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,
,§
*
Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97201; and
Department of Neurology,
Department of Biochemistry and Molecular Biology, and
§
Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1"
molecule included the ß1 domain of the rat RT1.B class II
molecule covalently linked to the amino terminus of the
1 domain. In association with the encephalitogenic
myelin basic protein (MBP) 69–89 peptide recognized by Lewis rat T
cells, the ß11/MBP-69–89 complex
specifically labeled and inhibited activation of MBP-69–89 reactive T
cells in an IL-2-reversible manner. Moreover, this complex both
suppressed and treated clinical signs of experimental autoimmune
encephalomyelitis and inhibited delayed-type hypersensitivity reactions
and lymphocyte proliferation in an Ag-specific manner. These data
indicate that the ß11/MBP-69–89 complex
functions as a simplified natural TCR ligand with potent inhibitory
activity that does not require additional signaling from the
ß2 and 2 domains. This new class of small
soluble polypeptide may provide a template for designing human
homologues useful in detecting and regulating potentially
autopathogenic T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ag-driven activation of CD4+ T cells is a multistep process initiated by coligation of the TCR and CD4 by the MHC class II/peptide complex present on APC (signal 1), as well as costimulation through additional T cell surface molecules such as CD28 or OX40 (signal 2). The classic experiment by Quill and Schwartz (8) demonstrated that stimulation through the TCR by cell-associated MHC class II/peptide in the absence of costimulation, rather than being a neutral event, induced a state of unresponsiveness to subsequent optimal Ag presentation, a phenomenon termed anergy (8, 9). Thus, a direct approach toward Ag-driven immunosuppression is to present the complete ligand, Ag plus MHC, in the absence of costimulatory signals that are normally provided by specialized APCs.
MHC class II molecules are heterodimeric membrane-bound glycoproteins
made up of noncovalently associated - and ß-polypeptide subunits.
Each subunit consists of a short cytoplasmic tail, a single
membrane-spanning sequence, and two extracellular domains. X-ray
crystallographic studies have demonstrated that peptides from processed
Ag bind to MHC molecules in the membrane distal pocket formed by the
ß1 and 1 domains (10, 11). Moreover, the
ß2 domain contains a CD4-binding site that coligates CD4
when the ß11 domains with associated
antigenic peptide interact with the TCR - and ß-chains (12, 13, 14, 15),
whereas the 2 domain appears to contribute to ordered
oligomerization in T cell activation (16). Complexes of MHC/Ag have
been purified as detergent extracts of lymphocyte membranes (17) and as
associated recombinant proteins using baculovirus and bacterial
expression systems (18, 19, 20, 21, 22). These two-chain, four-domain molecular
complexes, after loading with selected peptide epitopes, have been
demonstrated to interact with T cells in an Ag-specific manner (20, 23, 24, 25, 26, 27).
To develop a simple and effective agent that could bind selectively to
the TCR, we have designed molecules consisting of the 1
and ß1 domains of the rat RT1.B MHC class II molecule
genetically linked into a single polypeptide chain. Molecules with this
design would be useful for studying binding specificity in vitro for
exploring primary TCR signaling events independent of costimulatory
input associated with the MHC II ß22
domains or with other molecules expressed by APCs and for treating
CD4+ T cell-mediated autoimmune disease in an MHC
II/epitope-specific manner.
Experimental autoimmune encephalomyelitis (EAE) is a paralytic,
inflammatory, and sometimes demyelinating disease mediated by
CD4+ T cells specific for central nervous system (CNS)
myelin components including myelin basic protein (MBP). EAE shares a
number of immunologic abnormalities with the human demyelinating
disease MS (28) and has been a useful model for preclinical testing of
therapies for the human illness (29, 30, 31, 32, 33, 34, 35). In Lewis rats, the dominant
encephalitogenic MBP epitope resides in the 72–89 peptide (36). Onset
of clinical signs of EAE occurs on day 10–11, and the disease lasts
4–8 days. The majority of invading T lymphocytes are localized in the
CNS during this period (37). In this study, we demonstrate that
ß11 complexed with the MBP-69–89 peptide
administered in saline could successfully suppress and treat EAE by
blocking the activation of encephalitogenic T cells, thereby preventing
entry to the spinal cord and protecting the animals from EAE.
Lymphocytes from treated rats had a reduced response to MBP-69–89,
although this was reversible in vitro in the presence of IL-2. Thus,
Ag-specific anergy of encephalitogenic T cells may be the mechanism of
protection from EAE using this construct. By expressing the
single-chain ß11 molecules as inclusion
bodies in Escherichia coli followed by purification and
refolding in vitro, we can isolate biologically active material in very
high yield. In addition to demonstrating the potent biologic properties
of these ß11 molecules, we demonstrate
their utility in staining Ag-specific T cells by cytofluorometric
analysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 constructs
RT1B 1 and ß1 domain-encoding cDNAs
were prepared by PCR amplification of cloned RT1.B - and ß-chain
cDNA-coding sequences (6, ß118,
respectively) obtained from Dr. Konrad Reske (38, 39). The
primers used to generate ß1 were
5'-AATTCCTCGAGATGGCTCTGCAGACCCC-3' (XhoI 5' oligo);
5'-TCTTGACCTCCAAGCCGCCGCAGGGAGGTG-3' (3' ligation oligo). The
primers used to generate 1 were
5'-CGGCGGCTTGGAGGTCAAGACGACATTGAGG-3' (5' ligation oligo);
5'-GCCTCGGTACCTTAGTTGACAGCTTGGGTTGAATTTG-3' (KpnI 3' oligo).
Additional oligos used as primers were
5'-CAGGGACCATGGGCAGAGACTCCCCA-3' (NcoI 5' oligo); and
5'-GCCTCCTCGAGTTAGTTGACAGCTTGGGTT-3' (XhoI 3' oligo).
Step one involved production of cDNAs encoding the ß1 and
1 domains. PCR was conducted with Taq
polymerase (Promega, Madison, WI) through 28 cycles of denaturation at
94.5°C for 20 s, annealing at 55°C for 1.5 min and extension
at 72°C for 1.5 min, using ß118 as template and the
XhoI 5' oligo and 3' ligation oligo as primers; and
6 cDNA as template and the 5' ligation oligo and
KpnI 3' oligo. PCR products were isolated by agarose gel
electrophoresis and purified using Gene-Clean (Bio 101, La Jolla, CA).
In step two, these products were mixed together without additional
primers and heat denatured at 94.5°C for 5 min followed by two cycles
of denaturation at 94.5°C for 1 min, annealing at 60°C for 2 min
and extension at 72°C for 5 min. In step three, the annealed,
extended product was heat denatured at 94.5°C for 5 min and subjected
to 26 cycles of denaturation at 94.5°C for 20 s, annealing at
60°C for 1 min, and extension at 72°C for 1 min, in the presence of
the XhoI 5' oligo and KpnI 3' oligo. The final
PCR product was isolated by agarose gel electrophoresis and
Gene-Cleaned. This effectively produced a cDNA encoding the novel
ß11 molecule. The cDNA encoding the
ß11 molecule was moved into cloning vector
pCR2.1 (Invitrogen, Carlsbad, CA) using Invitrogen’s TA Cloning kit.
The cDNA in pCR2.1 was used as template and PCR was conducted through
28 cycles of denaturation at 94.5°C for 20 s, annealing at
55°C for 1.5 min and extension at 72°C for 1.5 min, using the
NcoI 5' oligo and XhoI 3' oligo. The PCR products
were cleaved with the relevant restriction enzymes and directionally
cloned into pET21d+ (Novagen, Madison, WI; see 40 .
The constructs have been confirmed by DNA sequencing. The
ß11 molecule used in these studies differs
from wild-type in that it contains a ß1 domain Q12R amino
acid substitution. E. coli strain BL21(DE3) cells were
transformed with the pET21d+ construct containing the
ß11-encoding sequence. Bacteria were grown
in 1-liter cultures to mid-logarithmic phase (OD600 =
0.6–0.8) in Luria-Bertani broth containing carbenicillin (50 µg/ml)
at 37°C. Recombinant protein production was induced by addition of
0.5 mM isopropyl ß-D-thiogalactoside (IPTG). After
incubation for 3 h, the cells were centrifuged and stored at
-80°C before processing. All subsequent manipulations of the cells
were at 4°C. The cell pellets were resuspended in ice-cold PBS, pH
7.4, and sonicated for 4 x 20 s with the cell suspension
cooled in a salt/ice/water bath. The cell suspension was then
centrifuged, the supernatant fraction poured off, and the cell pellet
resuspended and washed three times in PBS and then resuspended in 20 mM
ethanolamine/6 M urea, pH 10, for 4 h. After centrifugation, the
supernatant containing the solubilized recombinant protein of interest
was collected and stored at 4°C until purification. Recombinant
ß11 construct was purified and
concentrated by fast protein liquid chromatography ion-exchange
chromatography using Source 30Q anion-exchange media (Pharmacia
Biotech, Piscataway, NJ) in an XK26/20 column (Pharmacia Biotech),
using a step gradient with 20 mM ethanolamine/6 M urea/1 M NaCl, pH 10.
The homogeneous peak of the appropriate size was collected, dialyzed
extensively against PBS at 4°C, and concentrated by centrifugal
ultrafiltration with Centricon-10 membranes (Amicon, Beverly, MA). For
purification to homogeneity, a finish step used size-exclusion
chromatography on Superdex 75 media (Pharmacia Biotech) in an HR16/50
column (Pharmacia Biotech). The final yield of purified protein varied
between 15 and 30 mg/L of bacterial culture.
SDS-PAGE and Western blot analysis of recombinant
ß11 molecules
Aliquots of purified protein were electrophoresed by the
method of King and Laemmli (41). After electrophoresis, the gels
were stained with Coomassie blue for 15 min followed by destaining
overnight. Photos of the Coomassie-stained proteins were taken at this
point, and then the same gels were processed for Western blot analysis
(42). Protein was transferred to Immobilon-P membranes
(Millipore, Bedford, MA), blocked with powdered milk, and then
incubated for 1 h in a 1:500 dilution of mAb OX-6
(mouse-anti-RT1.B; PharMingen, San Diego, CA), washed and then
incubated with a 1:1000 dilution of -Mu IgG-alkaline phosphatase
conjugate (Sigma, St. Louis, MO) for 1 h and washed again. The
blot was developed using an aqueous stabilized alkaline phosphatase
substrate (Promega, Madison, WI) and stopped by rinsing in water.
Synthetic peptides
MBP-69–89 peptide (GSLPQKSQRSQDENPVVHF), MBP-55–69 peptide
(SGKDSHHAARTTHYG), MBP-87–99 peptide (VHFFKNIVTPRTP), and cardiac
myosin peptide CM-2 (KLELQSALEEAEASLEH) (43) were prepared by
solid-phase techniques (44). The MBP peptides are numbered according to
the bovine MBP sequence (35, 45). Peptides were loaded onto
ß11 at a 1:10 protein:peptide molar ratio
by mixing at room temperature for 24 h, after which all subsequent
manipulations were performed at 4°C. Free peptide was then removed by
centrifugal ultrafiltration with Centricon-10 membranes, serially
diluting and concentrating the retained solution until the free peptide
concentration was less than 2 µM.
Cell lines and the A1 hybridoma
Short-term T-lymphocyte lines were selected with MBP-69–89
peptide from lymph node (LN) cells of naive rats or from rats immunized
12 days earlier with Gp-MBP/CFA or CM-2 peptide/CFA. Details of this
procedure have been described previously (46). The rat
Vß8.2+ T cell hybridoma C14/BW12–12A1 (A1) used in this
study has been described previously (47). Briefly, the A1 hybridoma was
created by fusing an encephalitogenic LEW(RT1l) T cell
clone specific for Gp-MBP-72–89 (48, 49) and strongly cross-reactive
with Rt-BP-72–89 with a TCR (/ß)-negative thymoma, BW5147 (50).
Wells positive for cell growth were tested for IL-2 production after
stimulation with Ag in the presence of APCs (irradiated Lewis rat
thymocytes) and then subcloned at limiting dilution. The A1 hybridoma
secretes IL-2 when stimulated in the presence of APCs with whole Gp-MBP
or Gp-MBP-69–89 peptide, which contains the minimum epitope,
MBP-72–86.
Flow cytometry
Two-color immunofluorescent analysis was performed on a FACScan
instrument (Becton Dickinson, Mountain View, CA) using CellQuest
software (Becton Dickinson). Quadrants were defined using
non-relevant isotype-matched control Abs.
ß11 molecules with and without loaded
peptide were incubated with the A1 hybridoma (10 µM
ß11/peptide) for 17 h, 4°C, washed
three times, stained with fluorochrome (FITC or phycoerythrin
(PE))-conjugated Abs specific for rat class II (OX6-PE) and TCR Vß8.2
(PharMingen) for 15 min at room temperature and analyzed by flow
cytometry. The CM-2 cell line was blocked for 1 h with
unconjugated OX6, washed, and then treated as the A1 hybridoma.
Staining media was PBS containing 2% FBS and 0.01% azide.
Animals
Female Lewis rats (Harlan Sprague-Dawley, Indianapolis, IN), 8–12 wk of age, were used for clinical experiments in this study. The rats were housed under germfree conditions at the Veterans Affairs Medical Center Animal Care Facility (Portland, OR) according to institutional guidelines.
Induction of EAE
Active EAE was induced in rats by s.c. injection of 25 µg guinea pig MBP or 200 µg MBP-69–89 peptide or 200 µg MBP-87–99 peptide in CFA supplemented with 100, 400, or 150 µg Mycobacterium tuberculosis strain H37Ra (Difco, Detroit, MI), respectively. The clinical disease course induced by the two emulsions was essentially identical, with the same day of onset, duration, maximum severity, and cumulative disease index. The rats were assessed daily for changes in clinical signs according to the following clinical rating scale: 0, no signs; 1, limp tail; 2, hind leg weakness, ataxia; 3, paraplegia; and 4, paraplegia with forelimb weakness, moribund condition. A cumulative disease score was obtained by summing the daily disability scores over the course of EAE for each affected rat, and a mean cumulative disease index was calculated for each experimental group.
Analysis of CNS mononuclear cell infiltrates from control and protected animals
Spinal cord mononuclear cells were isolated by a discontinuous Percoll gradient technique and counted as previously described (51). The cells were stained with fluorochrome (FITC or PE)-conjugated Abs specific for rat CD4, CD8, CD11b, CD45ra, TCR Vß8.2, and CD134 (PharMingen) for 15 min at room temperature and analyzed by flow cytometry. The number of positive-staining cells per spinal cord was calculated by multiplying the percent staining by the total number of cells per spinal cord.
Histology
Control and
ß11/MBP-69–89-protected rats were
sacrificed at peak and recovery of clinical disease, and spinal cords
were dissected and fixed in 10% buffered formalin. The spinal cords
were paraffin-embedded and sections were stained with luxol fast
blue-periodic acid Schiff-hematoxylin for light microscopy.
Delayed-type hypersensitivity (DTH) reactions
DTH reactions were measured by the ear swelling assay using a pressure sensitive micrometer (35) before and 24 h after intradermal injection of 50 µg MBP-69–89 peptide or 20 µg purified protein derivative (PPD) into the ear pinna.
Ag-specific proliferation assays
Proliferation assays were performed in 96-well plates as described previously (46). Briefly, 4 x 105 cells in 200 µl/well (for organ stimulation assays) or 2 x 104 T cells and 1 x 106 irradiated APCs (for short-term T cell lines) were incubated in RPMI 1640 and 1% rat serum in triplicate wells with stimulation medium only, Con A, or Ag with or without supplemental IL-2 (20 U/ml) at 37°C in 7% CO2. The cultures were incubated for 3 days, the last 18 h in the presence of [3H]thymidine (0.5 µCi/10 µl/well). The cells were harvested onto glass fiber filters and [3H]thymidine uptake assessed by liquid scintillation.
In some experiments, the T cells were pretreated 24 h with
ß11 constructs (with and without loaded
peptides), washed, and then used in proliferation assays with and
without IL-2, as above. Mean cpm ± SD were calculated from
triplicate wells and differences between groups determined by
Student’s t test.
Computer modeling and graphics
The programs MidasPlus (52) and Sybyl (Tripos Associates, St. Louis, MO) and coordinates deposited in the Brookhaven Protein Data Bank (Brookhaven National Laboratories, Upton, NY) were used to generate graphic images using an O2 workstation (Silicon Graphics, Mountain View, CA). Structure-based homology modeling was based on the refined crystallographic coordinates of human DR1 (53), murine I-Ek molecules (54), and scorpion toxins (55, 56, 57) using side-chain substitution followed by local energy minimization.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 constructs
Genes were constructed encoding a single polypeptide chain with
the amino terminus of the 1 domain genetically linked to
the carboxyl terminus of the ß1 domain (Fig. 1
). Molecular modeling studies of rat MHC
class II RT1.B and its Ag-binding domain were conducted based on the
crystal structures of human DR (53, 58) and murine I-Ek
with covalently bound single peptides (54) (Fig. 2). Our analysis of these structures
focused on the solvent accessible surface of the ß-sheet
platform/anti-parallel helix Ag-binding domain. The crystal
structure of MHC class II I-EK showed a number of water
molecules between the membrane proximal surface of ß-sheet and the
membrane distal surfaces of the 2 and ß2
Ig-fold domains (54). The 1 and ß1 domains
of class II molecules showed an extensive hydrogen-bonding network and
a tightly packed and buried hydrophobic core similar to the molecular
interactions that provide structural integrity and thermodynamic
stability to the helix/ß-sheet scaffold characteristic of
scorpion toxins (55, 56, 57). These modeling studies predicted that the
Ag-binding domain would remain stable in the absence of the
2 and ß2 Ig-fold domains.
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1
constructs
Protein expression was tested in a number of different E.
coli strains, including a thioredoxin reductase mutant, which
allows disulfide bond formation in the cytoplasm (37). With such a
small molecule, it became apparent in our studies that the greatest
yield of material could be readily obtained from inclusion bodies,
refolding the protein after solubilization and purification in buffers
containing 6 M urea. This procedure avoided problems with proteases
associated with large scale production of recombinant protein in
bacteria. Pure ß11 protein was obtained by
fast protein liquid chromatography using a combination of ion exchange
and size-exclusion chromatography. After purification and refolding,
the protein was dialyzed against PBS at 4°C for our in vitro and in
vivo studies. The final yield of ß11
protein was approximately 15–30 mg/L culture. Conformational integrity
of the molecules was demonstrated by the presence of a disulfide bond
between cysteines ß15 and ß79 (Fig. 3; compare lanes 2 and
3) and authenticity of the purified protein was verified
using the OX-6 mAb specific for RT1.B (Fig. 3, lanes 4 and
5).
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1 Molecules bind T lymphocytes in
an epitope-specific manner
Epitope-specific binding was evaluated by loading the
ß11 molecule with various peptides and
incubating ß11/peptide complexes with the
A1 hybridoma derived from an encephalitogenic T cell clone that
recognized both guinea pig and rat sequences of the MBP-69–89 peptide
(59), or with a cardiac myosin CM-2-specific cell line. As is shown in
Fig. 4A, the
ß11 construct loaded with MBP-69–89
peptide (ß11/MBP-69–89) specifically
bound to the A1 hybridoma, with a mean fluorescence intensity (MFI) of
800 U, whereas the ß11 construct loaded
with CM-2 peptide (ß11/CM-2) did not stain
the hybridoma (MFI = 10–20 U). Conversely,
ß11/CM-2 specifically bound to the CM-2
line (MFI = 1, 800 U), whereas the
ß11/MBP-69–89 complex did not stain the
CM-2 line (MFI = 10 U). The ß11
construct without exogenously loaded peptide did not bind to either the
A1 hybridoma (Fig. 4A) or the CM-2 line (data not shown).
Thus, bound epitope directed the specific binding of the
ß11/peptide complex.
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1 constructs loaded with peptides
To evaluate the effect of the constructs on T cell activation, a
range of concentrations (10 nM to 20 µM) of peptide-loaded
ß11 complexes were preincubated with an
MBP-69–89-specific T cell line before stimulation with the MBP-69–89
peptide plus APC. As is shown in Fig. 5,
pretreatment of MBP-69–89-specific T cells with 10 nM
ß11/MBP-69–89 complex significantly
inhibited proliferation (>90%), whereas preincubation with 20 µM
ß11/MBP-55–69 complex produced a nominal
(27%) but insignificant inhibition. Of mechanistic importance, the
response inhibited by the ß11/MBP-69–89
complex could be fully restored by including 20 U/ml of IL-2 during
stimulation of the T cell line (Fig. 5).
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1 constructs loaded with MBP-69–89
peptide
The ß11/MBP-69–89 complex was
evaluated for its ability to suppress the induction, as well as to
treat existing signs of EAE in Lewis rats. Intravenous injection of 300
µg of the ß11/MBP-69–89 complex in
saline on days 3, 7, 9, 11, and 14 after injection of Gp-MBP/CFA or
MBP-69–89 peptide/CFA suppressed the induction of clinical (Fig. 6 and Table I) and histologic (not shown) signs of
EAE. Injection of as little as 30 µg of the
ß11/MBP-69–89 complex following the same
time course was also effective, completely suppressing EAE in four of
six rats, with only mild signs in the other two animals. All of the
control animals that were untreated, that received 2 µg MBP-69–89
peptide alone (the dose of free peptide contained in 30 µg of the
complex), or that received 300 µg of the empty
ß11 construct developed a comparable
degree of paralytic EAE (Table I). Interestingly, injection of 300 µg
of a control ß11/CM-2 peptide complex
produced a mild (about 30%) suppression of EAE (Fig. 6 and Table I).
In parallel with the development of clinical signs, rats with EAE
showed a 15% loss in body weight (Fig. 6), whereas animals treated
with the ß11/MBP-69–89 complex showed no
significant loss of body weight throughout the course of the
experiment. In contrast to the epitope-dependent effect of
ß11/MBP-69–89 vs
ß11/CM-2 for inhibiting EAE induced by
MBP-69–89/CFA, there was no difference between these two constructs
(cumulative disease index = 2.8 ± 1.7 and 2.7 ± 1.5,
respectively) in treating rats developing EAE induced with
MBP-87–99/CFA, a secondary encephalitogenic determinant for Lewis rats
(35). These data indicate that the
ß11/MBP-69–89 complex did not exert an
epitope-dependent inhibition of clinical EAE induced by a different MBP
determinant.
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1/MBP-69–89 complex on the first day of
disease onset, with follow-up injections 48 and 96 h later. EAE in
the control rats progressed to complete hind limb paralysis, whereas no
progression of the disease occurred in any of the treated animals (Fig. 7). The mild course of EAE (mean
cumulative index, 3 ± 0.13) in the treated group was
significantly less than the severe course of EAE in the control group
(mean cumulative index, 11.2 ± 2.7, p = 0.013),
although the duration of disease (6 days) was the same in both groups.
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1/MBP-69–89 complex essentially
eliminated the infiltration of activated inflammatory cells into the
CNS. Mononuclear cells were isolated from the spinal cords of control
and protected animals at peak and recovery of clinical disease and
examined by FACS analysis. The total number of mononuclear cells
isolated from spinal cords of control animals at peak of clinical
disease (day 14) was 40-fold higher than from protected animals
evaluated at the same time point (Table II). Moreover, protected animals had 72%
fewer activated (OX40+) Vß8.2+ T cells in the
spinal cord when compared with control animals (Table II).
CD4+ and CD8+ T cells, macrophages, and B cell
numbers were also significantly reduced in protected animals (not
shown). The number of mononuclear cells isolated after recovery from
EAE was reduced 4.5-fold in protected animals (0.64 x
105 cells/spinal cord) compared with control animals
(2.9 x 105 cells/spinal cord). Protected animals also
had 10-fold fewer activated (OX40+), Vß8.2+ T
cells in the spinal cord than control animals after recovery from
disease.
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In addition to inhibiting clinical EAE, treatment with
ß11/MBP-69–89 complex specifically
inhibited DTH response to MBP-69–89, as measured by changes in ear
thickness 24 h after i.d injection of Ag into the ear pinna. As is
shown in Fig. 8B, treatment of
MBP-69–79/CFA-immunized rats with the "empty" construct or
ß11 complexed with the CM-2 peptide had no
effect on the DTH response, whereas treatment with the
ß11/MBP-69–89 complex reduced the DTH
response to MBP-69–89 by 80%. In contrast, the DTH response to PPD,
evaluated on the other ear in the same rats, was not affected by any of
the constructs, including ß11/MBP-69–89
(Fig. 8A). This result strongly corroborates the Ag
specificity of the inhibition induced by the
ß11/MBP-69–89 construct in vivo.
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1/MBP-69–89 complex also specifically
inhibited proliferation responses of LN T cells cultured ex vivo. As is
shown in Fig. 9, LN cells from
MBP-69–89/CFA-immunized rats treated with
ß11/MBP-69–89 were inhibited two- to
fourfold in response to MBP or the MBP-69–89 peptide compared with
control rats. In contrast, response to PPD (stimulated by the CFA
injection) was not affected by treatment with
ß11/MBP-69–89.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 domains of MHC class
II. These molecules lack the 2 domain, the
ß2 domain known to bind to CD4, and transmembrane and
intracytoplasmic sequences. The reduced size and complexity of the
ß11 construct gave us the ability to
express and purify the molecules from bacterial inclusion bodies in
high yield (15–30 mg/L cell culture). The
ß11 molecules refolded in a manner that
allowed binding of allele-specific peptide epitopes and had excellent
solubility in aqueous buffers. The specificity of the
ß11/peptide complexes for the TCR could be
visualized directly by FACS, with the specificity of binding determined
by the peptide Ag. The ß11/69–89 complex
exerted powerful and selective inhibitory effects on T cell activation
in vitro and in vivo. Because of its simplicity, biochemical stability,
biologic properties, and structural similarity with human class II
homologues, the ß11 construct represents a
template for producing a novel class of TCR ligands. Moreover,
development of this construct separates for the first time the
peptide-binding ß11 domains from the
platform ß22 domains that may provide
additional signaling through CD4 or other cell surface molecules.
Direct binding studies using the A1 hybridoma specific for MBP-72–89
showed distinct staining with
ß11/MBP-69–89, with a 40- to 80-fold
increase in MFI over background or cells stained with either
ß11/CM-2 or the "empty"
ß11 construct (Fig. 4
). In a reciprocal
manner, binding studies using a CM-2 specific T cell line showed strong
staining with ß11/CM-2 but background
staining with ß11/MBP-69–89 or the
"empty" construct. Thus, bound epitope directed specific
interaction of the ß11/peptide complexes
with TCR. Identification of Ag-specific T cells has been possible in a
few systems, using labeled anti-idiotypic TCR Abs as specific
markers (60, 61, 62, 63), but the general approach of staining specific T cells
with their ligand has failed because soluble peptide-MHC complexes have
an inherently fast dissociation rate from the TCR (64, 65, 66). Multimeric
peptide-MHC complexes containing four-domain soluble MHC molecules have
been used to stain Ag-specific T lymphocytes (67), with the ability to
bind more than one TCR on a single T cell presumably giving the
multimeric molecules a correspondingly slower dissociation rate.
Staining with ß11/peptide complexes, while
specific, did take an incubation period of approximately 10 h to
saturate (data not shown). The distinct staining pattern of the A1
hybridoma with the ß11/MBP-69–89 complex
and the CM-2 line with ß11/CM-2 (Fig. 4),
coupled with the length of time it takes to achieve binding saturation,
suggests that this molecule might have a very slow off-rate once bound
to the TCR. An evaluation of binding constants is currently in
progress. These complexes, perhaps with some modifications, may be
unusually well suited to directly label Ag-specific T cells for
purposes of quantification and recovery.
The ß11/peptide complex was highly
specific in its ability to bind to and inhibit the function of T cells.
In vitro proliferation of MBP-specific T cells was inhibited >90%
with the ß11/MBP-69–89 complex (Fig. 5),
and in vivo there was a nearly complete peptide-specific inhibition of
clinical and histologic EAE, DTH response, and proliferation of
cultured LN cells. However, there was a nominal (but not statistically
significant) decrease in T cell proliferation with a much higher
concentration of the ß11 molecule loaded
with MBP-55–69 (Fig. 5) and a small but significant in vivo inhibition
of EAE using the control ß11/CM-2 complex
(Fig. 6; Table I). These data suggest a contribution to overall binding
affinity of the ß11/peptide complexes for
the TCR by the allele-specific residues on the solvent exposed ridges
of MHC II that may interact with any TCR that was selected
intrathymically with the same MHC. Thus, besides the antigenic
peptide-directed binding indicated by FACS analysis (Fig. 4), the
ß11 construct loaded with any antigenic
peptide might be expected to bind weakly to any RT1.B-restricted T
cell, possibly with some associated inhibitory activity. In a
concurrent study, we observed a similar peptide-independent inhibition
of Th1 but not Th2 cell activation by ethyl
carbodiimide-cross-linked APC (75), indicating that MHC loaded
with any compatible peptide may deliver a degree of negative signaling
to effector Th1 cells.
The most profound biologic activity demonstrated for
ß11/MBP-69–89 was its ability to almost
totally ablate the encephalitogenic capacity of MBP-69–89 specific T
cells in vivo. Injection of this complex after initiation of EAE nearly
completely suppressed clinical and histologic signs of EAE (Fig. 6, Table I), apparently by directly inhibiting the systemic activation of
MBP-69–89-specific T cells (Fig. 8) and preventing recruitment of
inflammatory cells into the CNS (Table II). Moreover, injection of
ß11/MBP-69–89 after onset of clinical
signs arrested disease progression (Fig. 7), demonstrating the
therapeutic potential of this molecular construct. On the other hand,
the ß11/MBP-69–89 complex did not exert
peptide-dependent effects on EAE induced with MBP-87–99 or on DTH or
proliferation responses to PPD.
Mechanistically, the powerful and selective inhibitory effects of the
ß11/MBP-69–89 complex on T cell
activation appeared to be reversible with IL-2 (Fig. 5). This stands in
sharp contrast to the mechanism of Fas-mediated cell death (apoptosis)
that is known to be induced by pretreating T cells with four-domain MHC
class II/peptide complexes before TCR stimulation (68), cross-linking
CD3 with Abs before TCR stimulation, or cross-linking CD4 with Abs
before binding the HIV glycoprotein (gp) 120 (69). The unique feature
that distinguishes the ß11 construct from
its progenitor class II molecule is the deletion of the
ß2 and 2 domains. The ß2
domain of MHC class II contains a solvent-exposed loop necessary for
binding CD4 and for eliciting CD4 coreceptor activity (12, 13, 14, 70) and
an 2 domain that allows ordered oligomerization in T
cell activation (13, 71).
CD4 is a membrane glycoprotein on T lymphocytes that binds to MHC class
II and TCR, promoting the localization of the scr family tyrosine
kinase p56lck into the receptor complex (72). The novel
class II-derived ß11 molecule described
here (Fig. 2) remained stable in the absence of the 2
and ß2 Ig-fold domains (Fig. 3 and 4) and clearly still
interacted with the TCR in an Ag-directed manner (Fig. 4). While it has
been clearly demonstrated that there are zones of molecular contact
with CD4 located on the ß2-domain of class II (12, 13, 14, 15, 16, 70), the data does not exclude a priori the existence of other sites of
CD4 interaction on class II molecules. Early work showed that chimeric
class I/class II MHC molecules containing only the class II
ß1 domain could interact with T cells whose function
could be blocked by anti-CD4 Ab (50). Whether this indicated the
existence of a direct interaction between CD4 and the ß1
domain is still unknown.
Thus, a plausible explanation for the mechanistic differences observed
with ß11/peptide treatment is that the
ß11/MBP-69–89 complex may not bind CD4,
and recruitment of the CD4 coreceptor into the TCR signaling complex
may be critical for Fas-mediated signaling, which, in the absence of
costimulatory inputs, leads to cell death. Experiments are in progress
to explore the contribution of ß11/peptide
complexes in combination with coreceptors and costimulatory molecules
in the ultimate development of anergy (8, 9), activation-induced cell
death/apoptosis (68, 69) and Ag-specific activation.
From a drug engineering and design perspective this prototypic molecule
represents a major breakthrough. The demonstrated biologic efficacy of
the ß11/MBP-69–89 complex in EAE raises
the possibility of using this construct as a template for engineering
human homologues for treatment of autoimmune diseases such as MS that
likely involves inflammatory T cells directed at CNS proteins. One
candidate molecule would be HLA-DR2/MBP-84–102, which includes both
the disease-associated class II allele (73) and a known immunodominant
epitope that has been reported to be recognized more frequently in MS
patients than controls (74). However, because of the complexity of T
cell response to multiple CNS proteins and their component epitopes, it
is likely that a more general therapy may require a mixture of several
MHC/Ag complexes. The precision of inhibition induced by the novel
ß11/MBP-69–89 complex reported herein
represents an important first step in the development of potent and
selective human therapeutic reagents. With this new class of reagent,
it may be possible to directly quantify the frequency and prevalence of
T cells specific for suspected target autoantigens, and then to
selectively eliminate them in affected patients. Through this process
of detection and therapy, it may then be possible for the first time to
firmly establish the pathogenic contribution of each suspected T cell
specificity.
|
Acknowledgments |
|---|
- and
ß-chains. We also acknowledge the expert technical assistance of Dr.
Keith Wegmann, Dr. Botond Siklodi, and Jeff Stevens. Finally, we thank
Eva Niehaus for assistance in preparing the manuscript. |
Footnotes |
|---|
2 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomylitis; CNS, central nervous system; MBP, myelin basic protein; PE, phycoerythrin;; DTH, delayed-type hypersensitivity; PPD, purified protein derivative; MFI, mean fluorescence intensity; LN, lymph node.
Received for publication March 16, 1998. Accepted for publication August 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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