatively charged side chains (Lys⁵⁸  Asp, Lys⁵⁸  Glu).

We studied the hydrogen bond network formed between the wild type Lys⁵⁸, or mutations of this residue, to CD8-1 as shown in Table I. The perturbation to the conformation of the contact residues (CD8-1:Arg⁴_, Asp⁷⁵, Val²⁴-Val²⁶, ₂m:Trp⁶⁰, Lys⁵⁸) (1) caused by various Lys⁵⁸ mutations is presented as the root mean square (r.m.s.) deviation of these residues to that observed in the crystal structure.

View this table: [in this window] [in a new window]   Table I Molecular dynamics results for the interaction between the CD8 and 2m Lys58/mutants

Neutralizing the Lys⁵⁸ positive charge in silico leads to the loss of two H-bonds to CD8-1 Asp⁷⁵ O2 and Val²⁴ O atoms, as illustrated in Fig 1a and Table I, and formation of alternative hydrogen bonds with water molecules in the cavity of the HLA-A2/CD8/₂m complex. A mutation that preserves the positive charge (Lys⁵⁸  Arg) has a less dramatic effect: the H-bond to CD8-1 Asp⁷⁵ O2 is preserved, and the r.m.s. value is similar to that of the wild type structure and smaller than that of the neutral Lys⁵⁸ side chain. Therefore, eliminating the positive charge on the side chain is likely to reduce the binding affinity to the CD8.

View larger version (59K): [in this window] [in a new window]   Fig. 1.   The effect of various 2m Lys58 mutations on the interaction with the CD8 and HLA-A2 molecules. H-bonds are shown in hashed lines, atom colors: carbon (C) = green; nitrogen (N) = blue; oxygen (O) = red; hydrogen (H) = white; sulfur (S) = yellow. a, the importance of the positive charge on Lys58 side chain. While the native Lys58 forms two H-bonds to CD8-1 Asp75 and Val24, neutral lysine (side chain in yellow) does not interact with the CD8-1. In the Lys58  Arg mutant (side chain in purple) the H-bond to CD8-1 Asp75 is preserved. b, in the Lys58  Ser and Lys58  Cys mutants the interaction with the CD8 is mediated through a water molecule: the side chain donates its hydroxyl or thiol hydrogen to a water molecule, which donates its hydrogen to CD8 Val24 carbonyl oxygen. c, the native structure 2m Lys58 and CD8-1 Asp75 (blue) compared with steric hindrance via a bulky side chain mutants: Lys58  Tyr (side chain in green) and Lys58  Trp (side chain in purple). The bulky side chain fills the cavity between the HLA-A2/2m CD8, and the interaction with the CD8 is poor. d, comparison between the native 2m DE loop consisting of residues 58-60 (Lys58-Asp59-Trp60), shown in blue, to insertions: Lys58  SES (green) and Lys58  GRG (purple). 2m residues Leu40-Ala79 are shown in azure. e, comparison between the native structure to a Lys58  Val mutation (blue). The hydrophobic valine side chain repels CD8-1 Asp75 carboxylate. f, mutations to a negatively charged side chain: Lys58  Asp (yellow), Lys58  Glu (purple) compared with the native lysine (gray). In both Lys58  Asp and Lys58  Glu mutations the positive charge on HLA-A2 Arg6 heavy chain attracts the mutant's carboxylate. Only Lys58  Glu carboxylate is in close proximity to CD8-1 Asp75 O2.

In short polar side chain mutants, Lys⁵⁸  Ser and Lys⁵⁸  Cys, the interaction with CD8 is mediated through a water molecule as shown in Fig. 1b and Table I. The side chain donates its hydroxyl or thiol hydrogen to a water molecule, which donates its hydrogen to CD8 Val²⁴ O. Unlike Lys⁵⁸  Ser, where the H-bond network is stable, the H-bond between the thiol moiety and the water molecule in the Lys⁵⁸  Cys mutant fluctuates during the simulation. This has an impact on the r.m.s. values shown in Table I, which increase from 0.70 for Lys⁵⁸  Ser to 0.93 for Lys⁵⁸  Cys. These results suggest that removing the H-donor group from the side chain at position 58 should reduce the binding affinity to CD8.

Mutations introducing steric hindrance via a bulky side chain, Lys⁵⁸  Tyr and Lys⁵⁸  Trp, are illustrated in Fig. 1c. The bulky side chain fills the cavity between HLA-A2/₂m and CD8, and the interaction with CD8 is impaired by the lack of H-bonding, resulting in high r.m.s. values (Table I). Lys⁵⁸  GRG and Lys58  SES insertions (Fig. 1d) perturb the tertiary structure and lead to increased r.m.s. values of 1.21 and 1.78, respectively. The higher r.m.s. value of the Lys⁵⁸  SES insertion is due to reversal of the positive charge and the fact that serine has a side chain that contributes to the overall steric hindrance.

The Lys⁵⁸  Val mutation yielded a higher r.m.s. value than all other single substitution mutations. In bulkier mutations, such as Lys⁵⁸  Trp, the side chain can orient itself toward the HLA-A2/₂m-CD8 cavity, whereas the valine side chain is not long enough to have this effect. As a result, the hydrophobic side chain repels the polar CD8-1 Asp⁷⁵ (Fig. 1e).

Fig. 1f shows mutations to a negatively charged side chain (Lys⁵⁸  Asp, Lys⁵⁸  Glu). Repulsion between the carboxylates leads to an average distance of 6.27 Å between the CD8-1 Asp⁷⁵ O2 and Lys⁵⁸  Asp O1 oxygens. Strikingly, the distance between CD8-1 Asp⁷⁵ O2 and Glu⁵⁸ O1 oxygens in the Lys⁵⁸  Glu is only 4.09 Å. Both Lys⁵⁸  Asp and Lys⁵⁸  Glu are attracted to the positive charge on HLA-A2 Arg⁶ heavy chain: in 52.9% of the frames taken from the MD simulation, the distance between the mutated Glu⁵⁸ O2 and HLA-A2 Arg⁶ N1 atoms was lower than 5 Å, fluctuating to a minimum of 3.18 Å. This interaction stabilizes the HLA-A2/₂m complex and orients the Lys⁵⁸  Glu side chain toward CD8-1 Asp⁷⁵. HLA-A2 Arg⁶ heavy chain attracts the Lys⁵⁸  Asp carboxylate as well; however since Lys⁵⁸  Asp side chain is shorter, it is distant from Asp⁷⁵ O2.

We defined the free energies for the association reactions as following: G₁ = HLA-A2/₂m_(native) complex + CD8 HLA-A2/₂m_(native)/CD8 complex and G₂ = HLA-A2/ ₂m_(LysGlu) complex + CD8  HLA-A2/₂m_(KE)/CD8 complex. We employed free energy perturbations to calculate the relative association free energy: G₂  G₁ = +14.95 Kcal/mol. These results show that the complex containing ₂m_(LysE) has a considerable lower affinity for CD8 than the wild type complex.

The theoretical results show that, although the contribution of the ₂m Lys⁵⁸ to CD8 binding is relatively small (1), its mutation should have a marked effect upon CD8 binding.

To test these predictions, we used SPR to measure the binding of soluble CD8 (sCD8) and soluble JM22 T cell receptor (sTCR) to HLA-A2-influenza matrix peptide complex, containing wild type or mutant ₂m (Fig. 2). The affinity of sTCR for pMHC was similar for all of the complexes studied (Table II), indicating that the active material on the chip surface was correctly folded. However, the absolute measurements of response varied markedly between the different complexes, indicating varying proportions of active material on the chip surface. This implies that some mutant ₂m complexes are more stable than others. In particular, HLA complexes containing Lys⁵⁸ to Tyr, Trp, SES, and GRG mutations show reduced stability (data not shown). These observations correlate with refolding efficiency, with yields of Lys⁵⁸  Arg and Lys⁵⁸  Glu complexes being several times higher than those for Lys⁵⁸  SES and Lys⁵⁸  GRG complexes (data not shown). This difference agrees with the MD simulations, which predict that the mutant Lys⁵⁸  Arg complex structure is closest to that of the wild type (Table I), and that the complex containing the ₂m Lys⁵⁸  Glu mutant is stabilized by the interaction with HLA-A2 heavy chain R₆.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   The affinity of CD8 binding to HLA-A2/2m complexes. a, CD8 was injected at increasing concentrations for 1 min through flow cells where HLA-A2/2m wild type (positive control), HLA-A2/2m mutant Lys58  Glu were coupled via biotinylated heavy chain and through a flow cell with no protein immobilized (negative control). The amount of CD8 that bound to the HLA-A2/2m complexes at each concentration was calculated as the difference between the responses at equilibrium in the HLA-A2/2m complexes and the negative control flow cells and is plotted against the CD8 concentration. b, a similar experiment was performed with the HLA-A2/2m mutant Lys58  Ser complex. In a and b, the solid lines represent nonlinear fits of the Langmuir binding to the data. In a, the responses obtained for the HLA-A2/2m mutant Lys58  Glu were too low, and it was not possible to fit the data (blue dotted line). These experiments were performed at 5 µl min1. Insets, responses obtained in the blank cell (black dotted line), in the HLA-A2/2m wild type cell (red line), and in the HLA-A2/b2m Lys58  Glu for a and Lys58  Ser for b cell (blue line) during the CD8 (80 µM) injection.

There is a strong correlation between the measured sCD8 binding (Table II) and the predicted H-bonds network formed between Lys⁵⁸ and Asp⁷⁵O2 or Val²⁴O (Table I). The wild type structure forms two hydrogen bonds and shows the highest affinity for sCD8. Complexes containing ₂m mutants Lys⁵⁸  Arg, Ser, or Cys are predicted to form only a single H-bond (mediated through a water molecule for Lys⁵⁸  Ser and Lys⁵⁸  Cys) and show decreased binding affinity for sCD8 (Fig. 2, Table II). In complexes containing other ₂m mutants (Lys⁵⁸ mutations to Val, Asp, Tyr, Trp, GRG, SES, and Glu), where no H-bond was predicted, the sCD8 binding was negligible (K_d > 1 mM). Similarly, mutations of Asp⁵⁹ and Trp⁶⁰ also greatly reduced sCD8 binding (data not shown). The BIAcore binding data showed that sCD8 binding is undetectable in HLA complexes containing ₂m in which Lys⁵⁸ is mutated to Glu (Fig. 2, Table II), yet this complex behaves identically to complexes containing wildtype ₂m in terms of sTCR binding, refolding yield, and stability.

The strong correlation between the predictions of the molecular dynamics simulations, free energy calculations, and biophysical measurements of sCD8 binding led us to conclude that the ₂m mutant containing a glutamate residue at position 58 (Lys⁵⁸  Glu) was the most promising candidate for further investigation.

The impact of mutating ₂m on the interaction between soluble antigen and TCR expressed on the cell surface was investigated using tetrameric pMHC I complexes. Fluorochrome-labeled tetramers containing either wild type or Lys⁵⁸  Glu forms of ₂m-stained CD8⁺ CTL at equivalent levels across a range of concentrations (Fig. 3a). However, tetrameric complexes containing the Lys⁵⁸  Glu form of ₂m were substantially impaired in their ability to activate CTL. This effect was titratable and most apparent at lower concentrations (Fig. 3b). These data indicate that, at similar levels of interaction with cell surface TCR, the impaired ability of complexes containing the Lys⁵⁸  Glu form of ₂m to engage the CD8 coreceptor translates into a biologically significant effect. This is consistent with previous work demonstrating that the major role of the CD8 coreceptor is the provision of signaling moieties that allow an enhanced sensitivity to antigen (2).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Effects of soluble wild type and mutant 2m proteins on CD8+ CTL activation in vitro. a, peptide-HLA-A2 tetramers containing either wild type or Lys58  Glu 2m were folded around the HIV-1 p17-8 Gag-derived epitope SLYNTVATL (residues 77-85). Equivalent staining of the 003 CTL clone (17) at the concentrations of tetramer shown is demonstrated, consistent with previous results (2). Tetramer containing wild type 2m folded around the cytomegalovirus-derived HLA-A2-restricted peptide NLVPMVATV was included as a control for nonspecific binding. b, tetramer-induced activation of 003 CTL. Tetramers folded around SLYNTVATL containing either wild type or Lys58  Glu 2m were incubated in 96-well plates with 104 CTL per well at the concentrations shown. Release of RANTES into the culture supernatant was measured after 30 min by enzyme-linked immunosorbent assay. The tetramer containing Lys58  Glu 2m induces less activation compared with the tetramer containing wildtype 2m at low concentrations. The control tetramer containing wild type 2m folded around the NLVPMVATV peptide failed to activate CTL at 107 g/ml. c, antagonism of CTL activation by soluble Lys58  Glu 2m. Target T2 hybridomas were plated at 5 × 103 per well in 96-well round-bottomed plates with soluble proteins at 60 µg/ml as indicated and cognate peptide at 106 M. After incubation for 20 min at room temperature, CTL were added at an effector:target (E:T) ratio of 2:1. Effector CTL (clone 4D5) were specific for the HLA-A2-restricted MAGE-3 tumor antigen epitope FLWGPRALV (17). The assay was harvested after 6 h. Similar results were obtained with the HLA-A2-restricted SLYNTVATL-specific CTL clone 5D8 (data not shown). d, inhibition of direct ex vivo fresh PBMC response to endogenously processed antigen. Target cells were autologous SC21 B-LCL infected with either Nef or control WR rVV as described and incubated to allow expression in the presence of 50 µg/ml protein as indicated for 6 h. Proteins were present at this concentration throughout the 51Cr labeling process and for the entire duration of the assay. The assay was harvested after 12 h. Vaccinial expression of HLA class I-restricted antigens obviates the problems associated with potential effects of 2m or mutants thereof on peptide exchange at the cell surface when external loading protocols are followed. The inhibitory effect of the mutant Lys58  Glu protein is controlled by the addition of equimolar concentrations of wild type 2m. Similar results were seen in direct ex vivo assays with RPMTYKGAL-pulsed SC21 B-LCL (data not shown). The Lys58  Glu mutant also inhibited a primary CTL line (data not shown) specific for the HLA B35-restricted parvovirus B19-derived epitope QPTRVDQKM (NS1, residues 391-399) (22).

The effects of soluble exogenous wild type and mutant ₂m proteins on CTL interactions with target cells expressing pMHC I antigenic complexes were assessed using standard ⁵¹Cr release cytolytic assays. At concentrations of pMHC on the target cell surface insufficient to saturate the maximal lytic capacity of the CTL, exogenous wild type ₂m, and those mutants that efficiently refold with soluble MHC class I heavy chain to form stable complexes, consistently enhanced CTL-mediated lysis and interferon- production (data not shown). This effect likely relates to the ability of these proteins to stabilize pMHC class I on the target cell surface in a TCR-cognate form and is consistent with previous work (3, 20). At higher concentrations of pMHC class I on the target cell surface, exogenous mutant forms of ₂m that impair CD8 binding to pMHC class I were found to inhibit CTL-mediated lysis (Fig. 3c). In all experiments with CD8-dependent CTL, the Lys⁵⁸  Glu mutant was the most potent inhibitor of CTL activation. These results indicate that mutant forms of ₂m can, through inhibition of CD8 binding to complexed pMHC I, antagonize CTL activation.

The potential use of such reagents in a therapeutic setting requires an effect on whole blood antigen-specific responses mounted by polyclonal unmanipulated CTL. We examined this using peripheral blood mononuclear cells (PBMC) isolated from donor SC21, an HIV-1-infected individual with a potent CTL response to the viral Nef protein that maps predominantly to the HLA B7-restricted epitope RPMTYKGAL (residues 75-83). The Lys⁵⁸  Glu mutant was found to inhibit this fresh PBMC lytic response to endogenously presented pMHC class I antigen in comparison to equimolar levels of wild type ₂m (Fig. 3d).

In conclusion, we have developed stable ₂m mutants that inhibit CD8 coreceptor binding to pMHC I and exert an inhibitory effect on CTL activation. These data suggest that such mutant forms of ₂m could be used to selectively modulate the CD8⁺ cellular immune response, a principle that could be applied therapeutically (21).

	FOOTNOTES

^* This work was supported by the Medical Research Council (to D. A. P. and J. I. B.), the Wellcome Trust (to A. K. S., B. L., M. G., and S. L. H.), EMBO (to A. O.), and Avidex Ltd. (to A.-L. V., B. K. J., J. M. B., and T. B. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Medical Research Council Clinician Scientist.

^** To whom correspondence should be addressed: Avidex Ltd., 57 Milton Park, Abingdon, Oxon OX14 4RX, UK. Tel.: 44-1235-438603; Fax: 44-1235-438601; E-mail: bent.jakobsen@avidex.com.

Published, JBC Papers in Press, March 25, 2002, DOI 10.1074/jbc.M201819200

	ABBREVIATIONS

The abbreviations used are: MHC, major histocompatibility complex; pMHC, peptide-MHC class I complex; CTL, cytotoxic T lymphocyte(s); ₂m, ₂-microglobulin; TCR, T cell receptor; r.m.s., root mean square; MD, molecular dynamic; B-LCL, B-lymphoblastoid cell line(s); rVV, recombinant vaccinia virus; RANTES, regulated on activation normal T cell expressed and secreted; SPR, surface plasmon resonance; PBMC, peripheral blood mononuclear cell(s); HIV-1, human immunodeficiency virus, type 1.

	REFERENCES

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