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,§
Departments of
*
Neurology,
Biochemistry and Molecular Biology, and
Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, OR 97201; and
§
Neuroimmunology Research, Veterans Affairs Medical Center, Portland, OR 97201
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1
domains loaded with free antigenic peptides with potent inhibitory
activity on encephalitogenic T cells. We have now produced single-chain
constructs in which the peptide Ag is genetically encoded within the
same exon as the linked ß1 and 1 domains, overcoming the problem
of displacement of peptide Ag from the peptide binding cleft. We here
describe clinical effects of recombinant TCR ligands (RTLs) comprised
of the rat RT1.B ß11 domains covalently linked to the 72–89
peptide of guinea pig myelin basic protein (RTL-201), to the
corresponding 72–89 peptide from rat myelin basic protein (RTL-200),
or to cardiac myosin peptide CM-2 (RTL-203). Only RTL-201 possessed the
ability to prevent and treat active or passive experimental autoimmune
encephalomyelitis. Amelioration of experimental autoimmune
encephalomyelitis was associated with a selective inhibition of
proliferation response and cytokine production by Ag-stimulated lymph
node T cells and a drastic reduction in the number of encephalitogenic
and recruited inflammatory cells infiltrating the CNS. The exquisitely
selective inhibition could be observed between molecules that differ by
a single methyl group (the single amino acid residue difference between
RTL-200 (threonine) and RTL-201 (serine) at position 80 of the myelin
basic protein peptide). These novel RTLs provide a platform for
developing potent and selective human diagnostic and therapeutic agents
for treatment of autoimmune disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of CD4+ T cells in vivo 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
(signal 2). Ligation of the TCR in the absence of costimulatory signals
disrupts normal T cell activation, inducing a range of responses from
anergy to apoptosis (4, 5, 6). Thus, a direct approach toward
Ag-driven immunosuppression is to present the complete TCR ligand, Ag
in the context of MHC, in the absence of costimulatory signals that are
normally provided by specialized APCs. Toward the long-term goal of
targeted Ag-driven immunosuppression of pathogenic T cells, we have
developed a family of novel recombinant TCR ligands (RTLs) encoding
portions of the ß1 and 1 domains of MHC class II
molecules.4 In a
previous study, we demonstrated that RTLs loaded with soluble peptide
Ag had potent inhibitory activity on encephalitogenic T cells
(7). Recently, we described the biochemical
characterization of single-chain constructs in which the peptide Ag was
genetically encoded within the same exon, a design that favored
specific loading and high occupancy of peptide Ag in the MHC binding
cleft, and the ability to alter the encoded Ag of interest using
standard molecular biology techniques (8). We developed
our initial RTL constructs for testing in experimental autoimmune
encephalomyelitis (EAE), a paralytic, inflammatory, and sometimes
demyelinating disease mediated by CD4+ T cells
specific for CNS myelin components, including myelin basic protein
(MBP). EAE shares a number of immunological similarities with the human
demyelinating disease MS (9) and has been a useful model
for preclinical testing of therapies for the human illness
(10, 11, 12, 13, 14, 15, 16). In Lewis (LEW) rats, the dominant
encephalitogenic determinant resides in the 72–89 peptide of guinea
pig MBP (Gp-MBP-72–89) (17), and active immunization with
this peptide in CFA can induce a severe paralytic episode starting on
day 10–11 and lasting 5–7 days with associated formation of
inflammatory perivascular lesions within the CNS (18).
Interestingly, the corresponding 72–89 peptide of rat MBP
(Rt-MBP-72–89) that differs by a single conservative residue (T
instead of S at position 80) has greatly reduced encephalitogenic and
tolerogenic activity in LEW rats. We produced RT1.B-derived RTLs
covalently linked to Gp-MBP-72–89, Rt-MBP-72–89, or CM-2 peptides and
investigated the regulatory effects of these novel constructs on
actively or passively induced EAE. We here demonstrate that RTL-201
(ß11-Gp-MBP-72–89), but not RTL-200 (ß11-Rt-MBP-72–89) nor
RTL-203 (ß11-CM-2), could both suppress and treat clinical signs
of EAE through a mechanism that inhibited T cell activation and
resulted in a striking reduction of CNS-infiltrating cells. These
results illustrate the ability of RTL constructs (with covalently
coupled Ag) to inhibit clinical and histological EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods for cloning, expression, purification, and
biochemical analysis of these molecules have been described previously
(7, 8). In brief, genes encoding single-chain RTLs were
constructed by splicing the sequence encoding the amino terminus of the
rat RT1.B 1 domain to the sequence encoding the carboxyl terminus of
the ß1 domain. The 5' end of the gene contained an insertion
sequence that encoded a covalently coupled antigenic peptide and a
thrombin cleavage site embedded within a flexible linker
(19). Within this linker, a unique SpeI
restriction endonuclease site allowed production of RTLs with different
covalently coupled peptides by simply cutting the construct and
directionally cloning in the DNA fragment of interest. The organization
and logic of the RTL design has been previously described
(8). There is only a single amino acid difference between
RTL-200 (encoding rat-MBP-72–89) and RTL-201 (encoding Gp-MBP-72–89).
This difference is at position 80 (MBP numbering) within the antigenic
peptide derived from MBP (PQKSQR(S/T)QDENPVVHF). Residue 12 is a
threonine in RTL-200 and a serine in RTL-201. RTL-203 contains a
covalently coupled cardiac myosin-derived peptide, CM-2
(KLELQSALEEAEASLEH), that has been described previously
(8).
Synthetic peptides
Gp-MBP-69–89 peptide (GSLPQKSQRSQDENPVVHF) was prepared using solid-phase techniques (20). The MBP peptide is numbered according to the bovine MBP sequence (21, 22).
Animals
Female LEW rats (Harlan Sprague-Dawley, Indianapolis, IN), 8–12 wk of age, were used for clinical experiments in this study. The rats were housed under specific pathogen-free 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 (Gp-MBP) or 300 µg Gp-MBP-69–89 peptide in CFA supplemented with 100 µg Mycobacterium tuberculosis strain H37Ra (Difco, Detroit, MI). 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. For passive transfer of EAE, short-term T lymphocyte lines were selected with Gp-MBP-69–89 peptide from lymph node (LN) cells of naive rats or from rats immunized 10 days earlier with Gp-MBP-69–89/CFA. After 72 h stimulation, 10 x 106 blasting T cells were transferred i.v. into the tail vein on day 0. Details of this procedure have been described previously (23). 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 (CDI) 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 (17). The cells were stained with fluorochrome (FITC or PE)-conjugated Abs specific for rat CD4, CD8, CD11b, CD45ra, TCR BV8S2, and CD134 (PharMingen, San Diego, CA) 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.
Ag-specific proliferation assays
T cell recovered from the LN of control and RTL-treated animals were suspended at 2 x 104 cells in 200 µl/well and cocultured with 1 x 106 irradiated APC plus Gp-MBP-69–89 Ag as described previously (23). 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 was assessed by liquid scintillation. Mean cpm ± SD were calculated from triplicate wells, and differences between groups determined by Student’s t test.
Cell lines and the A1 hybridoma
Short-term T lymphocyte lines were selected with MBP-69–89 and
MBP-87–99 peptide from lymph node cells of rats immunized 12 days
earlier with Gp-MBP/CFA. Details of this procedure have been described
previously (23). The rat BV8S2+
(RT1.B (I-A)-restricted) T cell hybridoma C14/BW12–12A1 (A1) used in
this study has been described previously (7, 24). Briefly,
the A1 hybridoma was created by fusing an encephalitogenic
LEW(RT1l) T cell clone specific for Gp-MBP-72–89
(25, 26) and strongly cross-reactive with rat-MBP-72–89
with a TCR (/ß)-negative thymoma, BW5147 (27). Wells
positive for cell growth were tested for IL-2 production after
stimulation with Ag in the presence of APCs (irradiated LEW rat
thymocytes) and then subcloned at limiting dilution. The A1 hybridoma
secretes IL-2 when stimulated in the presence of APCs with whole MBP or
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. Cells were stained with fluorochrome (FITC or PE)-conjugated Abs specific for rat CD4 and the BV8S2 TCR (OX-35 and R-78; PharMingen, San Diego, CA) for 1 h, after which the cells were washed three times with PBS containing 2% FBS and then analyzed by flow cytometry. Quadrants were defined using irrelevant isotype-matched control Abs. Staining media was PBS containing 2% FBS.
5- (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) staining of passively transferred cells
For tracking passively transferred cells, short-term T cell lines were stained with the fluorescent tracking dye CFSE (C-1157; Molecular Probes, Eugene, OR) at 0.5 µM concentration for 15 min at 37°C in RPMI 1640, washed two times with 10-fold excess RPMI 1640, and then injected into the animals at 1 x 107 cells/300 µl RPMI 1640. On days 2, 4, and 7, animals were sacrificed, organs harvested as described previously (8), and cells from each organ analyzed by FACS.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-Rt-MBP-72–89), RTL-201
(ß11-Gp-MBP-72–89), and RTL-203 (ß11-CM-2). RTL-200 and
RTL-201 differ at a single amino acid position (T (RTL-200) for S
(RTL-201) at position 80 of the MBP peptide). Suppression and treatment of EAE using recombinant TCR ligands
RTL constructs were evaluated for their ability to suppress
the induction of, as well as to treat existing signs of, actively
induced EAE in LEW rats. Intravenous injection of 300 µg of RTL-201
in saline on days 3, 7, 9, 11, and 14 after injection of Gp-MBP or
Gp-MBP-69–89 peptide/CFA suppressed the induction of clinical (Fig. 1
and Table I) and histological (not shown) signs of
EAE. All of the control animals that were untreated, that received 300
µg of RTL-200, or that received 300 µg of RTL-203 developed
paralytic EAE (Table I). All of the control animals that received 20
µg Gp-MBP-69–89 peptide alone (the dose of free peptide contained in
the 300 µg dose assuming complete cleavage of the peptide from
RTL-201 in vivo) developed paralytic EAE (Table I), effectively ruling
out the possibility that free peptide cleaved from RTL-201 accounted
for protection. RTL-200 and RTL-203 produced a mild (about 25%)
suppression of EAE (Fig. 1 and Table I), similar to that previously
reported using noncovalent complexes of "empty" RTLs loaded with
peptide (7). In parallel with the development of clinical
signs, untreated rats with EAE showed a 15% loss in body weight (not
shown), whereas animals treated with RTL-201 showed no significant loss
of body weight throughout the course of the experiment.
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and Table II). The mild course of EAE (mean
cumulative index (MCI) = 3.6 ± 0.7) in the RTL-201-treated
group was significantly less than the severe course of EAE in the
control group (MCI = 15.8 ± 0.52), the RTL-200-treated group
(MCI = 13.6 ± 0.77), or the RTL-203-treated group (MCI
= 14.4 ± 0.6), although the duration of disease (7 ± 1
days) was the same in both groups (Table II).
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). Mononuclear cells were isolated
from the spinal cords of control and protected animals at recovery of
clinical disease and examined by FACS analysis. The number of
mononuclear cells isolated after recovery from EAE was reduced 5-fold
in RTL-201-protected animals (0.24 x 106
cells/spinal cord) compared with control animals (1.10 x
106 cells/spinal cord) or animals protected with
RTL-203 (1.17 x 106 cells/spinal cord) and
4-fold lower than animals protected with RTL-200 (0.88 x
106 cells/spinal cord). RTL-201-protected animals
also had 10-fold fewer activated (OX40+) T cells
in the spinal cord than control animals after recovery from disease and
8-fold fewer activated T cells than seen with animals protected with
RTL-200 and RTL-203 (Table III).
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The effect of RTL-200, RTL-201, and RTL-203 on passively
transferred disease was evaluated in recipient rats after i.v. transfer
of 10 x 106 blasting Gp-MBP-69–89-specific
T cells (23). Intravenous injection of 300 µg RTL-201
(encoding Gp-MBP-72–89) in saline on days 1, 3, and 5 after passive
transfer of encephalitogenic T cells completely blocked the induction
of clinical (Table IV) and histological
(not shown) signs of EAE. In contrast, 19 of 20 of the control animals
that were untreated, 5 of 6 animals that received 300 µg of RTL-200
(encoding Rt-MBP-72–89), and 3 of 3 animals that received 300 µg of
RTL-203 (encoding Rt-CM-2) developed paralytic EAE (Table IV). RTL-200
produced a marked but statistically insignificant decrease in the CDI
of EAE (Table IV). The specificity of the effect of RTL-201 on
passively transferred disease was further evaluated after i.v. transfer
of 15 x 106 activated
Gp-MBP-87–99-specific T cells to naive recipient rats. Intravenous
injection of 300 µg RTL-201 (encoding Gp-MBP-72–89) in saline on
days 1, 3, and 5 after passive transfer of encephalitogenic Gp-MBP-87-
99-specific T cells had no effect on the induction of clinical (Table IV) or histological (not shown) signs of EAE. Similarly, all five
untreated rats developed paralytic EAE (Table IV).
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Consistent with inhibition of EAE in vivo, treatment of
animals with RTL-201 also specifically inhibited proliferation
responses of T cells cultured ex vivo. Draining LN (DLN) cells were
recovered from both treated and control animals at the peak of actively
induced EAE and stimulated in vitro with Gp-MBP-72–89 (50 µg/ml) or
whole Gp-MBP (10 µg/ml). The proliferative response was measured
72 h later using a standard [3H]thymidine
incorporation assay. T cells from RTL-201-treated animals showed an
3-fold decrease in their proliferative response to Gp-MBP-72–89 or
whole Gp-MBP compared with T cells from untreated animals (Fig. 3). T cells from RTL-200- and
RTL-203-treated animals showed a mild (about 25%) decrease in
proliferative response to Gp-MBP-72–89 or whole Gp-MBP (Fig. 3),
consistent with the effect of these molecules on suppression of EAE in
vivo (Fig. 1
and Table I).
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Passive transfer experiments were repeated after labeling
activated Gp-MBP-72–89-specific T cells with the fluorescent dye CFSE.
Intravenous injection of 300 µg RTL-201 (encoding Gp-MBP-72–89) in
saline inhibited infiltration of donor-derived
CD4+ T cells (CFSE+) into
the CNS (Fig. 4, upper right quadrants;
Table V). In passively transferred EAE,
onset of disease typically occurred between days 3 and 5. Two days
after passive transfer (before disease induction), there was a
>10-fold decrease in the number of labeled CD4+
T cells in the CNS of RTL-201 (encoding Gp-MBP-72–89)-treated animals
vs RTL-203-treated or untreated controls (40 vs 450–500 cells/spinal
cord) (Table V), and at onset of disease (Day 4) there was a 300-fold
difference (200 vs 45–60,000 cells/spinal cord) (Table V).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 domains derived from MHC class II
RT1.B possessed a potent and exquisitely selective ability to suppress
and treat active and passive EAE mediated by T cells specific for this
peptide. We have previously demonstrated that "empty" RTLs loaded
with Gp-MBP-72–89 peptide suppressed and treated actively induced EAE
(7). The major limitation when using these complexes was
controlling the specific loading of the empty RTLs and maintenance of
this complex in vivo. To overcome this limitation, we have added a
sequence encoding an amino-terminal linker and the antigenic peptide of
interest. These relatively small (200 aa residues) RTLs can be
produced in Escherichia coli in large quantities and
refolded from inclusion bodies, with a final yield of purified protein
between 15 and 30 mg/L of bacterial culture. The design of the
constructs allows for substitution of sequences encoding different
antigenic peptides using restriction enzyme digestion and ligation of
the constructs. Structural characterization using circular dichroism
demonstrated that these molecules retained the anti-parallel
ß-sheet platform and antiparallel -helices observed in the native
class II heterodimer, and the molecules exhibited a cooperative
two-state thermal folding-unfolding transition. The RTLs with the
covalently linked Ag-peptide showed increased stability to thermal
unfolding relative to "empty" RTLs (8).
The design of this second generation of constructs containing
covalently linked Ag favors equimolar loading of peptide Ag within the
binding cleft of the MHC class II ß11-derived RTL moiety, yet
allows ready exchange of the encoded Ag of interest using
straightforward molecular biology techniques. Of crucial importance to
the RTL concept, covalently bound Ag minimizes potential extraneous
biological effects of peptide that might disassociate from our
previously described "empty" RTLs (7). In experiments
described here, the ability of RTL-201 to protect and treat EAE could
not be accounted for by antigenic peptide released from the construct,
because an equivalent molar concentration of free Gp-MBP-72–89 peptide
had no inhibitory effect on EAE (Table I), nor did the construct alone
or in association with a different noncovalently bound peptide
(7). Thus, inhibition of EAE required the combination of
both MHC and peptide components of the RTL.
The Gp-MBP-72–89 epitope represents the dominant encephalitogenic determinant in the LEW rat, whereas the corresponding 72–89 peptide of rat MBP differs by a single conservative residue (T instead of S at position 80). However, this seemingly minor difference in sequence has profound immunological effects in LEW rats, with the Rt-MBP-72–89 peptide possessing about 10-fold less encephalitogenic activity on a molar basis (28, 29). Most T cells induced after immunization with Gp-MBP-72–89 are RT1.B restricted, but are only weakly stimulated with Rt-MBP-72–89 (30). Conversely, most T cells induced after immunization with Rt-MBP-72–89 are also RT1.B restricted, but can still be stimulated efficiently with Gp-MBP-72–89 (28). These findings suggest that TCR interactions are stronger with the Gp than with the rat epitope. This could occur if the hydroxyl side chain at position 80 of serine provided a dominant interaction with hydrogen accepting TCR residues of Gp-MBP-72–89-specific T cells; in Rt-MBP-72–89, the presence of the extra methyl group in threonine might limit the mobility and hence the binding of the hydroxyl group, thus impeding this potentially important interaction. Studies are now underway in our laboratory to directly examine the differences in Rt- and Gp-MBP-72–89 peptide binding to the RT1.B-derived RTLs.
The clinical effects of RTL-200 and RTL-201 on EAE contrasted
strikingly. RTL-201 (encoding Gp-MBP-72–89) possessed potent
suppressive and therapeutic activity for actively induced disease,
substantially reducing the proliferative response of DLN T cells (Fig. 3). Even more striking, RTL-201 completely suppressed passive EAE
induced after transfer of Gp-MBP-72–89-specific T cells (Table IV),
but had no effect on passive EAE induced with a different I-E
(RT1.D)-restricted T cell line specific for a distinct encephalitogenic
determinant, MBP-87–99 (Table IV). Finally, treatment with RTL-201
prevented infiltration of both CSFE-labeled donor T cells as well as
host recruited inflammatory cells into the CNS (Tables III and IV). In
contrast, RTL-200 (encoding Rt-MBP-72–89) and a second control,
RTL-203 (encoding rat CM-2), showed only a very mild suppression of
actively and passively induced disease (Fig. 1; Table I and IV), a
slight reduction of infiltrating cells into the CNS (Table III), and
only a mild decrease in the proliferative response of DLN T cells (Fig. 3). The remarkable decrease in the number of inflammatory cells that
penetrated the blood-brain barrier after RTL- 201 treatment may provide
important insights into the mechanism of action of these molecules in
vivo. We are currently exploring the possibility that RTL treatment
alters expression of cell-surface adhesion markers that are
up-regulated after Ag challenge and that are required for crossing the
blood-brain barrier.
Our results demonstrating selective inhibition of EAE with the Gp- but not Rt-MBP-72–89 RTL are in full agreement with a previous study (31) that showed that orally administered Gp-MBP-68–88 conferred resistance to an encephalitogenic challenge with Gp-MBP-68–88 or Rt-MBP-68–88. However, Rt-MBP-68–88 did not confer oral tolerance to either molecule. These observations suggest that some MHC/peptide interactions with TCR can both stimulate and tolerize Ag-specific T cells, whereas other interactions may stimulate but not tolerize. Perhaps the simplest explanation for this disparity in tolerizing activity might be a difference in ligand binding avidity to the TCR, with tolerance induction occurring only in high-avidity T cells. In accordance with the differential avidity model of self-tolerance (32), we would speculate that T cells with a high avidity for RT1.B/Rt-MBP-72–89 have been deleted in LEW rats by negative selection in the thymus, leaving TCRs with moderate to low avidity for native Rt-MBP-72–89 but high avidity for Gp-MBP-72–89. However, an important implication is that the moderate- to low-avidity TCR repertoire that remains after thymic selection to self-Ags may not be amenable to tolerization with self-sequences but would require altered peptide ligands with higher avidity for the TCR. Although differences in avidity for the Gp- and Rt-MBP-72–89 epitopes have not been assessed, the development of specifically labeled RTLs may now make such studies feasible. Our data demonstrated the clinical utility of RTLs with covalently coupled Ag in regulating pathogenic T cells in EAE. These studies provide a template for engineering human homologues that may be useful in treatment of autoimmune diseases such as MS that likely involves inflammatory T cells directed at CNS proteins.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gregory G. Burrows, Department of Neurology L-219, Oregon Health Sciences University, 3181 Southwest Sam Jackson Park Road, Portland, OR 97201.
3 Abbreviations used in this paper: MS, multiple sclerosis; RTL, recombinant TCR ligand; MBP, myelin basic protein; EAE, experimental autoimmune encephalomyelitis; LN, lymph node; LEW, Lewis; CDI, cumulative disease index; MCI, mean cumulative index; CFSE, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester; DLN, draining LN.
4 Patent No. 09/153,586; Recombinant MHC Molecules Useful for the Detection and Purification of Antigen-Specific T-Cells, filed September 15, 1998.
Received for publication April 13, 1999. Accepted for publication April 3, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 integrin. Nature 356:63.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
5) were injected with 25 µg of
Gp-MBP/CFA/100 µg MTB to induce active EAE. On days 3, 7, 9, 11, and
14 after disease induction, rats were given 300 µg RTL-200 (encoding
rat-MBP-72–89), 300 µg RTL-201 (encoding Gp-MBP-72–89), 300 µg
RTL-203 (encoding rat-CM-2), or were left untreated, as indicated. A
single representative experiment is shown; the experiment was performed
three times. Values indicate mean clinical score ± SEM on each
day of clinical disease.
0.05;
**, p 