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Department of Allergy and Rheumatology, University of Tokyo Graduate School of Medicine, and
Department of Hematopoietic Factors, The Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that T cells recognizing organ-specific autoantigens are involved in the pathogenesis of autoimmune diseases (2). These autoreactive T cells migrate to and accumulate in the target organs, inducing inflammation and leading to the destruction of tissues. Because it is the Ag-specific TCR that endows autoreactive T cells with the ability to recognize the autoantigen and to migrate into the target organs, circulating T cells with specificity for the autoantigen would be one of the most effective ways for transporting therapeutic agents.
The concept of employing T cells recognizing an organ-specific
autoantigen as vehicles for delivering therapeutically useful agents
was first demonstrated in experimental autoimmune encephalomyelitis
(14). A T cell line specific for P2 protein, which is a
peripheral myelin protein, is neuritogenic when transferred adoptively.
A P2 protein-specific T cell line transduced with nerve growth factor
was no longer neuritogenic, because locally released nerve growth
factor exhibited an anti-inflammatory effect. This concept was
confirmed by a more refined study in which an anti-myelin basic
protein T cell clone transfected with TGF-
1 protected against
experimental autoimmune encephalomyelitis induced by immunization with
either myelin basic protein or proteolipid protein (15).
These successes prompted us to test the concept that T cells specific
for a joint Ag might be an efficient therapeutic vehicle in
experimental arthritis.
IL-10 is known to mediate immunosuppressive effects predominantly
through down-regulation of macrophage functions and inhibition of
proinflammatory cytokines produced by Th1 cells, such as IL-1, IL-6,
IL-8, TNF-
, IFN-
, and GM-CSF (16). Earlier studies
demonstrated that IL-10 was able to prevent disease expression and
development in collagen-induced arthritis by i.p. injection of mouse
IL-10 (mIL-10) or an adenovirus vector encoding murine IL-10
(17, 18, 19, 20). Viral IL-10, which is a biological homologue of
mIL-10, was demonstrated to exert similar effects (18). It
has also been demonstrated that anti-collagen type II
(anti-CII) T cells transfected with viral IL-10 can reduce disease
severity (19). These abilities of IL-10 in ameliorating
disease seem to depend on systemic suppression of the immune response
to the induced Ag, because, for example, anti-CII Ab was suppressed
in these manipulated mice. Systemic immunosuppression could present the
risk of severe infection. Therefore, it is desirable that the
immunosuppressive effect of IL-10 is exerted only at the inflammatory
lesion as a result of an efficient local delivery system.
In the present study to evaluate the effect of Ag-specific T cells transduced with IL-10 on locally inflamed joints, we used an Ag-induced arthritis (AIA) model that is frequently employed as a model of RA. BALB/c mice were immunized with OVA, followed by intra-articular injection of OVA. The immune response against OVA causes inflammation in the joints, leading to destruction of the joints. Adoptively transferred OVA-reactive DO11.10 mouse splenocytes retrovirally transduced with IL-10 migrated selectively to arthritic joints and reduced disease severity without impairing the systemic immune response to the Ag. Although highly efficient transduction and selection of the transduced cells are both rather difficult to achieve for splenocytes, compared with transformed cell lines that can be selected by drug resistance, we overcame these problems by employing green fluorescent protein (GFP) selection. Our results suggest that Ag-specific T cells have the potential to serve as a therapeutic vehicle in autoimmune diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice were purchased from Japan SLC (Shizuoka, Japan), and DO11.10 transgenic mice, whose T cells express receptors specific for OVA (21), were bred in our animal facility under specific pathogen-free conditions. All mice were used at the age of 8–10 wk.
Antibodies
Anti-CD4 Ab (L3T4) was purchased from PharMingen (San Diego, CA). A DO11.10 TCR-specific mAb, KJ1-26 (21), was purified from the supernatant of a hybridoma culture and labeled with biotin (Immunoprobe Biotinylation Kit; Sigma, St. Louis, MO) according to the manufacturer’s protocol.
Induction of AIA
Female BALB/c mice were immunized with 100 µg of OVA (Sigma) in CFA (Difco, Detroit, MI) by injection into the base of the tail and were boosted 2 wk later with 100 µg of OVA in IFA. Before the immunization, the mice were injected i.p. with 200 ng of pertussis toxin (Wako, Tokyo, Japan). Two weeks after the booster, 100 µg of OVA dissolved in 20 µl of PBS was intra-articularly (i.a.) injected into the left ankle joint. The right ankle joint was injected with 20 µl of PBS as a negative control. The joint thickness was measured with a dial gauge caliper calibrated with 0.01-mm graduations (Mitsutoyo, Tokyo, Japan). The net increase in joint thickness attributable to the antigenic challenge was calculated by subtracting the increase in thickness of the right ankle from the increase in thickness of the left ankle. There was no net joint swelling after i.a. injection of OVA in nonimmunized mice. Induction of arthritis with methylated BSA (mBSA; Sigma) was performed in almost the same manner as OVA-induced arthritis (22). Twenty micrograms of mBSA was injected into the ankle joint of mice immunized with 100 µg of mBSA in CFA. To see the effect of transferred cells, 20 µg of mBSA and 100 µg of OVA were coinjected into the left ankle joint. The same volume of PBS (20 µl) was injected into the right ankle joint.
Production of replication-defective retrovirus
pMFGmIL-10, a retroviral plasmid containing the mIL-10 gene, was obtained from Riken Gene Bank (Tsukuba, Japan) with the approval of Dr. H. Hamada (23, 24). The production of retrovirus was performed as described previously (25). Briefly, an ecotropic retrovirus was produced in the BOSC23 packaging cell line. BOSC23 cells were seeded onto 100-mm dishes at 5 x 106 cells/plate 1 day before transfection. Transfection using Lipofectamine reagent (Life Technology, Gaithersburg, MD) was performed according to the manufacturer’s protocol using 10 µg/plate of the pMFGmIL-10 plasmid. Cells were cultured for 48 h, with fresh medium replacement at 24 h. The supernatant was harvested as a replication-defective retrovirus source and centrifuged twice at 1000 x g for 5 min to remove nonadherent producer cells. The replication-defective retrovirus was concentrated to 1 ml by centrifugation at 6000 x g at 4°C for 16 h and then stored at -80°C until use.
Infection of the retrovirus
Splenocytes from the DO11.10 transgenic mice were cultured in the presence of 10 nM OVA peptide with RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10% heat-inactivated FCS, and 5 x 10-5 M 2-ME. After 48-h culture, 4 x 106 cells were resuspended in 1 ml of the concentrated retrovirus stock in the presence of 4 µg/ml Polybrene (Sigma) and incubated for 2 h. After replacement of the medium, the cells were cultured for 48 h in the presence of 10 µg/ml Con A (Sigma). Infection of Ag nonspecific splenocytes was performed using PHA (5 µg/ml; Difco) instead of OVA peptide.
Adoptive transfer
Before adoptive transfer, CD4+ T cells were enriched using MACS (Miltenyi Biotech, Bergisch Gladbach, Germany) as previously described (26). Analysis by flow cytometry revealed that the purity of CD4+ T cells was >96%. Various numbers (5 x 105, 1 x 106, 3 x 106) of CD4+ DO11.10 splenocytes were transferred i.v. into nonirradiated OVA-primed BALB/c mice the day before i.a. antigenic challenge.
Quantitation of IL-10 by ELISA
IL-10 in the supernatant of transduced T cells or the sera from AIA mice was quantitated using a sandwich ELISA kit (Endogen, Woburn, MA).
GFP selection
An internal ribosome entry site (IRES) and the GFP gene were inserted downstream of the mouse IL-10 gene, designated pMFGmIL10-IRES-GFP. GFP-positive CD4+ cells were sorted with FACS Vantage (Becton Dickinson, San Jose, CA).
Ab assay
The level of anti-OVA Ab was measured by ELISA as described previously (26), except that OVA was employed as a coated Ag instead of human U1snRNP-A.
T cell proliferation assay
Spleen cells and LN cells were cultured at 1 x 105 cells/well with various concentrations of OVA in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10% heat-inactivated FCS, and 5 x 10-5 M 2-ME for 5 days, followed by a final 16 h of culture in the presence of 1 µCi of [3H]TdR/well. The incorporated radioactivity was counted with a gamma scintillation counter. The proliferative response was expressed as the stimulation index (the mean cpm of test cultures/the mean cpm of control cultures without Ag) ± SD.
TCR RT-PCR/single-strand conformation polymorphism (SSCP) analysis
A TCR RT-PCR/SSCP study was performed as described previously
(27, 28), except that only V8.2-specific primer, which
DO11.10 T cells use, was employed in this study. In brief, after
synthesizing cDNA using a random primer, the cDNA was amplified by PCR
with a common C primer and a V8.2-specific primer. The amplified
DNA was electrophoresed on nondenaturing 4% polyacrylamide gel. After
transfer onto a nylon membrane, the cDNA hybridized with a biotinylated
internal common C oligonucleotide probe was visualized by subsequent
incubations with streptavidin, biotinylated alkaline phosphatase, and a
chemiluminescent substrate system (Phototope-Star Chemiluminescent
Detection Kit; New England Biolabs, Beverley, MA).
Histology
Excised ankles and knees were fixed in 10% phosphate-buffered formalin and decalcified. The tissues were then dehydrated in a gradient of alcohols, paraffin-embedded, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.
Flow cytometric analysis of infiltrating cells into arthritic lesions
Both arthritic and control knee joints were excised and minced, followed by digestion with 1 mg/ml of collagenase (Sigma) and 1 mg/ml of DNase (Sigma) for 2 h. After passing through a nylon mesh, cells were collected by centrifugation. GFP-transduced CD4-positive DO11.10 T cells were detected by staining with either anti-CD4-PE Ab or KJ1-26-biotin in combination with streptavidin-PE (PharMingen).
Statistical analysis
Statistical evaluation was performed with one-way ANOVA or Mann-Whitney’s U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although OVA is frequently used in rabbits to induce AIA
(29, 30, 31), to date there have been no reports of use of OVA
as an induction Ag in mice. The BALB/c mice we immunized with OVA
developed severe arthritis after intra-articular challenge (see the
positive control in Figs. 1
and 2, left). Neither the joints
challenged with PBS nor the joints of mice immunized with PBS exhibited
obvious inflammation. Thus, we successfully established an OVA-induced
murine AIA model.
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To generate functionally modulated Ag-specific T cells, we
transduced murine IL-10 into splenocytes from DO11.10 transgenic mice,
which bear TCRs specific for OVA. Almost 70–80% of
CD4+ T cells from the DO11.10 transgenic
splenocytes were positive for KJ1-26, which is specific for clonotype
DO11.10 (data not shown). After 48 h of infection by pMFGmIL-10,
the culture supernatant of 106
CD4+ T cells from DO11.10 splenocytes was found
to contain IL-10 at 5.2 ± 0.5 ng/ml, whereas the supernatant of
both nontransfected and mock-transfected DO11.10 splenocytes did not
contain a detectable level of IL-10. The transduction efficiency into
splenocytes of our retrovirus vector, examined using a GFP-expressing
construct, was 
5%.
DO11.10 splenocytes transduced with IL-10 reduce the severity of joint swelling in AIA
We next examined the effect of IL-10-transduced DO11.10
splenocytes on AIA. One day after the adoptive transfer of DO11.10
CD4+ T cells (3 x
106) transduced with either the IL-10 or control
vector, the ankle joints of mice primed with OVA were challenged with
OVA. Mice that underwent i.v. transfer of CD4+ T
cells producing IL-10 exhibited less joint swelling than the
nontransferred positive control mice (Fig. 1
). When we transferred
three different numbers (5 x 105, 1 x
106, and 3 x 106) of
CD4+ DO11.10 splenocytes infected with
pMFGmIL-10, the percent inhibition of hind paw thickness increased as
the number of transferred cells increased. The transfer of 3 x
106 cells exhibited the greatest inhibition,
i.e., as much as 96.0 ± 1.92% on day 4. These results indicate
that mIL-10-transduced OVA-specific T cells reduce the disease severity
and that the disease amelioration depends on the number of
IL-10-transduced cells transferred.
Histopathologic analysis of AIA
To confirm the effect of DO11.10 T cells transduced with mIL-10,
we examined ankle sections from animals sacrificed 21 days after
disease induction and transfer of the cells. As shown in Fig. 2, left, severe arthritis was observed in the ankle joints of
arthritic mice to which mock-infected T cells were transferred, because
the periarticular tissues were accompanied by synovial hyperplasia and
massive cell infiltration. In contrast, the ankle joints of mice
transferred with mIL-10-transduced T cells demonstrated almost the same
histology as ankles derived from healthy mice (Fig. 2, right).
Number of transduced cells sufficient to ameliorate the disease after GFP selection
Interestingly, mice transferred with mock-transduced DO11.10
CD4+ T cells demonstrated more severe swelling,
indicating that functionally nonaltered Ag-specific T cells contribute
to exacerbation of the arthritis (see Fig. 1). Considering that only a
small fraction of the transferred DO11.10 splenocytes was successfully
transduced with the vector and that the larger population remained
nontransduced, the reduction of the disease severity by this composite
population indicates that the IL-10 produced by the small population of
transduced T cells is sufficient to ameliorate the disease despite the
presence of a larger number of nontransduced DO11.10 splenocytes.
Therefore, we postulated that even a smaller number of Ag-specific T
cells transfected with pMFGmIL-10 would be sufficient to control
disease severity in the absence of nontransfected DO11.10 T cells. To
select only successfully transduced DO11.10 T cells, we employed
pMFGmIL10-IRES-GFP. We sorted GFP-expressing CD4
T+ cells by FACS and transferred them to the
mice. Although the degree of amelioration decreased as the number of
transferred cells decreased, as few as 1 x
104 GFP+
CD4+ cells was found to reduce disease severity
(Fig. 3). Because the purity of the
sorted cells was still about 60%, <104
transfected cells would be sufficient to control the disease.
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Although it seems that the Ag specificity of the transferred
CD4+ T cells is required for efficient
recruitment to the inflamed joints and that locally released IL-10
might be involved in the reduction of disease severity, it might also
be possible that IL-10-producing T cells migrate into the joint in
response to the inflammation regardless of their Ag specificity. To
examine these points, we transferred wild-type BALB/c splenocytes
transfected with IL-10. The mice developed AIA as severe as that in the
positive control, whereas splenocytes from DO11.10 mice resulted in
amelioration (Fig. 4). This result
indicates that the Ag specificity of the CD4+ T
cells is indispensable for efficient reduction of disease severity,
supporting the concept that the T cells should recognize the Ag in the
joints and migrate to such joints.
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To confirm that transferred DO11.10 T cells migrate into arthritic
joints preferentially, we employed the TCR RT-PCR/SSCP method
(27, 28). Because each CDR3 sequence demonstrates unique
mobility on nondenatured gel due to its unique single-strand
conformation, the identical mobility of the amplified V CDR3 region
on a gel indicates that the clones are identical. This rule has always
been confirmed by sequencing (32).
Whereas splenocytes from BALB/c mice immunized with OVA exhibited a
smear pattern that indicates a heterogeneous T cell population,
splenocytes from mice which were transferred with DO11.10 cells shared
a distinct band with the positive control in addition to a
heterogeneous smear pattern (Fig. 5). The
arthritic left ankle joint exhibited the unique distinct band,
indicating that the transferred DO11.10 cells selectively migrated to
this site. Other noninflamed joints also exhibited a faint band
corresponding to DO11.10 T cell, probably due to contamination by
peripheral blood.
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GFP-positive cells were not detected either in joints from the negative
control or in arthritic joints of mice transferred with BALB/c mice
splenocytes transduced with GFP. Thus, we confirmed the selective
accumulation of the transduced DO11.10 cells in the arthritic joints by
both clonotype analysis and flow cytometric analysis.
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IL-10 could not be detected in sera from mice transferred with
IL-10-transduced DO11.10 splenocytes (data not shown). Considering this
together with the selective accumulation of the transferred cells and
the restricted space of the joint, it is conceivable that the
suppressive effect of IL-10 could be observed preferentially in the
joint. To examine whether suppression of the disease was induced
locally or systemically, we examined both the Ab titer to OVA and the T
cell proliferative response to OVA after adoptive transfer of the
IL-10-transduced T cells selected by GFP. Neither the T nor the B cell
response of the splenocytes from mice transferred with
104 transduced T cells demonstrated a significant
difference compared with that of the splenocytes from control mice
(Fig. 7). This result indicates that the
transferred T cells express their suppressive activity locally.
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Finally, we examined whether IL-10-transduced DO11.10 T cells are
able to ameliorate arthritis induced by another Ag, using mBSA as an
arthritis-inducing Ag (33, 34). Arthritis induced by mBSA
in BALB/c mice showed severer joint swelling than that induced by OVA.
DO11.10 splenocytes (5 x 104) that were
transduced with mIL-10 and selected by GFP were transferred to BALB/c
mice immunized with mBSA. We observed a reduction of the swelling in
the joint that was coinjected intra-articularly with OVA, but not in
the joint that was not coinjected with OVA (Fig. 8). Mock-transfected DO11.10 T cells
exacerbated the swelling of joints that were coinjected with OVA. These
results indicate that DO11.10 T cells transduced with mIL-10 migrate
into the joint in response to the intra-articularly injected OVA and
that they are able to exhibit bystander suppression of mBSA-induced
arthritis. Taken together, the IL-10-transduced DO11.10 splenic T cells
preferentially migrated into the joint, responding to the Ag, and
reduced disease severity quite efficiently without impairing the
systemic Ag-specific immune response. These results suggest that
Ag-specific T cells could serve as a therapeutic vehicle for
arthritis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-10 has already been demonstrated to exert a therapeutic effect on arthritis in a collagen-induced arthritis (CIA) model (17, 18, 19, 20). To date, the effect of IL-10 seems to be mediated by suppression of the systemic immune response to CII, although contradictory results has been reported. Walmsley et al. demonstrated that the disease severity of CIA was reduced in mice treated with i.p. injection of mIL-10 (35). No difference in the amount of anti-CII IgG was observed between the treatment and control groups. Similarly, the ratio of anti-CII IgG1 to anti-CII IgG2a level revealed no significant differences. Ma et al. demonstrated successful inhibition of CIA by systemic administration of a viral IL-10-encoding adenovirus (Av(vIL-10)) (19). They found that T cells from mice treated with Av(vIL-10) showed a decreased proliferative response to CII, whereas the serum level of Ab against CII was not affected. Apparailly et al. also demonstrated that viral IL-10 gene transfer mediated by adenovirus inhibits CIA (18). They did not examine the T and B cell responses against CII, except for the ratio of IgG1/IgG2a of anti-CII Abs, which increased in adenovirus-vIL-10-treated mice, suggesting that the Th2 response is dominant. Therefore, although IL-10 also possesses some immunostimulatory activities, including enhanced expression of class II MHC molecules on B cells and induction of CTL differentiation, in these experiments IL-10 seems to mediate immunosuppression of the T cell response. This immunosuppression, however, appeared to be Ag nonspecific, because they could detect viral IL-10 in the sera from adenovirus-vIL-10-treated mice. By suppressing the immune responses to a wide variety of foreign Ags, Ag nonspecific immune suppression might increase the risk of immunocompromised infections.
Interestingly, quite recently it was demonstrated that viral
IL-10-expressing adenovirus injected periarticularly into mouse paws
suppressed the development of CIA in both the injected and the
uninjected contralateral paws (36, 37). Such a distal
anti-arthritic effect has also been reported using OVA-induced
arthritis in rabbits to which IL-1 and TNF- soluble receptors were
delivered by adenovirus-mediated gene transfer (9). It was
demonstrated that certain cells infected with the delivered viral
vectors moved to other joints (37). It would be
interesting to know whether they are T cells.
Ex vivo gene delivery using T cells in experimentally induced arthritis
has been reported by Chernajovsky et al. (38). Splenocytes
from DBA/1 mice immunized with CII that were transduced with either
soluble TNF receptor or TGF- were able to inhibit development of
disease in SCID mice transferred with anti-CII-responding
splenocytes. In their experiments the significance of Ag-specific
receptors on T cells remained ambiguous. Although they demonstrated
that T cells infected with TGF- were able to reduce the anti-CII
Ab level, they did not mention whether the transduced T cells
ameliorated the disease. The inability of their T cells to reduce
disease severity might be due to the low infection efficiency, because
in their system only between 0.2 and 0.3% of the transferred
lymphocyte population was infected.
Adoptive transfer of IL-10-transduced T cells has been reported in nonobese diabetic (NOD) mice. Islet-specific Th1 cells that were transduced with IL-10 and selected by G418 abrogated the diabetes in NOD mice (39). The number of transferred cells needed to ameliorate the disease was 2–6 x 106. Although the affected organ is different, considering that the number of transferred cells needed to reduce disease activity was smaller in our experiments (1 x 104 cells were needed), GFP selection would be one of the best strategies for selecting transduced peripheral blood cells that could be frequently used in ex vivo gene therapy for patients.
Joint Ag-specific T cells transduced with the IL-10 gene might fit the idea of regulatory T cells (40), which were demonstrated to suppress experimentally induced colitis. Ag specificity of the engineered regulatory T cells is indispensable for Ag-specific immunomodulation as well as for cell migration into arthritic lesions, where the regulatory T cells exhibit bystander suppression (41). We are now planning an experimental system in which the authentic joint-specific Ag serves as the target of the transporter.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshikata Misaki, Department of Allergy and Rheumatology, University of Tokyo Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
3 Abbreviations used in this paper: RA, rheumatoid arthritis; AIA, Ag-induced arthritis; GFP, green fluorescent protein; IRES, internal ribosomal entry site; CII, type II collagen; CIA, collagen-induced arthritis; i.a., intra-articularly; mBSA, methylated BSA; SSCP, single-strand conformation polymorphism; mIL-10, mouse IL-10; NOD, nonobese diabetic.
Received for publication September 27, 1999. Accepted for publication August 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(cA2) versus placebo in rheumatoid arthritis. Lancet 344:1105.[Medline]
soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects. Proc. Natl. Acad. Sci. USA 95:4613. monoclonal antibody combined with low-dose weekly methotrexate in rheumatoid arthritis. Arthritis Rheum. 41:1552.[Medline]
1. Proc. Natl. Acad. Sci. USA 95:12516.v3 antagonist. J. Clin. Invest. 103:47.[Medline]
1 ameliorate disease in a collagen-induced arthritis model. Gene Ther. 4:553.[Medline]
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) or mock-transfected DO11.10 splenocytes
(•) were transferred the day before arthritis induction. Mice
transferred only with PBS (
) were included as a reference. The
severity was evaluated by hind paw thickness. Mock-transfected
CD4+ DO11.10 splenocytes exacerbated the disease. Bars show
the mean ± SD (n = 10/group). *,
Significant difference (p < 0.01) between each
group and the control group.
), 5 x 104 (
), and 1 x 105
(
). Bars show the mean ± SD (n =
10/group). The difference between the control group and the groups of
mice transferred with GFP-selected cells was statistically significant
(*, p < 0.01; 
, p <
0.05), whereas the difference between the control group and the groups
of mice transferred with mock-transfected cells was not
significant.
