(Received for publication, November 30, 1995; and in revised form, February 8, 1996)
From the
Major histocompatibility (MHC) class II molecules are cell
surface heterodimeric () glycoproteins that display processed
antigens to T cell receptors (TCRs) of CD4-positive T cells. The
present study describes that individual recombinant
and
chains of human MHC class II molecules lacking the transmembrane region
(
-Tm and
-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
-Tm and the
-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 DR2
-Tm and
DR2
-Tm was demonstrated with biotinylated and fluoresceinated
MBP-(83-102)Y
peptide. The specificity of the
MBP-(83-102)Y
peptide binding to both DR2
-Tm
and DR2
-Tm was demonstrated in a competitive peptide binding
assay. When exposed to a transformed T cell clone (SS8T) restricted to
DR2(DRB5
0101) and MBP-(84-102) peptide, complexes of
DR2
-Tm and DR2
-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
-MBP-(83-102)Y
and
-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.
MHC ()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
polypeptide and a
28-30-kDa
polypeptide chain noncovalently associated with
each other. Furthermore, each polypeptide contains two distinct
extracellular domains (
1/
2 in the
chain and
1/
2 in the
chain), a transmembrane region and a small
cytoplasmic C terminus region. The crystal structure of MHC class II
molecules reveals that the extracellular
1 and
1 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
and
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
and
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
-specific cytotoxic T lymphocytes
to specifically lyse transfected L cells expressing either
A
b1/D
c2 (17) or
Ak
1/Dd
2 (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 and
polypeptides of
human HLA-DR2 (DRB5*0101) lacking the transmembrane region are capable
of binding immunodominant epitopes from MBP. In addition, monomeric
complexes of DR2
-Tm and DR2
-Tm chains and MBP peptide can
induce antigen-specific apoptosis in cloned T cells to a degree
comparable with that of native
/
dimer-peptide complexes.
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.
MHC class II molecules consist of two individual polypeptide
chains (an and a
) of similar size that are noncovalently
associated with each other. Recently reported crystal structure of MHC
class II molecules shows that the extracellular
1 and
1
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
and
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
and
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 and
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 DR2
-MBP-(83-102)Y
and
DR2
-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. (
)The increased binding of N terminus
tyrosine containing peptide was also observed in several other MHC
class II-peptide complexes. (
)
The cloning and expression
of human HLA-DR2 -Tm and
-Tm was carried out as described
recently(19) . The expression of recombinant individual
and
chain of HLA-DR2 represent 30% of the total cell protein. In
contrast to individual
and
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
and
chains were dialyzed against PBS buffer. Binding of various
biotinylated MBP peptides to purified
and
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
and
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
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
chain at basic pH and
weakly to the
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
or
chain like the native
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 -Tm and
-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
-Tm and
-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
-Tm and
-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
and
chain was demonstrated in a competitive binding experiment. Purified
or
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 -MBP-(83-102)Y
and
-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, DR2
and DR2
, 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
and
chains like the native
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
-Tm and
-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,
-Tm, and
-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
10
cpm/µg.
Further characterization of single chain-peptide
complexes with respect to aggregation level was performed by
size-exclusion HPLC analysis. Although the recombinant and
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
polypeptide preparation appeared in the aggregated
state (600 kDa). In contrast, most of the
polypeptide appears to
be in a state of
-
homodimers (60 kDa) as shown in Fig. 3A. The existence of
-
and
-
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
and
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
- and
-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. (
)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 -Tm (A) and
-Tm (D) before peptide loading were
compared with that of
-MBP-(83-102)Y
(B) and
-MBP-(83-102)Y
complexes (E). Complexes of
and
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
-MBP-(83-102)Y
and
-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 -
and
-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
- and
-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,
or
polypeptide chain with the
MBP-(83-102)Y
peptide. In various control
experiments, cells incubated with
or
chain alone and
complexes containing irrelevant peptide (
-MBP-(124-143) or
-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, -Tm, and
-Tm with
MBP-(83-102)Y
peptide was shown by increase in
-IFN using SS8T cloned T cells. Complexes of DR2, DR3,
-Tm,
and
-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,
-Tm, and
-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 and
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
,
-MBP-(83-102)Y
, and
-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,
chain
alone,
chains alone, DR2-MBP-(124-143),
-MBP-(124-143) and
-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
-MBP-(83-102)Y
or
-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 10
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 10
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
-MBP-(83-102)Y
, and
-MBP-(83-102)Y
complexes showed typical
apoptotic cells (Fig. 7, B-D).
Figure 7:
Electron microscopy of complex treated
SS8T cells. 2 10
T cells were incubated with PBS (A), 50 µg/ml of DR2-MBP-(83-102)Y
complex (B), 25 µg/ml of
-MBP-(83-102)Y
complex (C), and 25
µg/ml of
-MBP-(83-102)Y
complex. Cells were
fixed, sectioned, and visualized by JEOL-100CX TEM at a magnification
of
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 chain complexed with rat MBP-(90-101) peptide
was highly effective in preventing experimental allergic
encephalomyelitis in mice, an animal model for human MS. (
)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) . (
)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.