©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Functionally Active Recombinant and Chain-Peptide Complexes of Human Major Histocompatibility Class II Molecules (*)

(Received for publication, November 30, 1995; and in revised form, February 8, 1996)

Bishwajit Nag(§)(¶) Subhashini Arimilli (§) Prabha V. Mukku Irina Astafieva

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

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Major histocompatibility (MHC) class II molecules are cell surface heterodimeric (alphabeta) glycoproteins that display processed antigens to T cell receptors (TCRs) of CD4-positive T cells. The present study describes that individual recombinant alpha and beta chains of human MHC class II molecules lacking the transmembrane region (alpha-Tm and beta-Tm) are capable of binding antigenic peptide and that these complexes of chain-peptide are recognized by TCRs to induce antigen-specific apoptosis in restricted T cells. The alpha-Tm and the beta-Tm of human HLA-DR2 (DRB5*0101) were cloned, expressed in Escherichia coli, and purified in large scale by conventional chromatographic methods. The in vitro binding of an immunodominant epitope from the myelin basic protein (MBP-(83-102)Y) to purified DR2alpha-Tm and DR2beta-Tm was demonstrated with biotinylated and fluoresceinated MBP-(83-102)Y peptide. The specificity of the MBP-(83-102)Y peptide binding to both DR2alpha-Tm and DR2beta-Tm was demonstrated in a competitive peptide binding assay. When exposed to a transformed T cell clone (SS8T) restricted to DR2(DRB50101) and MBP-(84-102) peptide, complexes of DR2alpha-Tm and DR2beta-Tm with MBP-(83-102)Y peptide were able to specifically recognize TCRs as measured by the increase in -interferon (-IFN) cytokine. Such recognition of TCRs by soluble alpha-MBP-(83-102)Y and beta-MBP-(83-102)Y complexes led to the induction of antigen-specific apoptosis in SS8T cells as measured by double fluorescence flow cytometry and electron microscopy. These results provide the first evidence that soluble complexes of antigenic peptide and individual chains of human MHC class II molecules lacking the transmembrane region can recognize TCRs and induce antigen-specific apoptosis in T cells. Since activated CD4-positive T cells are involved in pathogenesis of various autoimmune diseases, the apoptosis triggered by individual soluble chain-peptide complexes has significant potential for eliminating autoreactive T cells.


INTRODUCTION

MHC (^1)class II proteins are heterodimeric glycoproteins that bind peptides within the cell and present them at the cell surface for interaction with T cells(1, 2) . Several in vitro studies have demonstrated that peptides can bind to affinity purified MHC class II molecules (3, 4, 5, 6, 7) and that these complexes stimulate specific T cell responses(8, 9, 10) . In general, MHC class II molecules consist of a 34-kDa alpha polypeptide and a 28-30-kDa beta polypeptide chain noncovalently associated with each other. Furthermore, each polypeptide contains two distinct extracellular domains (alpha1/alpha2 in the alpha chain and beta1/beta2 in the beta chain), a transmembrane region and a small cytoplasmic C terminus region. The crystal structure of MHC class II molecules reveals that the extracellular alpha1 and beta1 domains of both chains are involved in creating the peptide binding groove of MHC class II molecules (11, 12, 13) .

The first observation for the binding of antigenic peptide to individual alpha and beta polypeptide of MHC class II molecules appeared in a study where fluorescence peptide was found to be associated with both chains when murine class II-peptide complexes were subjected to SDS-gel electrophoresis under reduced conditions(14) . Recent results from our laboratory have showed that electroeluted purified native individual alpha and beta polypeptide chains isolated from affinity-purified murine MHC class II proteins are capable of binding antigenic peptide (15) and that purified chain-peptide complexes can stimulate T cells in vitro as measured by an increase in extracellular acidification rate in a sensor-based assay(16) . In earlier studies, the possibility of T cell activation by MHC chain-peptide complexes was also suggested by the ability of alloreactive IA^k-specific cytotoxic T lymphocytes to specifically lyse transfected L cells expressing either A^kb1/D^dc2 (17) or Ak^a1/Dd^c2 (18) MHC class II/class I hybrid molecules.

Although these studies show that native individual chains of murine MHC class II containing the transmembrane region are capable of binding peptide and can trigger T cells, no evidence of peptide binding to individual polypeptides of human MHC class II exists. In this report we describe that Escherichia coli expressed individual recombinant alpha and beta polypeptides of human HLA-DR2 (DRB5*0101) lacking the transmembrane region are capable of binding immunodominant epitopes from MBP. In addition, monomeric complexes of DR2alpha-Tm and DR2beta-Tm chains and MBP peptide can induce antigen-specific apoptosis in cloned T cells to a degree comparable with that of native alpha/beta dimer-peptide complexes.


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, GMO 3107 expressing HLA DR2 and GMO 8067 expressing HLA DR3, were obtained from the National Institute of General Medical Sciences (NIGMS) human genetic mutant cell repository (Coriell Institute of Medical Research, NJ). Immunopure biotinylated bovine serum albumin containing known amount of biotin molecules was purchased from Pierce Chemicals. Anti-human -IFN monoclonal antibody and rabbit anti-human -IFN polyclonal antibody were obtained from Endogen, Inc. Peroxidase-conjugated rabbit IgG was purchased from Jackson Immunoresearch Laboratories. Human -IFN was obtained from Boehringer Mannheim. 3,3`,5,5`-Tetramethyl benzidine was obtained from Moss, Inc.

Cloning, Expression, and Purification of DR2alpha-Tm and DR2beta-Tm

Cloning and expression of alpha-Tm and beta-Tm of DR2 (DRB50101) in E. coli were described in our earlier report(19) . Briefly, the plasmids p329129 and p33425 expressing DR2alpha-Tm and DR2beta-Tm chains were transfected into the E. coli expression host W3310/DE3. 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 were harvested for inclusion body preparations.

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(20) . 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 an Pharmacia XK50 (2.5-cm diameter times 100-cm height) column. Both alpha and beta chain fractions were collected and analyzed by SDS-gel electrophoresis using 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 Epstein-Barr virus transformed lymphoblastoid cells was carried out as described earlier (21) 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 similar method in 0.01% Tween 80 detergent.

Synthesis and Modifications of MBP Peptides

The N-acetylated myelin basic protein peptide analogs MBP-(83-102)Y with the sequence Ac-YDENPVVHFFKNIVTPRTPP, MBP-(124-143) peptide with the sequence Ac-GFGYGGRASDYKSAHKGFKG, and MBP-(1-14) with the sequence Ac-ASQKRPSQRHGSKY 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. The deprotected, crude peptides were purified by reverse-phase HPLC, and the homogeneity and identity of the purified peptides were confirmed by mass spectrometry.

Biotinylation of various peptides was carried out as described previously(22) . For the synthesis of carboxyfluoresceinated MBP-(83-102)Y (CF-MBP-(83-102)Y) peptide, 220 mg of 6-carboxyfluorescein (Molecular Probes) was dissolved in 10 ml of dimethylformamide, and 0.12 mmol of peptide resin was added to this solution. After mixing the suspension for 1 min, 75 µl of diisopropylcarbodiimide was added, followed by gently mixing the slurry at room temperature for 2 h. The resin was then filtered and washed with dimethylformamide and methanol alternately, followed by washing with dichloromethane and vacuum drying for 2 h. The modified peptide was cleaved from the resin by suspending the resin in 10 ml of trifluoroacetic acid containing 0.7 g of 4-methylmercaptophenol and 1 ml of 4-methoxybenzenethiol. After 2 h, the resin was separated by filtration, and the filtrate was collected in 1 liter of pentane:acetone (8:1, v/v) mixture. The precipitated CF-peptide was separated by decantation and centrifugation and washed with pentane/acetone followed by pentane. Crude peptide was purified by reverse-phase HPLC using C18 column (Vydac, CA) with acetonitrile gradient (0.1% trifluoroacetic acid in water to 0.1% trifluoroacetic acid in 70% aqueous acetonitrile) and characterized by mass spectroscopy.

Complex Preparation and Peptide Binding Assay

For the quantitative detection of bound peptide, affinity-purified HLA-DR2 at a concentration of 2 µg/ml was incubated with biotinylated MBP peptides at 37 °C for 96 h at optimized pH. The resulting complex preparations were analyzed by antibody capture plate assay using an enzyme-conjugated avidin system as described recently (7) with minor modifications. Briefly, purified polyclonal antibodies against HLA-DR2, DR2alpha-Tm chain, and DR2beta-Tm chain at a concentration of 20 µg/ml were immobilized in a 96-well microtiter plate. Bovine serum albumin-biotin with 8 biotin molecules per bovine serum albumin was used as a standard with a concentration range of 0.014-1.8 pmol (0.117-15 ng). The bound biotinylated peptide in complex preparations were detected colorimetrically using alkaline phosphatase-conjugated streptavidin and p-nitrophenyl phosphate in 0.1 M diethanolamine as a substrate.

Dissociation Kinetics of Chain-Peptide Complexes by SDS-Gel Electrophoresis

Stability of single chain-peptide complexes was measured by SDS-polyacrylamide gel analysis of various complexes prepared with radioiodinated MBP peptide. Complexes of HLA-DR2 and I-MBP peptides were prepared under optimized binding conditions and purified from unbound peptide by G-75 size exclusion gel filtration chromatography. Resulting complexes were then incubated at 4 and 37 °C, and at various time points complex samples were removed and frozen at -20 °C. At the end of 72 h, the complexes were applied on 13.5% SDS gels under nonreduced conditions. Gels were stained, dried, and autoradiographed. Each lane containing the alphabeta dimer, alpha-Tm, or beta-Tm polypeptide chain was cut and counted in a counter.

Size-exclusion HPLC Analysis and Fluorescence Peptide Binding

Solutions of 5 µM alpha-Tm or beta-Tm and 50-fold molar excess of CF-MBP-(83-102)Y peptide in PBS buffer, pH 9, were incubated at 37 °C for 72 h. Complexes were purified from the excess of peptide by SE-HPLC using a Toso Haas TSK SW3000 column (0.75 times 60 cm) at the flow rate of 0.5 ml/min in PBS buffer at pH 9. The protein fraction has been collected from 20 to 40 ml, concentrated using a Centricon-10 microconcentrator (10-kDa cut-off membrane), and then analyzed by size-exclusion HPLC using the same column connected to UV diode array and fluorescence detectors in series. Two signals have been collected for each SE-HPLC chromatogram: fluorescence with excitation wavelength of 448 nm and emission wavelength of 525 nm and a UV signal at 278 nm. The fluorescence signal was used to measure the bound peptide, and the UV detection was used to calculate the protein concentration.

T Cell Receptor Occupancy Assay

The herpes saimiri virus-transformed SS8T human T cell clone restricted to DR2 (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 IL-2 (rIL-2) at 37 °C. Every alternate day cells were transferred to fresh media. Various complex preparations were incubated in a microtiter tissue culture plate at a density of 20,000 cells/200 µl/well in the absence of rIL-2. After 48 h of incubation at 37 °C, the supernatants were collected from each well to test for the increase in -IFN cytokine level. The detection of -IFN levels was performed by antibody enzyme-linked immunosorbent assay as described recently(19) .

In Situ Terminal Deoxynucleotidyl Transferase Assay and Flow Cytometry

The quantitative detection of DNA strand break was performed by end labeling of 3`-OH end of fragmented DNA using biotinylated dUTP followed by a fluorescein isothiocyanate-conjugated avidin detection system in a flow cytometer as described earlier with some modifications(23) . Briefly, the transformed SS8T cloned T cells at a density of 1 times 10^6 cells/ml were incubated with equimolar amounts of DR2 dimer-peptide or chain-peptide complex preparations. Cells were fixed in 1% buffered formaldehyde and stored at -20 °C in 70% ethanol. Cells were rehydrated in Hanks' balanced salt solution and resuspended in 50 µl of terminal deoxynucleoside transferase reaction mixture containing 10 µl of terminal deoxynucleoside transferase buffer (1 M potassium cacodylate, 125 mM Tris-HCl, pH 6.6, 1.25 mg/ml bovine serum albumin), 0.2 µl terminal deoxynucleotide transferase, 5 µl of CoCl(2), and 0.5 nmol of biotin-16-dUTP. The reaction mixture was incubated at 37 °C for 30 min, and cells were rinsed in Hanks' balanced salt solution and resuspended in 100 µl of staining solution containing 2.5 µg/ml fluorescein isothiocyanate-avidin in saline sodium citrate buffer. Cells were incubated for an additional 30 min at room temperature in the dark and resuspended in 1 ml of Hanks' balanced salt solution containing 5 µg/ml propidium iodide in the presence of 0.1% RNase. Double fluorescence measurements were carried out in Becton-Dickinson flow cytometer using LYSYS II software.

Transmission Electron Microscopy

SS8T cells at a density of 2 times 10^6 cells/ml were incubated with 50 µg/ml of freshly prepared native DR2-MBP-(83-102)Y complexes or 25 µg/ml of DR2alpha-MBP-(83-102)Y or DR2beta-MBP-(83-102)Y complexes at 37 °C for 18 h. T cells were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, at 25 °C for 2 h. Following a rinse in the buffer, cells were post fixed in 1% osmium tetraoxide at 25 °C for 1 hour. Cells were then dehydrated in a graded series of ethanol, treated with propylene oxide, embedded in a quick-mix epoxy resin, and polymerized at 60 °C for 16 h. The sample blocks were thin-sectioned with a diamond knife and sections were stained with 2% uranyl acetate in the presence of 0.4% lead citrate. Cells were characterized using JEOL-100CX TEM at an accelerating voltage of 80 kV.


RESULTS AND DISCUSSION

MHC class II molecules consist of two individual polypeptide chains (an alpha and a beta) of similar size that are noncovalently associated with each other. Recently reported crystal structure of MHC class II molecules shows that the extracellular alpha1 and beta1 domains are involved in creating the peptide binding domain that can accommodate peptides of varied length(11, 12, 13) . In the heterodimeric structure, it has been shown that the electrophoretically resolved alpha and beta chains independently bind the same peptides, although the two chains may or may not bind identical amino acid residues in the peptides(14, 24) . In our previous reports, we demonstrated that electroeluted purified alpha and beta chains of murine MHC class II polypeptides can bind antigenic peptides like the native heterodimer, and equimolar amounts of single chain-peptide complexes can trigger T cell response as measured by a sensor-based assay(15, 16, 25) .

The limited availability of purified native chains by the tedious electroelution method led us to investigate recombinant MHC class II chains for further studies. In this report we describe our successful effort to demonstrate that (i) E. coli expressed individual alpha and beta polypeptide chains of human MHC class II (HLA-DR2) lacking the transmembrane region are equally capable of binding an immunodominant epitope from myelin basic protein (MBP-(83-102)Y) and (ii) complexes of DR2alpha-MBP-(83-102)Y and DR2beta-MBP-(83-102)Y can induce antigen-specific apoptosis in restricted cloned T cells. The selection of human HLA-DR2 antigen is based on its predominant involvement in the autoimmune disorder multiple sclerosis (MS)(26) . Similarly, the peptide of MBP (MBP-(84-102)) used in this study is considered a major immunodominant epitope for human MS(27) . The peptide analog of MBP used in our study contains a tyrosine residue at position 83 and was found to have increased binding affinity to HLA-DR2 without loss of TCR recognition. (^2)The increased binding of N terminus tyrosine containing peptide was also observed in several other MHC class II-peptide complexes. (^3)

The cloning and expression of human HLA-DR2 alpha-Tm and beta-Tm was carried out as described recently(19) . The expression of recombinant individual alpha and beta chain of HLA-DR2 represent 30% of the total cell protein. In contrast to individual alpha and beta polypeptide chain, the recombinant expression of heterodimeric MHC class II molecules in E. coli was totally unsuccessful. The insoluble denatured inclusion body preparations were solubilized in 8 M urea, purified in a scale of 50-100 mg by conventional chromatography methods as described earlier(20) , and stored in PBS containing an 8 M urea solution. Prior to peptide loading, the alpha and beta chains were dialyzed against PBS buffer. Binding of various biotinylated MBP peptides to purified alpha and beta polypeptide chains were carried out by antibody captured plate assay using chain-specific rabbit polyclonal antibodies and enzyme-conjugated streptavidin as described recently(7) .

The optimum pH for maximum peptide occupancy was measured by incubating a known amount of each chain with 50-fold molar excess of MBP peptides. Three MBP peptides were selected for the optimization of peptide binding to individual chains. Their affinities toward HLA-DR2 have been shown in the order of MBP-(84-102) > MBP-(124-143) > MBP-(1-14) (28) . The epitope MBP-(1-14) had no affinity toward HLA-DR2 and was used as a negative control. As shown in Fig. 1, the binding of MBP-(83-102)Y peptide to both alpha and beta chain was maximum at pH 9.0. In contrast, the binding of MBP-(83-102)Y peptide to native DR2 heterodimer was found to be maximum at acidic pH(7) . In case of the alpha chain, the binding pH consistently appears to be critical below or above pH 9. The second high binding epitope of MBP, (MBP-(124-143)) bound strongly to the beta chain at basic pH and weakly to the alpha chain. Further increase in peptide concentration beyond 50-fold molar excess as well as increase in incubation period did not provide additional binding (data not shown). In negative controls, the MBP-(1-14) peptide did not bind to either the alpha or beta chain like the native alphabeta DR2 heterodimer. Although the maximum binding of the MBP peptide to each individual chain was at pH 9.0, a significant amount of peptide remained bound at around physiological pH between 7 and 8.


Figure 1: Binding of biotinylated MBP peptide to alpha-Tm and beta-Tm chains. Binding of biotinylated MBP peptides to purified chains was carried out using chain specific polyclonal antibody and enzyme conjugated streptavidin. A and B represent the pH-dependent binding of biotinylated MBP-(83-102)Y (closed circles), MBP-(124-143) (open circles), and MBP-(1-14) (closed squares) peptides to alpha-Tm and beta-Tm, respectively. C and D represent the competitive inhibition of biotinylated MBP-(83-102)Y peptide binding in the presence either nonbiotinylated MBP-(83-102)Y (closed circles) or MBP-(124-143) peptide (open circles) to alpha-Tm and beta-Tm polypeptides, respectively. Each data point represents an average of triplicate determinations and arrows in A and B indicate the optimum pH for maximum binding.



The specificity of the binding of MBP-(83-102)Y peptide to individual alpha and beta chain was demonstrated in a competitive binding experiment. Purified alpha or beta chain was incubated with 50-fold molar excess of biotinylated MBP-(83-102)Y peptide in the presence of increasing concentration of nonbiotinylated MBP-(83-102)Y or MBP-(124-143) peptide. As shown in Fig. 1, C and D, the binding of biotinylated MBP-(83-102)Y peptide was 75-80% inhibited at 2-4-fold excess concentration of nonbiotinylated MBP-(83-102)Y peptide. The binding of biotinylated MBP-(83-102)Y peptide to chains was partially inhibited by MBP-(124-143) peptide. In other words, the MBP-(83-102)Y peptide appears to be more inhibitory than MBP-(124-143) peptide, which correlates with their affinities to DR2 (28) .

The stability of the bound peptide in complexes of alpha-MBP-(83-102)Y and beta-MBP-(83-102)Y was examined using I-labeled MBP-(83-102)Y peptide in SDS-gel electrophoresis under nonreduced conditions. Complexes of native DR2 heterodimer, DR2alpha and DR2beta, with I-MBP-(83-102)Y peptide were prepared under fully optimized binding conditions and purified from unbound labeled peptide by G-75 gel filtration chromatography. The stability of three complexes was then monitored at 4 and 37 °C for 72 h. As shown in Fig. 2, the bound I-MBP-(83-102)Y peptide remained associated with both alpha and beta chains like the native alphabeta DR2 heterodimer.


Figure 2: Dissociation kinetics of chain-peptide complexes at 4 and 37 °C. The stability of the bound MBP-(83-102)Y peptide to individual alpha-Tm and beta-Tm polypeptide chains was measured by SDS-gel electrophoresis using I-MBP-(83-102)Y peptide. A-C represent the dissociation kinetics of complexes of native DR2, alpha-Tm, and beta-Tm with MBP-(83-102)Y peptide, respectively. Closed and open circles represent stability at 4 and 37 °C. Each data point is an average of triplicate determinations. Specificity of I-MBP-(83-102)Y peptide was 4.4 times 10^5 cpm/µg.



Further characterization of single chain-peptide complexes with respect to aggregation level was performed by size-exclusion HPLC analysis. Although the recombinant alpha and beta polypeptide chains used in our study lack the hydrophobic transmembrane region, due to inclusion body preparation of these polypeptides in E. coli, purified proteins tend to aggregate in solution in the absence of denaturing agent. The HPLC result presented in Fig. 3D shows that, prior to the peptide loading, almost all of beta polypeptide preparation appeared in the aggregated state (600 kDa). In contrast, most of the alpha polypeptide appears to be in a state of alpha-alpha homodimers (60 kDa) as shown in Fig. 3A. The existence of alpha-alpha and beta-beta homodimers in purified native polypeptide chains was also observed in our earlier studies(15, 16) . Upon peptide binding, however, a significant amount of purified complexes of both alpha and beta chain-peptide shifted to the monomeric state with a molecular size of 30 kDa (Fig. 3, B and E). In these experiments, carboxyfluorescein labeled MBP-(83-102)Y peptide was used to monitor the bound peptide associated with various molecular size protein fractions. Results presented in Fig. 3, C and F, show that almost no fluorescence intensity was associated with highly aggregated proteins or with homodimers. In fact, the fluorescence intensity was only found to be associated with the monomeric form of both alpha- and beta-MBP-(83-102)Y peptide complexes. These results clearly demonstrate that bound peptide prevents aggregation of purified chains. Such prevention of aggregation of MHC class II dimers on cell surface by peptide binding has been reported recently(29) . Similarly, in a separate study we have observed that bound peptide significantly inhibits aggregation of purified MHC class II heterodimers in solution. (^4)Calculated percent aggregation and associated bound peptide by size-exclusion HPLC of individual chains and their complexes are also presented in Fig. 3. The molar percent of total bound peptide data observed with CF-MBP-(83-102)Y peptide in HPLC experiment correlates well with the biotinylated peptide binding results obtained with the antibody captured plate assay.


Figure 3: Size-exclusion HPLC analysis of purified chains and complexes. Size-exclusion HPLC profiles of purified alpha-Tm (A) and beta-Tm (D) before peptide loading were compared with that of alpha-MBP-(83-102)Y (B) and beta-MBP-(83-102)Y complexes (E). Complexes of alpha and beta chains with peptide were prepared using 50-fold molar excess of CF-MBP peptide and purified from unbound peptide. CF-MBP-(83-102)Y peptide was used to monitor bound peptide associated with various peaks. Fluorescence measurement of alpha-MBP-(83-102)Y and beta-MBP-(83-102)Y complexes are shown in C and F, respectively. For fluorescence measurements, 10 times less amount of complexes was injected. The quantitative numbers in A, B, D, and E represent percentage of aggregates, homodimers, and monomers. The numbers in C and F represent the percent of monomers with bound CF-MBP peptide.



The recognition of alpha- and beta-MBP-(83-102)Y peptide complexes by TCR was performed using herpes saimiri virus-transformed SS8T cloned T cells. The SS8T cell clone was generated from an MS patient and was fully characterized for its restriction to HLA-DR2 (DRB5*0101) and MBP-(84-102) peptide(30) . The TCR engagement by soluble alpha- and beta-MBP-(83-102)Y peptide complexes was monitored by an increase in -IFN cytokine in a dose-dependent manner. Such increase in -IFN production by T cells was correlated with the occupancy of TCRs on the surface of T cells in an earlier study (30) and was adopted for SS8T cells in our laboratory(19) . As shown in Fig. 4, specific increase in -IFN was observed when SS8T cells were exposed to complexes of native DR2, alpha or beta polypeptide chain with the MBP-(83-102)Y peptide. In various control experiments, cells incubated with alpha or beta chain alone and complexes containing irrelevant peptide (alpha-MBP-(124-143) or beta-MBP-(124-143)) did not show any significant increase in -IFN level. Human T cells are known to express a low levels of MHC class II molecules on their surfaces and can be stimulated in the presence of antigenic peptide(31, 32) . 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 MBP-(83-102)Y peptide was complexed with irrelevant HLA-DR3 as a control and showed no increase in -IFN level (Fig. 4A). Similarly, in a mock experiment, equivalent amount of MBP-(83-102)Y peptide incubated and passed through Sephadex G-75 column under identical purification conditions in the absence of chains, did not show any increase in -IFN level (data not shown).


Figure 4: T cell recognition by single-chain peptide complexes. Occupancy of TCRs by complexes of native DR2, alpha-Tm, and beta-Tm with MBP-(83-102)Y peptide was shown by increase in -IFN using SS8T cloned T cells. Complexes of DR2, DR3, alpha-Tm, and beta-Tm with MBP-(83-102)Y83 (or MBP-(124-143)) peptides were prepared, purified, and incubated with SS8T cells in equimolar amounts. A-C represent increased -IFN by SS8T cells in the presence of DR2, alpha-Tm, and beta-Tm complexes, respectively. Complexes of DR2 or chains with: MBP-(83-102)Y peptide (closed circles), MBP-(124-143) peptide (open circles), and DR2 or chains alone (closed squares). Open squares in A represent -IFN level with DR3-MBP-(83-102)Y complexes. Each data point is an average of triplicate determinations.



Prolonged incubation of SS8T cells with relevant complexes of individual alpha and beta chains led to the induction of apoptosis. Typically apoptosis is characterized by chromatin condensation and is associated with endonuclease activity. The endonuclease activity in apoptotic cells can be demonstrated by cleavage of cellular DNA. The quantitative detection of DNA strand breaks in this study was demonstrated by labeling the 3`-OH end of the fragmented DNA with biotinylated dUTP followed by fluorescein isothiocyanate-conjugated avidin detection system in a flow cytometer. Induction of apoptosis in T cells by DR2-MBP-(83-102)Y, alpha-MBP-(83-102)Y, and beta-MBP-(83-102)Y complexes as measured by fluorescence-activated cell sorter analysis is shown in Fig. 5. In this flow cytometry assay, the DNA degradation is directly related with the biotin-dUTP incorporation and can be utilized in quantitative measurement of apoptosis. Cells incubated with DR2 alone, alpha chain alone, beta chains alone, DR2-MBP-(124-143), alpha-MBP-(124-143) and beta-MBP-(124-143) complexes were used as controls. Minimal incorporation of biotinylated dUTP was observed in various control experiments, whereas T cells incubated with either recombinant alpha-MBP-(83-102)Y or beta-MBP-(83-102)Y complexes showed approximately 30-35% of the cells labeled with biotinylated dUTP similar to the native DR2-MBP-(83-102)Y complexes. The calculated percent T cell apoptosis by relevant chain-peptide complexes with respect to various controls is presented in Fig. 6A. Apoptosis of T cells by chain-peptide complexes appeared to be time-dependent as shown in Fig. 6B.


Figure 5: Flow cytometry analysis of DNA strand break. Quantitative detection of fragmented DNA was carried out by 3`-end labeling of DNA by biotinylated dUTP in the presence of terminal deoxynucleoside transferase enzyme as described under ``Material and Methods.'' 1 times 10^6 T cells/ml were incubated with 50 µg/ml of DR2-MBP-(83-102)Y complex or 25 µg/ml of chain-MBP-(83-102)Y complex at 37 °C for 18 h. Cells were fixed with paraformaldehyde, labeled with biotinylated dUTP and propidium iodide, and analyzed in a flow cytometer. The contour graphs represent cells treated with DR2 or chains alone (a), complexes of DR2 or chains with MBP-(83-102)Y peptide (b), and complexes of DR2 or chains with MBP-(124-143) peptide (c).




Figure 6: Apoptosis of T cells by chain-peptide complexes. The percent apoptosis of SS8T cells was calculated from the fluorescence-activated cell sorter data using the LYSYS II software. A represents the calculated percent cell death from Fig. 5along with various controls. B represents percent cell death with time where 1 times 10^6 T cells/ml were incubated with 50 µg/ml of DR2 complexes or 25 µg/ml of chain-peptide complexes. Cells were analyzed at different time intervals by flow cytometry.



Finally, the chromatin condensation and cell shrinkage characteristics of apoptotic cells were demonstrated by transmission electron microscopy (Fig. 7). As compared with untreated T cells (Fig. 7A), SS8T cells treated with native DR2-MBP-(83-102)Y alpha-MBP-(83-102)Y, and beta-MBP-(83-102)Y complexes showed typical apoptotic cells (Fig. 7, B-D).


Figure 7: Electron microscopy of complex treated SS8T cells. 2 times 10^6 T cells were incubated with PBS (A), 50 µg/ml of DR2-MBP-(83-102)Y complex (B), 25 µg/ml of alpha-MBP-(83-102)Y complex (C), and 25 µg/ml of beta-MBP-(83-102)Y complex. Cells were fixed, sectioned, and visualized by JEOL-100CX TEM at a magnification of times 3,500.



In summary, results presented in this report describe that individual recombinant polypeptide chains of human MHC class II molecules are capable of binding antigenic peptide and that complexes of single-chain peptide can recognize TCR to induce antigen-specific apoptosis in T cells. Furthermore, this study demonstrates that the transmembrane region of human MHC class II molecules are not involved in either peptide binding or TCR recognition. The physiological significance of single chain-peptide complexes is unknown at present and requires further investigation. In a parallel study, we observed that recombinant murine IA^salpha chain complexed with rat MBP-(90-101) peptide was highly effective in preventing experimental allergic encephalomyelitis in mice, an animal model for human MS. (^5)Prevention and treatment of several autoimmune diseases in animal models by soluble native MHC class II-peptide complexes were demonstrated in our laboratory(33, 34) . (^6)The T cell apoptosis reported here by recombinant soluble chain-peptide complexes may have significant clinical relevance in developing therapeutics for the elimination of autoreactive T cells in various autoimmune diseases in an antigen-specific manner.


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.

§
These authors contributed equally to this work.

To whom all correspondence should be addressed. Tel.: 415-361-8901; Fax: 415-361-8958.

(^1)
The abbreviations used are: MHC, major histocompatibility complexes; alpha-Tm, recombinant alpha chain lacking the transmembrane domain; beta-Tm, recombinant beta chain lacking the transmembrane domain; TCR, T cell receptor; MBP, myelin basic protein; -IFN, -interferon; MS, multiple sclerosis; CF, carboxyfluorescein; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography.

(^2)
P. Mukku, S. Arimilli, I. Astafieva, and B. Nag, unpublished results.

(^3)
B. Nag, S. Arimilli, P. V. Mukku, and I. Astafieva, unpublished results.

(^4)
P. Mukku, I. Astafieva, S. Arimilli, and B. Nag, unpublished results.

(^5)
B. Nag, S. Arimilli, and S. Sriram, unpublished results.

(^6)
H. Bhayani and B. Nag, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. H. Wekerly for providing SS8T cloned T cells, Dr. Shrikant Deshpande for synthesizing various MBP peptides, Eric Rhodes for cloning and expression of recombinant chains, Cristina Cardoso for technical assistance, and Melina Dunsavage for editing the manuscript.


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