©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Refolding and Reconstitution of Functionally Active Complexes of Human Leukocyte Antigen DR2 and Myelin Basic Protein Peptide from Recombinant and Polypeptide Chains (*)

(Received for publication, September 23, 1994; and in revised form, November 7, 1994)

Subhashini Arimilli Cristina Cardoso Prabha Mukku Varsha Baichwal Bishwajit Nag (§)

From the From Anergen, Inc., Redwood City, California 94063

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Major histocompatibility complex (MHC) class II molecules are cell surface heterodimeric glycoproteins consisting of one alpha and one beta polypeptide chain of similar size. These molecules play a critical role in immune recognition by displaying processed antigens to CD4-positive T helper cells. Several attempts to express the MHC class II molecules by recombinant methods in various systems resulted in either failure or poor recovery of the intact heterodimer. The present study describes our successful effort to refold and reconstitute HLA DR2 heterodimer from individually expressed alpha and beta polypeptide chains lacking the transmembrane hydrophobic regions in Escherichia coli, in the presence of an immunodominant epitope analog from human myelin basic protein (b-MBP(83-102)Y). The reconstituted DR2 heterodimer complex was selectively purified from unfolded alpha and beta chains using heterodimer-specific monoclonal antibody (L243) coupled to a solid support. The detection of two polypeptide chains in the purified refolded DR2-peptide complex preparations was accomplished by Western blot analysis and enzyme-linked immunosorbent assay using heterodimer- and chain-specific polyclonal antibodies, and the presence of equimolar amounts of both alpha chain and beta chain in the reconstituted complex preparation was confirmed by a double label experiment. The quantitation of the bound peptide in complex preparation was measured by incubating two chains in the presence of I-labeled peptide. An increase in the yield of refolded and reconstituted DR2-peptide complexes was observed with increasing peptide concentration in the reaction mixture. Finally, the functional activity of the reconstituted DR2 complexes was measured by their ability to stimulate -interferon production by SS8T cloned T cells in an antigen-specific and dose-dependent manner. These results demonstrate that biologically active complexes of human DR2bulletb-MBP(83-102)Y can be prepared by proper folding of human leukocyte antigen DR2 alpha and beta chains in the presence of antigenic peptide. The yield of such DR2 heterodimers with bound peptide is several thousand-fold higher over native DR2 purified from transformed B cells. Since purified MHC class II-peptide complexes have been shown to prevent autoimmune diseases in various animal models, reconstituted heterodimer complexes may have significant clinical relevance in antigen-specific treatment of various autoimmune diseases. In addition, such complexes with increased yield will provide better understanding of the trimolecular interactions between MHC-peptide and T cell receptor.


INTRODUCTION

The presentation of antigens to CD4-positive T helper cells involves the binding of processed antigenic peptides to MHC (^1)class II molecules on the surface of antigen-presenting cells(1, 2, 3, 4, 5) . Purified MHC class II molecules isolated from cell surfaces are also known to bind antigenic peptides in vitro(6, 7, 8) . The yields of purified MHC class II molecules from various cell sources are usually very poor and represent less than 0.5% of the total protein pool(9, 10, 11) . The low yield of purified MHC class II molecules has always been an important limiting step in elucidating structure-function correlation studies of these molecules. Various attempts to clone and express MHC class II molecules have been reported recently in prokaryotic (12) and eukaryotic systems (13, 14, 15, 16) . The recombinant heterodimers prepared by these methods also represent very low yields due to difficulties in refolding.

Structural information of MHC class II-peptide interaction has become available recently from x-ray crystallographic studies(17, 18) . The peptide binding groove of MHC class II molecules consists of the alpha1 and beta1 domains with eight-stranded beta-pleated sheets as the floor of the peptide binding groove with extended alpha helices. Peptides play a related and distinct role in the structure and conformational maturation of MHC class II molecules(19) . Approximately 2000 different peptides may bind to MHC class II molecules(20) . Therefore it is difficult to obtain a peptide-free preparation of MHC heterodimeric molecules because of the slow dissociation rate constant of the bound peptides(13) . To overcome this problem some of the MHC class II molecules are expressed in eukaryotic systems which lack the peptide processing machinery(13, 14, 15, 16) .

The folding of MHC class II heterodimer also appears to be an inherently difficult problem. Unlike MHC class I molecules, where the alpha1 and alpha2 domains from the same polypeptide form an intramolecular dimer(21, 22) , the peptide binding site of class II molecules consists of two separate domains, alpha1 and beta1, from two individual polypeptides(17) . The MHC class II protein, therefore, differs from other heterodimeric proteins of the immune system that have been successfully folded in vitro, where each domain is composed of protein segments of a single polypeptide chain(23, 24, 25) . Reconstitution of functionally active murine MHC class II peptide complexes from Escherichia coli-expressed, unpurified individual alpha and beta chains have been demonstrated recently(12) . The yield of such active heterodimer represents 0.5-2% of the starting protein. In this report, we describe the refolding of E. coli-expressed recombinant human alpha and beta chains lacking the transmembrane regions followed by reconstitution of biologically active HLA DR2. MBP peptide complexes with a yield of over 20% of the starting protein concentration.


MATERIALS AND METHODS

Cell Lines, Antibodies, and Chemicals

The hybridoma cell line L243, producing monoclonal antibodies against monomorphic human HLA DR molecules, was obtained from American Type Culture Collection, Bethesda, MD. Homozygous lymphoblastoid cell lines, GM 03107 expressing HLA DR2 and GM 08067 expressing HLA DR3, were obtained from the National Institute of General Medical Sciences (NIGMS) human genetic mutant cell repository (Corinell Institute of Medical Research, NJ). Polyclonal antibodies against individual alpha and beta chains were raised in rabbits using recombinant purified alpha and beta chains lacking the transmembrane region as antigens. Both anti-alpha and anti-beta polyclonal antibodies were purified by affinity chromatography where purified antigens were coupled to activated Sepharose 4B column. Anti-human -IFN monoclonal antibody and rabbit anti-human -IFN polyclonal antibody were obtained from Endogen. HRP-conjugated rabbit IgG was purchased from Jackson Immunoresearch Laboratories. Human -IFN was obtained from Boehringer Mannheim. The color developing substrate 3,3`,5,5`-tetramethyl benzidine (TMB) was obtained from Moss, Inc.

Cloning and Expression of DR2Dw2 (-TM) Single Chains

The vector for expression of single chain MHC was derived from the pET system of plasmids(26) . The vector pET-11a (Novagen) was used to construct an expression vector p27313 by modifications of cloning sites. DR2 alpha and DRB5*0101 chains lacking the transmembrane regions were PCR-amplified using poly(A) mRNA from GM 03107 lymphoblastoid cells. The PCR primers were synthesized based on the sequences obtained from the GeneBank data base for human DR2Dw2 alleles. The top strand primer for both chains included a 5- amino acid sequence of the -10 gene of bacteriophage T7 followed by a translational stop codon and the initiator methionine for the chain of interest(27) . The amplified sequences were cloned into p27313 using BamHI and EcoRI restriction enzymes whose sites were tailored in the PCR primers. Insert-containing clones were identified and sequenced. The tetracycline resistance gene was cloned into the plasmids containing DR2 alpha and beta chains in order to facilitate scale-up culturing. The resulting plasmids - p329129 and p33425 expressing DR2 alpha-TM and DRB5*0101-TM chains were transformed into the E. coli expression host W3310/DE3. Induction cultures were grown at 37 °C in L-broth containing 0.4% glucose, 100 µg/ml ampicillin, and 15 µg/ml tetracycline. Cells were induced in mid-log growth by addition of isopropyl-beta-D-thiogalactopyranoside to a final concentration of 0.4 mM and allowed to grow for an additional 2 h. Cells were harvested and used for inclusion body preparation.

Purification of DR2 alpha and beta Chains Lacking the Transmembrane Region

The detailed procedure for the purification of alpha and beta chains lacking the transmembrane region from E. coli inclusion body preparations has been described recently(28) . Briefly, the alpha chain E. coli inclusion bodies were solubilized in 25 mM phosphate buffer, pH 7.4, containing 8 M urea and 20 mM dithiothreitol and purified by ion exchange chromatography using High Q-50 resin (Bio-Rad). The recombinant beta chain was purified by one-step gel filtration chromatography using Sephacryl S-100 resin packed in a Pharmacia XK50 (2.5-cm diameter times 100-cm height) column. Both alpha and beta chain fractions were collected and analyzed by SDS-PAGE electrophoresis using a LabLogix silver staining kit (Belmont, CA). Individually pooled alpha and beta chains showed purity levels greater than 95% with recovery of 52 and 86%, respectively.

Purification of Human HLA DR2 and DR3 from Lymphoblastoid Cells

Purification of HLA DR2 from Ebstein-Barr virus-transformed lymphoblastoid cells was carried out as described earlier (29) with some minor modifications. Triton X-100 cell lysate was applied onto L243-coupled Sepharose-4B column, and the bound DR2 was eluted in phosphate buffer containing 0.05% n-dodecyl-beta-D-maltoside detergent at pH 11.3. Fractions were immediately neutralized with 1 M acetic acid, and the DR2 pool was collected through a DEAE ion exchange column in a phosphate buffer containing 0.5 M NaCl and 0.05% n-dodecyl-beta-D-maltoside, pH 6.0. Purified protein was then filtered through a 180-kDa membrane, dialyzed against PBS for 24 h at 4 °C and characterized by 13.5% SDS-polyacrylamide gel electrophoresis followed by silver staining. Affinity purified HLA DR3 was obtained by a similar method in 0.01% Tween-80 detergent.

Synthesis of MBP Peptides and Conjugation of Biotin Tag

The N-acetylated myelin basic protein peptide analogs MBP(83-102)Y with the sequence Ac-YDENPVVHFFKNIVTPRTPP and MBP(124-143) peptide with the sequence Ac-GFGYGGRASDYKSAHKGFKG were synthesized by the standard solid phase method using side chain-protected Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acids on an Applied Biosystems 431A automated peptide synthesizer. A tyrosine residue at the N-terminal end of the MBP(83-102)Y peptide was introduced for radiolabeling of this peptide with I. The deprotected, crude peptides were purified by reverse-phase high performance liquid chromatography, and the homogeneity and identity of the purified peptides were confirmed by mass spectrometry. Biotinylation of peptide was carried out as described recently(30) .

In Vitro Folding, Reconstitution, and Purification of DR2bulletMBP Peptide Complexes

Purified alpha and beta polypeptides at a concentration of 0.5 mg/ml were dialyzed against PBS for 18 h at 25 °C. One milligram of alpha and beta chains in the presence of 1-50-fold molar excess of b-MBP(83-102)Y peptide was incubated for 96 h at 25 °C in an optimized refolding/reconstitution buffer containing 50 mM sodium phosphate (pH 7.5), 1 mM EDTA, 3 mM reduced glutathione, 0.3 mM oxidized glutathione, 25% (v/v) glycerol, and 10 mM dithiothreitol in a total volume of 20 ml. During discovery of the best reconstitution condition, various components from the refolding/reconstitution buffer were deleted one at a time. The complex preparations were dialyzed against 4 liters of PBS at 4 °C with two changes. The reconstituted DR2bulletMBP peptide complexes were purified by immunoaffinity chromatography using immobilized L243 monoclonal antibody. The column was washed with 10 bed volumes of PBS containing 0.5% Triton X-100 followed by 10 bed volumes of PBS. Finally, bound complexes were eluted in 20 mM phosphate buffer containing 0.1 M NaCl at pH 11.3. Fractions were immediately neutralized by 1 M acetic acid and analyzed by 13.5% silver stain SDS-PAGE under nonreduced conditions.

Characterization of Reconstituted Complexes by Western Blot Analysis and ELISA

Four µg of reconstituted complex were transferred from 13.5% SDS-polyacrylamide gels on polyvinylidene difluoride membranes using a semi-dry transfer cell (Bio-Rad) at 25 V for 20 min. The membranes were incubated for 2 h with anti-DR2 polyclonal serum or purified anti-alpha and anti-beta polyclonal antibodies. The blots were finally developed following a second antibody incubation using 4-chloro-1-naphthol. For the ELISA, 96-well plates (Nunc) were coated with 50 µl of anti-DR2 polyclonal antibody (1:20) in PBS and incubated with 12.5, 25, 50, 100, and 200 ng of reconstituted DR2bulletb-MBP(83-102)Y complex for 2 h at 25 °C. The polyclonal antibody-captured complexes were then detected by HRP-coupled L243 monoclonal antibody using TMB as a substrate.

Quantitation of Equimolar Chains in Purified Complexes by Double Label Experiments

Quantitative measurement of the presence of equimolar amounts of both chains in the final complex preparation was assessed by radiolabeling each chain with separate tags. The labeling of the alpha chain with S using a sulfur-labeling reagent (Amersham Corp.) and of the beta chain with I using Pierce IODO-BEADS was accomplished by previously described methods (31) . The specific activity of the S-labeled alpha chain and I-labeled beta chains were 0.47 times 10^5 and 3.5 times 10^5 cpm/µg, respectively.

Detection of Bound Peptide in Reconstituted Complex Preparation

The detection of bound peptide in the purified, reconstituted complex preparations was carried out by incubating equimolar amounts of the two chains in the presence of I-labeled b-MBP(83-102)Y peptide. Radioactive labeling of MBP peptide was achieved by the standard chloramine-T labeling procedure(32) . The specific activity of the b-MBP(83-102)Y was 7.9 times 10^4 cpm/µg. Equivalent amounts of each alpha and beta chain were incubated with a 10, 50, and 100 molar excess of labeled peptide in 10 ml of refolding/reconstitution buffer at 25 °C for 4 days. Reaction mixtures were extensively dialyzed against PBS and applied to L243 columns. The columns were washed with 10 bed volumes of washing buffer and eluted as described above. The percent of DR2 heterodimer containing labeled peptide was calculated from the specific activity of the peptide.

T Cell Receptor Occupancy Assay

The Herpesvirus saimiri-transformed SS8T human T cell clone (33) restricted by DRB5*0101 and MBP(84-102) was cultured in RPMI 1640 medium supplemented with 2 mML-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 10% fetal bovine serum (HyClone), and 50 units/ml human recombinant interleukin-2 (ABI) at 37 °C. Every alternate day the cells were transferred to fresh medium. Various complex preparations were added at a final concentration of 10% v/v in a microtiter tissue culture plate and the cells were inoculated at a density of 20,000/well in 200 µl of medium without interleukin-2. After 48 h of incubation at 37 °C, the supernatants were collected from each well to test for the increase in -IFN level. For the detection of -IFN, Nunc Maxisorb 96-well plates were coated with anti-human -IFN monoclonal antibody at a concentration of 0.5 µg/well and incubated at 4 °C overnight. The wells were blocked with 0.1% bovine serum albumin, and samples were incubated at room temperature for 2 h. The standard curve was generated by using recombinant human -IFN with a dilution range of 1000, 500, 100, 50, 10, 5, 1, 0.5, and 0.1 units/ml (270 units/ml = 10.75 ng/ml). Rabbit anti-human -IFN was then added at a concentration of 1 µg/ml and plates were incubated at room temperature for an additional 2 h. Wells were extensively washed and incubated with HRP-conjugated goat anti-rabbit at a concentration of 800 ng/ml for 1 h at 37 °C, prior to color developed using TMB as a substrate. The reaction was stopped by 2 N sulfuric acid at 5 min, and the absorbance was measured at 450 nm.


RESULTS AND DISCUSSION

In an attempt to reconstitute human MHC class II heterodimeric protein in the presence of known antigenic peptide and without any endogenously bound peptide, individual alpha and beta chains lacking the transmembrane region were cloned and expressed in an E. coli system. The recombinant alpha and beta chains prepared by this method represent approximately 30% of the total cell protein. The insoluble denatured inclusion body preparations were solubilized in 8 M urea and purified in large scale quantities by conventional chromatographic methods as described previously(28) .

Individual chains were refolded by slow dialysis at a concentration of 0.5 mg/ml against PBS prior to reconstitution of the heterodimer. Equal amounts of alpha and beta chains were mixed at a concentration of 0.5 mg/ml in the presence of b-MBP(83-102)Y peptide. The addition of a biotin tag at the N terminus of the peptide was created for the detection of bound MBP peptide in the final complex preparation. In addition, a tyrosine residue at the N terminus of the MBP peptide allowed radiolabeling with I. In various quantitative peptide binding studies, the detection of the biotin moiety of the peptide by enzyme conjugated streptavidin resulted in either failure or poor detection of the bound peptide. The reason for an undetected biotin tag in the reconstituted complex preparation is unclear at present and could be due to steric hindrance of the large streptavidin molecule to bind biotin molecules in the peptide binding domain. Based on this unexpected result, the radiolabeled peptide was used to calculate the percent peptide occupancy.

The reconstitution of the heterodimeric molecule in the presence of 10-fold molar excess of b-MBP(83-102)Y peptide was carried out under several different conditions during optimization of the reconstitution procedure for maximum yield. Among the various conditions, a reconstitution buffer containing both dithiothreitol and glutathione gave the best results. The reconstituted DR2 heterodimers as well as complexes with b-MBP(83-102)Y peptides were selectively separated from the alpha and beta chains by antibody affinity chromatography using dimer-specific L243 monoclonal antibody. This antibody is known to recognize a monomorphic region of all human HLA DR molecules and the epitope for this monoclonal antibody has been mapped on the alpha chain of the DR molecule(34) . The lack of reactivity of the L243 antibody to individual alpha and beta chains by ELISA and Western blot analyses was also confirmed in our laboratory (data not shown). In various control experiments, we observed that the alpha chain alone incubated in the presence of 10-fold molar excess of the b-MBP(83-102)Y peptide showed very weak binding to the L243 antibody column and the eluted minor peak represents less than 2% of the total eluted heterodimer preparation. In contrast, incubation of the beta chain in the presence of 10-fold molar excess of the b-MBP(83-102)Y peptide showed absolutely no binding to the L243 monoclonal antibody. Since individual chains are known to form a low level of alpha-alpha and beta-beta homodimers in solution(35, 36) , the small fraction of alpha chain binding to L243 monoclonal antibody affinity column could be due to the formation of a minor fraction of alpha-alpha homodimers in solution.

The reconstitution of functionally active heterodimers carried out from crude unpurified preparations of alpha and beta chains showed substantially low yield as compared to purified chains. As shown in Fig. 1, reconstitution performed with purified chains in the presence of equal amount of peptide resulted in 20-fold increase in yield as compared to unpurified chains. Eluted peak fractions were analyzed by SDS-PAGE which showed the presence of both heterodimers, partially dissociated monomers along with various levels of aggregates (see Fig. 1B, inset). Like the native heterodimer, various levels of aggregation were also observed for reconstituted heterodimer under nonreduced gel electrophoresis conditions. Upon reduction, these bands of high molecular mass merged into the expected molecular sizes of 21 and 23 kDa for two chains. The unexpected high molecular aggregates of purified complexes lacking the transmembrane hydrophobic regions suggest that the aggregation level of various purified native MHC class II molecules observed consistently is not dependent upon the hydrophobic transmembrane regions of these molecules. Using dynamic multi-angle laser light scattering measurements, we observed that the peptide plays a critical role in destabilizing such aggregates of MHC class II molecules, (^2)suggesting that most of the heterodimer aggregates observed in our reconstituted samples are the populations containing no bound peptide.


Figure 1: Isolation of reconstituted DR2 heterodimer by L243 antibody affinity chromatography. Elution profiles of reconstituted DR2 heterodimer from unpurified alpha chain (-TM) and beta chain (-TM) inclusion body preparation in the presence of 10-fold molar excess of b-MBP(83-102)Y peptide is shown in Panel A. Panel B represents the reconstitution elution profiles of DR2 heterodimers from purified alpha and beta chains in the presence of 10-fold molar excess peptide. The inset figure in Panel B represents peak fractions analyzed by SDS-PAGE.



The presence of both alpha and beta polypeptide chains in the eluted, reconstituted peak was demonstrated by Western blot analysis and ELISA. Western blot analysis was performed using heterodimer and chain-specific polyclonal antibodies. As shown in Fig. 2A, the reconstituted heterodimer was recognized by all three polyclonal antibodies under both reduced and nonreduced conditions. Similarly, the presence of both alpha and beta chains was characterized by antibody capture ELISA (Fig. 2B). In this assay, the reconstituted and native DR2 were captured by heterodimer-specific polyclonal antibody and detected by HRP-conjugated, L243-purified monoclonal antibody. The alpha chain and the beta chain alone, incubated with equivalent amounts of b-MBP(83-102)Y peptide, did not show any reactivity in this assay.


Figure 2: Characterization of reconstituted DR2 by Western blot analysis and ELISA. Panel A represents the Western blot analysis of reconstituted DR2 by anti-DR2, anti-alpha and anti-beta polyclonal antibodies, under reduced and nonreduced conditions. Panel B represents antibody capture ELISA of heterodimer using dimer specific antibodies. Closed circles, native DR2; open circles, reconstituted DR2; closed squares, alpha alone; and open squares, beta alone.



To demonstrate the presence of two chains in equimolar amounts, the alpha and beta chains were separately radiolabeled with S and I, respectively. The selection of the labeling tag for each chain was based on their sequences. The alpha chain contains 9 lysine residues whereas the beta chain contains 9 tyrosine residues. Equimolar amounts of the two differently labeled chains were refolded and mixed with 10-fold molar excess amount of b-MBP(83-102)Y peptide. The reconstituted heterodimers were eluted from the L243 monoclonal antibody affinity column and analyzed for the presence of two chains. Fig. 3shows the absorbance of eluted fractions along with I and S counts of each fraction measured in and beta counters, respectively. The quantitation of each chain in the final eluted pool was carried out using the specific activities of the two chains. Results presented in Table 1show that both chains are present in almost equal amounts. A small increase in the total alpha chain correlates well with our observation that a small fraction of alpha-alpha homodimers are capable of binding L243 antibody.


Figure 3: Elution of double-labeled reconstituted DR2 heterodimer from L243 column. alpha and beta chains were radiolabeled with S and I, respectively, as described under ``Materials and Methods.'' Purified labeled chains were allowed to refold in the presence of 10-fold molar excess of unlabeled b-MBP(83-102)Y peptide. Panel A represents the elution profile of reconstituted DR2 by absorbency at 280 nm. Panel B represents the profile of radiolabeled I of beta chain, as measured by the radiation count. Panel C represents the profile of radiolabeled S of alpha chain as measured by the beta radiation count.





The effect of antigenic peptide concentration on the yield of reconstituted heterodimers was next examined by incubating equimolar amounts of the two chains in the presence of increasing peptide concentrations. As shown in Fig. 4, increasing the peptide concentration has a significant effect on the yield of reconstituted DR2 heterodimers as seen by the elution profile and SDS-PAGE analysis of peak fractions. At a peptide concentration of 50-fold molar excess, the yield of reconstituted DR2 was more than 20% (Fig. 4). We observed a further increase in yield up to 29% with 100-fold molar excess of the MBP peptide. Thus, the presence of peptide is critical in increasing the yield of reconstituted complexes. A low level of reconstituted DR2 heterodimer representing 1-3% of the starting protein was also observed in various experiments in the absence of b-MBP(83-102)Y peptide. The peptide-dependent refolding and assembly of complexes correlates well with various earlier reports with MHC class I refolding (37, 38) in vitro. In vivo, the presence of peptide is also a prerequisite for the correct folding and transport of MHC class II-peptide complexes to the surface of the antigen-presenting cells(39, 40, 41, 42, 43, 44) . In this regard, peptides are also known to stabilize the quaternary structure of MHC class II molecules(45) .


Figure 4: Effect of the peptide concentration on reconstitution of heterodimers. Equivalent amounts of alpha and beta chains were incubated with no peptide (Panel A), 1-fold molar excess (Panel B), 10-fold molar excess (Panel C), and 50-fold molar excess (Panel D) b-MBP(83-102)Y peptide. Reconstituted DR2 complexes were purified over L243 antibody column. Fractions of each elution profile (10 µl) were analyzed by 13.5% SDS-PAGE followed by silver staining.



The quantitation of bound peptide in the refolded and reconstituted DR2 heterodimer preparation was carried out by increasing concentrations of radiolabeled peptide. The b-MBP(83-102)Y peptide was radiolabeled by I using the chloramine-T method and was incubated at various concentrations with equivalent amount of purified alpha and beta chains. Although increasing the peptide concentration had significant effect on the yield of reconstituted DR2 heterodimer, the percent of DR2 occupied with bound peptide remained constant (Table 2).



Finally, the functional activity of reconstituted heterodimeric complexes was analyzed by measuring the increased level of -IFN produced by H. saimiri-transformed SS8T cloned T cells in a dose-dependent manner. An increase in -IFN production has been correlated with the occupancy of TCR on the surface of T cells(33) . The specificity and restriction of the SS8T transformed T cell clone for the HLA DR2 and MBP(84-102) peptide has been demonstrated recently (33) . As shown in Fig. 5, both native and reconstituted DR2bulletb-MBP(83-102)Y complexes were able to recognize TCR on the surface of transformed T cells. The reconstituted DR2bulletb-MBP(83-102)Y complexes appeared to be more effective in producing -IFN than native DR2bulletb-MBP(83-102)Y complexes in several experiments (data not shown). In various controls, DR2 alone, DR2 with irrelevant peptide b-MBP(124-143) and b-MBP(83-102)Y peptide complexed with irrelevant MHC class II (HLA DR3), showed no significant increase in the level of -IFN production. Human T cells are known to express low levels of MHC class II molecules on their surfaces and can be stimulated in the presence of antigenic peptide(46, 47) . In order to demonstrate that the observed level of increased -IFN is not due to the release of bound peptide in the culture medium, the b-MBP(83-102)Y peptide was complexed with HLA DR3 as a control which showed no increase in -IFN level. Similarly in mock experiment equivalent amount of b-MBP(83-102)Y peptide incubated under identical refolding conditions in the absence of chains did not show any increase in -IFN level.


Figure 5: Level of -IFN production of SS8T T cells in the presence of reconstituted DR2bulletb-MBP(83-102)Y complex. Occupancy of TCR by complexes of reconstituted DR2. b-MBP(83-102)Y was shown by increased level of IFN using SS8T transformed T cells, restricted to DR2 and MBP(84-102) complexes. Purified native DR2 and DR3 proteins at a concentration of 0.5 mg/ml were incubated with 50-fold molar excess of respective peptides in citrate buffer pH 6.0 at 37 °C for 96 h. The complexes of DR2bulletb-MBP(83-102)Y, DR2bulletb-MBP(124-143), and DR3bulletb-MBP(83-102)Y were prepared and the unbound free peptide was removed by passing through Sephadex G-75 gel filtration column. The level of -IFN was measured by ELISA as described under ``Material and Methods.'' Closed circles, native DR2bulletb-MBP(83-102)Y complex; open circles, DR2 alone; open squares, DR2 with irrelevant b-MBP(124-143) peptide; closed squares, DR3 with b-MBP(83-102)Y; and closed triangles, reconstituted DR2bulletb-MBP(83-102)Y complex.



In conclusion, results presented here demonstrate the formation of functionally active HLA DR2 heterodimeric complexes containing antigenic epitopes. The yield of such complexes is approximately 8000-fold higher than the native DR2 molecules. Present results from our laboratory showed that purified MHC class II-peptide complexes can be used for the prevention and treatment of experimental autoimmune disease in various animal models(48) . The reconstituted heterodimeric MHC class II containing known antigenic epitope may have significant clinical relevance in developing antigen-specific therapeutics for various autoimmune diseases. In addition, such complexes in sufficient quantities, will facilitate several structure-function studies including x-ray crystallographic studies for better understanding of the trimolecular interactions among MHC class II, peptide, and TCR.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom all correspondence should be addressed: Anergen, Inc., 301 Penobscot Dr., Redwood City, CA 94063. Tel.: 415-361-8901; Fax: 415-361-8958.

(^1)
The abbreviations used are: MHC, major histocompatibility complex(es); MBP, myelin basic protein; HLA, human leukocyte antigen; TCR, T cell receptor; IFN, interferon; TMB, 3,3`,5,5`-tetramethylbenzidine; (-TM), chains lacking transmembrane and cytoplasmically exposed regions; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay; PCR, polymerase chain reaction.

(^2)
B. Nag, unpublished results.

(^3)
E. Spack, B. Nag, and S. D. Sharma, unpublished results.

(^4)
H. Bhayani, B. Nag, and S. D. Sharma, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Shrikant Deshpande for providing various MBP peptides, Dr. H. Wekerle for providing SS8T transformed T cell clone, Dr. Chris Raymond for providing the polyclonal antibodies against native DR2 heterodimer, and Dr. Jeffery Winkelhake for critically reading the manuscript.


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