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,
,§
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Neuroimmunology Research R&D-31, Portland Veterans Affairs Medical Center, and Departments of
Neurology,
Biochemistry and Molecular Biology, and
§
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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secretion, and
encephalitogenic activity of MBP-specific T cells. These results
suggest that immune regulation occurs through a nondeletional
cytokine-driven suppressive mechanism. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of TCR transgenic (Tg) mice specific for MBP provides a
system uniquely useful for evaluating the mechanisms of disease
development and regulation in EAE. Goverman et al. (14) produced mice
that were Tg for either or both of the TCR 
(AV2)- and ß
(BV8S2)-chains of a T cell clone specific for
MBP-NAc1–11 peptide, which is encephalitogenic for
mice expressing the H-2u haplotype (15). Although
spontaneous proliferation responses to MBP-NAc1–11 were
not reported in single Tg mice, unimmunized double Tg mice did have T
cell responses to this peptide, and some mice, mostly juveniles, kept
in nonsterile conditions developed spontaneous clinical EAE (14, 16).
The rate of spontaneous EAE could be dramatically enhanced from 
15
to 100% by crossing TCRß Tg mice with RAG-1-deficient mice
that could not rearrange endogenous TCR genes (17). These results
suggested that endogenous rearrangement may provide a relatively small
pool of regulatory T cells, possibly including those involved in
network interactions, that appears somewhat later than the
encephalitogenic T cells and naturally prevents EAE. In double TCR Tg
mice, these regulatory T cells would be expected to rearrange either
one or both of their TCR chains and express only one or neither of the
TCRß transgenes.
In the current study, we addressed the question of whether immune
regulation directed at TCR determinants could be induced in single Tg
mice expressing a TCR ß-chain specific for MBP-NAc1–11
peptide (developed by Dr. Joan Goverman (14)). In these mice,
essentially all of the T cells express the Tg BV8S2 chain, paired with
naturally rearranged TCR -chains. T cells specific for
MBP-NAc1–11 are enriched and express AV2 or AV4
chains that are known to pair preferentially with BV8S2 (4, 5).
Conceivably, these mice could also produce a distinct population of TCR
peptide-specific T cells that express the Tg BV8S2 chain paired with a
different naturally rearranged TCR -chain. This would create a
situation in which a substantial portion of regulatory T cells would
express the same BV8S2 chain as the pathogenic T cell targeted for
regulation. Indeed, in a previous study in single Tg mice, sequence
analysis of T cells specific for the MBP-NAc1–11 peptide
demonstrated uniform expression of the transgene and preferential
expression of the AV2S3 gene (18). Moreover, a BV8S2-specific
regulatory T cell line derived from the same mice predominantly
expressed the BV8S2 transgene as well as the same AV2S3 gene as the
MBP-NAc1–11-specific T cells, differing only in the AV
CDR3 sequence. These data indicated the presence of multiple T cell
clonotypes that both expressed the BV8S2 transgene and recognized a
dominant BV8S2 epitope.
According to network theory, overexpression of the BV8S2 transgene would be expected to enhance the spontaneous recognition of BV8S2 determinants. In this genetically restricted model, it is intriguing to consider what kind of regulation would result when the regulatory T cells express the same BV gene as the pathogenic target T cells. Would the BV8S2 T cells self-regulate, and if not, what factors would contribute to a selective regulatory mechanism? In the current study, we have established that the BV8S2 transgene engenders an elevated response both to MBP-NAc1–11 peptide and to self or heterologous BV8S2 proteins. Responses to MBP-NAc1–11 or BV8S2 Ags were potentiated by immunization, leading either to a relapsing form of EAE or, in the case of the heterologous rat BV8S2 protein, to protection against EAE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tg mice bearing the functionally rearranged BV8S2 gene specific for MBP-NAc1–11 on the B10.PL background were kindly provided by Dr. Joan Goverman (University of Washington Seattle, WA). Male Tg mice were bred with B10.PL females and the offspring tested for expression of the Tg by FACS analysis of blood cells stained for BV8S2 as described previously (14). For some experiments, mice expressing the BV8S2 Tg were compared with littermates that did not express the Tg. The colony was housed and cared for at the Animal Resource Facility (Portland Veterans Affairs Medical Center) according to institutional guidelines.
Antigens
N-acetylated MBP-1–11 peptide (Ac-ASQKRPSQRSK) was synthesized using solid phase techniques and was purified by HPLC at the Beckman Institute, Stanford University (Stanford, CA). Mouse basic protein (Mo-BP) was extracted from mouse brains (Pel-Freeze Biologicals, Rogers, AK) and purified as previously described (19). Glutathione S-transferase (GST) and GST-BV8S2 proteins were expressed and purified as described previously (12). The GST-BV8S2 fusion protein contains the complete BV, BD, and BJ regions and the first 19 residues of the BC region from the TCR of an encephalitogenic rat T cell clone fused to the C-terminal end of GST. To control for the GST-BV8S2 protein, the GST protein was produced and purified using the same expression system. In previous experiments, neonatal injection of the GST protein in IFA did not alter the course of EAE induced with guinea pig MBP/CFA in rats (12), nor did GST affect the course of EAE induced with MBP-NAc1–11/CFA in BV8S2 Tg mice (see below). The GST protein was included as a control in all tissue culture experiments utilizing the GST-BV8S2 protein. The BV8S2-his6 protein contains the identical BV, BD, and BJ regions plus the first amino acid of the BC region followed by a C-terminal histidine tag. The mouse BV8S2 protein (JB9) consists of the mouse BV8S2 transgene product, including the BV, BD, and BJ regions, and the first 19 residues of the BC region. To control for the BV8S2-his6 proteins, the identical host harboring a plasmid lacking the coding sequence of the BV8S2 protein was used in the same expression and purification process to produce a fraction that did not contain detectable protein, but would contain contaminants. This fraction had no effect on the course of EAE or on lymphocyte proliferation responses in rats (our unpublished data). A truncated form of the rat AV2S3 protein (from an encephalitogenic T cell clone specific for MBP72–89 peptide) without the constant region was cloned and expressed in Escherichia coli as a control in tissue culture experiments (Buenafe et al., manuscript in preparation).
Induction of active EAE and protection with BV8S2 protein
EAE was induced in Tg male mice by injecting 400 µg of MBP-NAc1–11/CFA containing 200 µg of Mycobacterium tuberculosis s.c. over four sites on the flank. For protection experiments (six total), mice were injected with 12.5 µg of recombinant rat or mouse BV8S2 proteins/IFA (experimental) or saline/IFA (sham control) i.p. on days -7 and +3 relative to injection of the MBP-NAc1–11, according to the protocol developed by Kumar and Sercarz (9). In three experiments, mice were boosted weekly with 12.5 µg BV8S2 protein or saline given s.c. Mice were assessed daily for clinical signs of EAE according to the following scale: 0 = no signs; 1 = limp tail; 2 = moderate hind limb weakness (waddling gait); 3 = moderately severe hind limb weakness; 4 = severe hind limb weakness; 5 = paraplegia; 6 = quadriplegia, moribund condition.
The primary episode was defined as the period beginning on the first day of clinical signs and ending on the second day after the daily clinical scores returned to the lowest score before a second increase in clinical severity (relapse) or no further change in daily scores (chronic EAE). The cumulative disease index (CDI) was determined for each mouse for the primary episode and relapse by summing the daily clinical scores during each episode, and the mean CDI ± SEM was calculated for the control and experimental groups. For passive EAE, the CDI was determined for the first 10 days of clinical EAE (10-day CDI). The mean clinical score (MCS) was calculated for each mouse by dividing the CDI by the duration (days) of the primary episode or relapse, and the mean ± SEM was calculated for the control and experimental groups, or for the clinically affected mice only (see tables). Representative mice from control and protected groups were sacrificed for histologic analysis of spinal cords. Spinal cords were removed, fixed in formalin, and embedded in paraffin, and sections from throughout the cord were stained with hematoxylin and eosin.
Isolation of lymphocytes
Spinal cord cells were isolated as previously described (20). Briefly, spinal cords were isolated by insufflation, washed in RPMI 1640 to remove contaminating blood cells, then passed through a wire mesh screen to obtain a single-cell suspension. This suspension was then washed and resuspended in 80% isotonic Percoll. T cells were isolated at the 40/80% interface of a Percoll step gradient. Lymph nodes (LN) and spleens were removed surgically, and single cell suspensions were prepared as described previously (21).
T cell lines
MBP-NAc1–11-specific T cell lines were derived from the draining LN of BV8S2 Tg mice immunized with 400 µg NAc1–11 peptide/CFA over four sites on the flank (coculture experiments) or with 200 µg GST-BV8S2/CFA (cytokine experiments) as described (12). Both immunizations contained 200 µg M. tuberculosis strain H37Ra (Difco Laboratories, Detroit, MI). LN cells were isolated 14 to 16 days after immunization and cultured for 3 days in the presence of 50 µg/ml Ag at 5 x 106 LN cells/ml in stimulation medium containing 1% FBS, 0.05 mM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics in RPMI. T cell lines were then expanded in growth medium (stimulation medium supplemented with 10% FBS and 20 U/ml human rIL-2) for 7 to 10 days, followed by stimulation of 5 x 105 T cells/ml with Ag plus 107 irradiated Tg thymocytes/ml as APCs.
Proliferation assay
Proliferative responses of the T cell lines were evaluated in 96-well microtiter plates by incubating 4 x 105 LN or spleen cells or 2 x 104 T lymphocyte line cells/well with 106 irradiated thymocytes/well plus Ag at varying concentrations ranging from 1 to 100 µg/well. Cultures were incubated for 72 h at 37°C and 7% CO2, the last 18 h in the presence of 0.5 µCi [3H]thymidine. Cells were harvested onto glass fiber filters, and thymidine uptake was determined by liquid scintillation. Mean cpm ± SEM was calculated from triplicate wells. The stimulation index (SI) was obtained by dividing cpm from Ag-stimulated wells by cpm from wells with no Ag. SI in cultures stimulated with GST alone was subtracted from the SI induced with GST-BV8S2 protein.
Phenotyping
Phenotyping of T cells was performed on a FACScan (Becton Dickinson, Mountain View, CA) as previously described (22). Spinal cord T cells were double stained with soluble mouse OX40 ligand/human Ig fusion protein (a gift from Dr. Andrew D. Weinberg, Chiles Research Center, Providence Hospital, Portland, OR) for 15 min at room temperature, washed, and incubated with a FITC-conjugated anti-human Ig (mouse and rat preabsorbed, PharMingen, San Diego, CA) for 15 min at room temperature. OX40 ligand-stained cells were subsequently stained with anti-mouse CD4-phycoerythrin (PharMingen). Two-color immunofluorescence analysis was performed on a FACScan (Becton Dickinson). Quadrants were drawn based on background staining with control Abs.
Measurement of cytokine secretion
LN and spleen cells were suspended at 4 x 106
cells/ml in stimulation medium with and without specific Ags. Ag
specific T cell lines were suspended at 0.5 x 106
cells/ml and cocultured with 5 x 106 irradiated
spleen cells as APC in stimulation medium as above. Cell culture
supernatants were recovered at 72 h and frozen at -70°C until
needed for the cytokine assay. Measurement of cytokines was performed
by ELISA developed in our laboratory using cytokine specific capture
and detection Abs (PharMingen). Capture Abs for IFN-,
IL-5, and IL-10 were diluted to 2 µg/ml in bicarbonate
coating buffer (0.1 M NaHCO3, pH 8.2). Standard curves for
each assay were generated using recombinant mouse cytokines
(PharMingen), and the concentration of cytokines in the cell
supernatants was determined by interpolation from the appropriate
standard curve.
Assessment of Ab responses
Ab reactivity to Mo-BP, MBP-NAc1–11 peptide, and rat and mouse recombinant BV8S2 proteins was determined by indirect ELISA as described previously (22). Briefly, mouse antisera from BV8S2-protected and control Tg mice with EAE were incubated in Ag-coated wells, and bound Ab was detected spectrophotometrically with peroxidase-labeled rabbit anti-mouse Ab and o-phenylenediamine as a substrate. Differences between groups were determined using Student’s t test.
Coculture of MBP-NAc1–11- and rat (Rt)-BV8S2-specific T cell lines
To study the effects of coculture on proliferation of
encephalitogenic T cells, 1 or 2 x 104
MBP-NAc1–11-specific T cells were stimulated in the
presence or absence of 2 x 104 Rt-BV8S2-specific T
cells with NAc1–11 (0.5 µg/ml) or Mo-BP (5 µg/ml) plus
106 irradiated Tg thymocytes as APC in 96-well microtiter
plates. Likewise, 2 x 104 BV8S2-specific T cells were
stimulated with NAc1–11 plus APC. Cultures were incubated
for 72 h at 37°C with 7% CO2, and proliferation was
measured by thymidine uptake as described above. To determine the
effects of coculture on encephalitogenicity, 1.5 x
106 MBP-NAc1–11-specific T cells were cultured
in the bottom compartment of a transwell system with
NAc1–11 peptide (10 µg/ml) and 3 x 107
irradiated Tg splenocytes as APC in multiple wells; the top well, which
was separated from the bottom well by a semipermeable membrane,
contained 3 x 106 Rt-BV8S2 T cells plus Rt-BV8S2 (10
µg/ml) plus 3 x 107 APC. Wells were cultured in
parallel containing each cell type alone plus Ag plus APC. The cultures
were incubated for 72 h, 2 x 107
MBP-NAc1–11 T cells from control wells were transferred by
i.p. injection into three Tg and three parental non-Tg recipient mice,
and the same number of MBP-NAc1–11-specific T cells from
the bottom compartment of the cocultured transwells were transferred
into three Tg recipients. The recipient mice were boosted with
pertussigen at the same time (75 ng) and 48 h (200 ng) after
transfer of the T cells and were scored daily for signs of EAE as
described above. To evaluate the effects of coculture on IFN-
release, cell culture supernatants from cocultured and control wells
were recovered at 72 h, and measurement of IFN- was performed
by ELISA as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction of the BV8S2 Tg would be expected to limit the TCR BV repertoire and, depending on the choice of paired AV genes in the CD4 compartment, enrich the pool of circulating T cells specific for MBP-NAc1–11. In this study, we sought to determine whether there was an increased spontaneous recognition of BV8S2 determinants as a result of the overexpression of BV8S2 in the Tg mice. In a previous study in BV8S2 Tg mice (18), evaluation of the TCR BV repertoire revealed that >90% of T cells expressed BV8S2, in contrast to <20% BV8S2 T cells in non-Tg B10.PL littermates. A similar evaluation of the native TCR AV repertoire revealed no significant skewing in the BV8S2 Tg mice compared with the non-Tg B10.PL littermates, indicating no general preferential pairing of AV genes with the overexpressed BV8S2 gene.
To evaluate the effects of the BV8S2 Tg specific for
MBP-NAc1–11 on spontaneous T cell responses, LN and spleen
cells from naive BV8S2 Tg mice and non-Tg B10.PL littermates were
assessed for Ag-driven proliferation and cytokine release upon
stimulation with MBP-NAc1–11 peptide. As shown in Figure 1
, there were highly significant T cell
proliferation responses to the encephalitogenic
MBP-NAc1–11 peptide in the LN and spleens from naive Tg
mice but not from naive B10.PL littermates. As we have shown in a
previous study, the BV8S2 Tg mice favored positive selection of
MBP-NAc1–11-specific T cells expressing the BV8S2 Tg in
combination with the AV2S3 or AV4 genes (18). Despite the increased
natural recognition of MBP-NAc1–11, spontaneous EAE was
never observed in the BV8S2 single Tg mice.
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. Indeed,
MBP-NAc1–11-specific T cells from naive Tg mice produced
low to absent levels (<1 ng/ml) of both Th1 and Th2 cytokines in LN
and spleens (Table I), despite strong
proliferation responses (Fig. 1
).
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A). Lower but
comparable responses to both BV8S2 proteins were also observed in
spleens from both Tg and non-Tg mice (Fig. 1B). These data
indicate a background recognition of BV8S2 determinants in B10.PL mice
that is strongly and spontaneously enhanced in the LN of BV8S2 Tg mice.
However, unlike T cells specific for MBP-NAc1–11 peptide,
which produced very low levels of cytokines, spleen cells from naive Tg
mice secreted significantly elevated levels of both IFN- and IL-10
upon stimulation with either Mo- or Rt-BV8S2 proteins (Table I). Immunization of BV8S2 Tg mice with MBP-NAc1–11 in CFA induced a Th1 response and EAE
To induce clinical signs of EAE, BV8S2 Tg mice were immunized with
MBP-NAc1–11 peptide in CFA. This immunization, in the
absence of pertussigen, induced an initial severe episode of clinical
EAE in >80% of mice, followed by less severe relapses or chronic
disease in a majority of mice (saline/IFA sham-treated control groups,
Table II and Fig. 2). The proliferation response to
MBP-NAc1–11 of LN cells from immunized Tg mice was similar
to or less than that from naive unimmunized Tg mice. However, the
cytokine profile of MBP-NAc1–11-stimulated LN cells and T
cell lines from the immunized mice was clearly Th1-like, with vastly
elevated levels of IFN- (Table I). Interestingly, LN cells and T
cell lines from mice immunized with MBP-NAc1–11 peptide in
CFA also had strongly elevated (>10-fold) naturally induced
IFN--dominated Th1 responses to Mo- and Rt-BV8S2 proteins in vitro
(Table I), even though neither of these BV8S2 proteins was included in
the injection mixture. This result clearly demonstrated that expansion
and maturation of TCR-reactive T cells occurred as a consequence of the
activation of MBP-NAc1–11-specific T cells.
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The naturally induced response to BV8S2 determinants in BV8S2 Tg
mice was clearly unable to prevent induction of EAE upon immunization
with MBP-NAc1–11/CFA. To boost the response to Mo-BV8S2
determinants, 10- to 12-wk-old Tg mice were vaccinated with
heterologous Rt-BV8S2 proteins that had induced strong proliferation
and cytokine responses in naive Tg spleen cell cultures and that
boosted T cell-mediated IFN- and IL-10 cytokine responses when
injected with IFA (Table I). Mice vaccinated with Rt-BV8S2 proteins
(either the GST-BV8S2 or the BV8S2-his6 forms) in IFA on days -7 and
+3 relative to induction of EAE with MBP-NAc1–11/CFA had a
significantly lower incidence of EAE (38 vs 81%, composite of three
separate experiments, p < 0.05), and as a group, a
significantly lower CDI and MCS than control mice treated with
saline/IFA (Table II and Fig. 2A). Mice treated with GST/IFA
developed EAE indistinguishable from saline/IFA controls (not shown).
Vaccinated mice that were not protected developed an intensity of
clinical EAE similar to that in control mice, although in most
vaccinated mice the CDI and MCS values were nominally less.
To sustain responses to BV8S2 proteins, mice in subsequent
experiments were vaccinated on days -7 and +3 as before and then
boosted weekly with Rt-BV8S2 protein, injected s.c. in saline (Fig. 2B). As shown in Table II, this boosting regime improved the
degree of resistance to the primary episode of EAE (11 vs 100%
incidence, composite of two separate experiments), with highly
significant differences in group disease severity scores. Moreover,
mice vaccinated and boosted with Rt-BV8S2 proteins had significantly
less severe relapses (Table II). Mice protected after vaccination with
BV8S2 proteins had essentially no inflammatory lesions in spinal cord
sections (Fig. 3), as well as a threefold
reduction of both total and activated (OX40+) inflammatory
cells in the CNS compared with saline/IFA-treated controls (Fig. 4). Two of nine vaccinated mice that
eventually developed mild EAE had intermediate levels of inflammatory
cells in the CNS (Fig. 4).
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and Figure 2C, the severity of EAE in
control mice was extreme (four of six mice died), and the treatment
effect, although nominally less (only one of six mice died, with lower
CDI and MCS scores), was not significant. Responses to MBP-NAc1–11 peptide and BV8S2 proteins in vaccinated mice
The most meaningful immunologic difference between
Rt-BV8S2-protected and control mice with EAE was a striking 19-fold
reduction in IFN- release by LN cells stimulated with the
MBP-NAc1–11 peptide (Table I). This 95% reduction in
cytokine production was not reflected by differences in the
proliferation response to MBP-NAc1–11, which was
essentially the same in protected and control LN cells (Fig. 5). Similarly, there was no difference in
Ab response to Mo-MBP or MBP-NAc1–11 peptide in the two
groups of mice (Fig. 6). However, unlike
LN cells or an MBP-NAc1–11-specific T cell line from the
control mice with EAE, which produced very high levels of IFN- (69.3
ng/ml) and low levels of Th2 cytokines (IL-5, 1.2 ng/ml; IL-10, 0.8
ng/ml; Table I), an MBP-NAc1–11-specific T cell line
selected from mice vaccinated with Rt-BV8S2 protein had low
proliferation response to the encephalitogenic peptide (1.2x), but was
highly skewed toward production of Th2 cytokines (IL-5, >32 ng/ml, and
IL-10, 22.5 ng/ml; Table I) instead of inflammatory cytokines (IFN-,
5.8 ng/ml; Table I) associated with induction of EAE.
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). The
BV8S2-reactive LN T cells from protected and control mice produced
similar high levels of IFN- (>4–11 ng/ml) with relatively low
levels of IL-10 (Table I). However, the most obvious difference in
response to BV8S2 proteins occurred in the spleen cells from protected
mice, which produced 5x higher levels (3.2 ng/ml) of IL-10 than spleen
cells from control mice (0.6 ng/ml), even though proliferation
responses were not different (not shown). In addition to elevated
splenic IL-10 responses to BV8S2, protected mice had higher Ab levels
to both Rt- and Mo-BV8S2 proteins than control mice with EAE (Fig. 6). MBP-NAc1–11 T cells are inhibited when cocultured with Rt-BV8S2-specific T cells or supernatants
To evaluate the direct regulatory influence of BV8S2-specific T
cells, an MBP-NAc1–11-specific T cell line was activated
with the MBP peptide and APC in the presence of Rt-BV8S2-specific T
cells, but without added Rt-BV8S2 protein. In these coculture
experiments, the proliferation response to the MBP-NAc1–11
peptide was significantly inhibited (>50%) in the presence of the
BV8S2-specific T cells (Fig. 7).
Moreover, MBP-NAc1–11-specific T cells incubated in
transwells exposed to soluble factors produced by activated
Rt-BV8S2-specific T cells had a similar reduction (40%) in secretion
of IFN- and a significantly reduced ability to transfer clinical EAE
to naive recipient mice (Fig. 7). These data clearly demonstrate the
inhibitory capacity of soluble factors produced by BV8S2-specific T
cells on the activation and encephalitogenic activity of
MBP-NAc1–11-specific T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains. Such TCR -chains, when paired with
the BV8S2 Tg chain specific for MBP-NAc1–11, could form
new specificities, including that directed at a dominant determinant
within the BV8S2 molecule itself (8). The positive identification of T
cells expressing BV8S2 that are also specific for BV8S2 in the Tg mice
does not preclude regulatory T cell populations that express other BV
or AV genes, as was implied by enhancement of spontaneous EAE in
TCRß double Tg mice lacking a functional RAG-1gene (17). However, our demonstration of BV8S2-specific T cells
that differed from MBP-NAc1–11-specific T cells by only
the AV CDR3 sequence indicated that the single TCR Tg mice had
considerable capacity to form productive combinations of novel AV genes
with the BV8S2 transgene. BV8S2-specific T cells expressing other BV
genes may have been rearranged and may have contributed to regulation,
but did not constitute more than a small minority of the BV8S2-reactive
population. However, formal proof of autoregulation will require highly
characterized T cell clones. In this study, we characterized the induction and function of both encephalitogenic and regulatory T cell specificities in BV8S2 Tg mice. The results demonstrate conclusively that overexpression of the BV8S2 transgene 1) engendered a natural pre-effector T cell population specific for MBP-NAc1–11 that acquired encephalitogenic capability only after immunization with this peptide in CFA; and 2) naturally induced a second population of T cells specific for BV8S2 determinants that upon activation could directly inhibit the activation and encephalitogenic activity of MBP-NAc1–11-specific T cells by soluble secreted factors. Vaccination with heterologous Rt-BV8S2 protein induced strong cross-reactivity to Mo-BV8S2 determinants and appeared to be more effective than Mo-BV8S2 in inducing protection against EAE. This result suggests that there is some degree of tolerance to self BV8S2 determinants, a finding consistent with that of Falcioni et al. (23).
Protection against EAE was associated with a striking decrease in
IFN- production by LN T cells specific for the encephalitogenic
MBP-NAc1–11 determinant, an increased production of IL-10
by BV8S2-reactive T cells, a decrease in CNS lesions and
infiltrating activated inflammatory cells, and an elevated Ab
response to BV8S2 proteins. Vaccination with BV8S2 protein
induced specific T cells that directly inhibited the proliferation
response, IFN- secretion, and encephalitogenic activity of
MBP-NAc1–11-reactive T cells in vitro and resulted in the
skewed production of Th2 cytokines by an
MBP-NAc1–11-specific T cell line. However, the
BV8S2-specific T cells were not self-regulated, even though they
predominantly expressed the BV8S2 transgene. Taken together, these
results suggest that activated regulatory T cells specific for BV8S2
proteins, through a nondeletional mechanism involving skewed production
of soluble cytokines, prevented pre-effector T cells specific for
MBP-NAc1–11 from developing into Th1 effector cells
capable of inducing EAE. Although anti-BV8S2 Abs were also present
and may have contributed to protection against EAE, selective
regulatory effects of these Abs on Th1 cells have not as yet been
demonstrated. However, there was no evidence of deletion of
BV8S2+ T cells, unlike the study of Haqqi et al. (24), in
which immunization of BUB mice with a BV10 peptide induced deletion of
most of the BV10+ T cells.
The expression of the rearranged BV8S2 transgene specific for
MBP-NAc1–11 clearly favored T cell recognition of this
encephalitogenic determinant in naive mice not previously exposed to
exogenous MBP. Of importance, however, the relatively strong 5-18X T
cell proliferation response to MBP-NAc1–11 in these mice
was accompanied by a low to absent release of all lymphokines tested,
strongly suggesting a lack of maturation to effector cells that might
otherwise cause clinical EAE. The functional lack of development of
encephalitogenic effector cells specific for MBP-NAc1–11
may well have been influenced by naturally induced T cells specific for
Mo-BV8S2 determinants. Upon stimulation with Mo-BV8S2 protein, naive
splenocytes produced significantly higher levels of both IFN- and
IL-10 than cells stimulated with MBP-NAc1–11. Moreover,
elevated levels of secreted IFN- and IL-10 were also induced in
naive splenic T cells stimulated with the heterologous Rt-BV8S2
protein.
Immunization of the Tg mice with MBP-NAc1–11 in CFA was a
powerful stimulus for activating Th1 effector cells that were quite
clearly capable of inducing EAE, even in the presence of the naturally
induced BV8S2-specific T cell population. The activation process of
MBP-NAc1–11-specific T cells produced a 150-fold increase
in IFN-, with a negligible increase in IL-10, compared with naive LN
cells (Table I). Surprisingly, MBP-NAc1–11-specific LN
cells from immunized Tg mice had an even lower proliferation index than
LN cells from naive Tg mice, indicating that immunization in CFA caused
T cell maturation rather than simply an expansion of these T cells.
Immunization with MBP-NAc1–11 in CFA also induced profound
changes in T cells specific for Mo- and Rt-BV8S2 proteins. Although
proliferation responses (Fig. 5) were even less than in unimmunized Tg
mice (Fig. 1), activation of LN cells from
MBP-NAc1–11/CFA-immunized mice with Rt- or Mo-BV8S2
proteins induced >10-fold increases of IFN- (Table I), due probably
to the strongly inflammatory environment imposed by CFA. However,
without further exposure to exogenous BV8S2 proteins, these
BV8S2-specific T cells were unable to prevent the onset of EAE after
immunization with MBP-NAc1–11. Yet, neonatal tolerization
of these naturally induced BV8S2-specific T cells with BV8S2 protein
resulted in more severe EAE, clearly demonstrating their regulatory
activity in vivo (13).
In contrast to the strong influence of CFA in directing a Th1 response,
prior vaccination with BV8S2 proteins in IFA induced significant
increases in both IFN- and IL-10 (Table I). It is noteworthy that in
mice, IFN- can inhibit EAE if applied intraventricularly 7 to 8 days
after disease induction (25), and anti-IFN- Ab can enhance the
severity of EAE in susceptible as well as resistant strains (26, 27).
Additionally, IL-10 has been shown to directly inhibit Th1 responses
(28), including those specific for MBP-NAc1–11. Thus, the
increased secretion of both of these cytokines by BV8S2-specific T
cells was associated with and probably contributed to the inhibition of
MBP-NAc1–11-specific T cells in coculture and transwell
experiments and in EAE-protected mice, as well as the unusual
maturation of MBP-NAc1–11-specific T cell line responses
toward the Th2 subtype (Table I). The production of soluble suppressive
cytokines by BV8S2-specific T cells in mice is consistent with our
previous studies of TCR peptide-specific T cells in both rats (29) and
humans (30). However, the finding that vaccination with BV8S2 proteins
can redirect the maturation of pre-effector T cells specific for
MBP-NAc1–11 toward a Th2 cell type is novel, although a
similar outcome has been reported using altered encephalitogenic
peptide ligands (31) or IL-4 (32). In addition to these direct
regulatory effects, the BV8S2-specific T cells potentially could
activate a third subset of CD8+ effector T cells that have
been proposed by Kumar and Sercarz (33) to interact with naturally
processed, MHC I-associated TCR determinants thought to be expressed by
the target MBP-specific Th1 cells.
Taken together, these experiments reveal the emergence of a powerful regulatory mechanism specific for BV8S2 determinants that is enhanced rather than muted in Tg mice overexpressing the BV8S2 gene specific for MBP-NAc1–11. There are two major findings from this study that may have important therapeutic implications for humans, including: 1) the apparent enhanced protective activity of heterologous TCR sequences that are cross-reactive with homologous TCR proteins; and 2) the selective regulation of inflammatory Th1 cells through a cytokine-driven mechanism that could inhibit bystander as well as target T cells. These findings, which have parallels in human studies using TCR peptides to induce regulatory Th2 cells for treatment of multiple sclerosis (30), provide an instructive foundation for regulation of human autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Halina Offner, Neuroimmunology Research R&D-31, Portland Veterans Affairs Medical Center, 3710 SW Veterans Hospital Road, Portland, OR 97201. E-mail address:
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; Tg, transgenic or transgene; Mo-, mouse; Rt-, rat; GST, glutathione S-transferase; CDI, cumulative disease index; MCS, mean clinical score; SI, stimulation index; LN, lymph node; NAc, N-acetylated; RAG, recombination-activating gene.
Received for publication November 26, 1997. Accepted for publication April 30, 1998.
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References |
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