BRIEF DEFINITIVE REPORT:
Treatment of Experimental Autoimmune Encephalomyelitis
with Genetically Modified Memory T Cells
By
Peter M.
Mathisen,*
Min
Yu,*
Justin M.
Johnson,*
Judith A.
Drazba,
and
Vincent K.
Tuohy*
From * The Cleveland Clinic Foundation, Research Institute, Department of Immunology, FFb-1,
and
The Cleveland Clinic Confocal Core Facility, Cleveland, Ohio 44195
Summary
Materials and Methods
Results and Discussion
Acknowledgements
References
Summary
The migratory properties of memory T cells provide a model vector system for site-specific delivery of therapeutic transgene factors to autoimmune inflammatory lesions. Lymph node cells
from (SWR×SJL)F1 mice immunized with the p139-151 determinant of myelin proteolipid
protein (PLP) were transfected with a DNA construct that placed the anti-inflammatory cytokine interleukin-10 (IL-10) cDNA under control of an antigen-inducible IL-2 promoter region. Isolated T cell clones demonstrated antigen-inducible expression of transgene IL-10 and
expressed cell surface markers consistent with the phenotype of normal memory T cells. Upon
adoptive transfer, transfected T cell clones were able to inhibit onset of experimental autoimmune encephalomyelitis (EAE) and to treat EAE animals therapeutically after onset of neurologic signs. Semiquantitative immunocytochemistry showed a significant correlation between
decreased demyelination and treatment with the transfected T cells. Taken together, these data
indicate the autoreactive T cells can be genetically designed to produce therapeutic factors in
an antigen-inducible manner resulting in a decreased severity of clinical and histological autoimmune demyelinating disease.
Experimental autoimmune encephalomyelitis (EAE) is
an inflammatory neurologic disorder widely used as an
animal model for multiple sclerosis (MS) (1). EAE is mediated by CD4+ T cells of the Th1 phenotype (IL-2, IFN-
,
TNF-
) in response to encephalitogenic peptides of central
nervous system (CNS) myelin proteins. Recent studies have
indicated that CD4+ Th2 (IL-4, IL-5, IL-10) play an immunoregulatory role in inhibiting the disease process (5).
Thus, distinct native T cell subpopulations facilitate delivery of either proinflammatory or therapeutic factors to sites
of inflammation.
We hypothesized that the antigen specificity and migratory properties of T cells may serve as an endogenous
model system for site-specific delivery of therapeutic transgene factors during autoimmune disease. To test this hypothesis, primed LN cells from (SWR×SJL)F1 (SWXJ) mice
immunized with the immunodominant p139-151 determinant of myelin proteolipid protein (PLP) were transfected with a transgene designed to provide expression of the anti-inflammatory cytokine IL-10 cDNA gene under control of the
antigen-inducible IL-2 promoter (IL-2 Prom
IL-10cDNA).
IL-10 was chosen in the design of the transgene because increased expression of IL-10 mRNA occurs in the CNS of
mice recovering from EAE and because of the ability of
IL-10 to inhibit macrophage-dependent stimulation of T cells
and production of proinflammatory cytokines (6). In addition, treatment with IL-10 has been shown to inhibit induction of EAE in rats (10). The IL-2 promoter region was
selected for its ability to drive relatively high levels of expression of a reporter gene in Jurkat and EL4.E1 lymphoma
cell lines (11). Thus, our strategy was to modify T cells genetically for delivering therapeutic factors in a nonconstitutive, antigen-inducible manner during an autoimmune disease.
Materials and Methods
Transgene Construction and Transfection.
The mouse IL-2 promoter region (
1890 to +50) (a gift from Dr. E. Rothenberg)
was subcloned into a derivative of the pSI expression vector
(Promega, Madison, WI). The mouse IL-10 cDNA is from
pcD(SR
)-F115 (ATCC no. 68027; American Type Culture Collection, Rockville, MD) (7). 7-10 d after immunization of SWXJ
mice with PLP 139-151, primed LN cells were reactivated in
vitro with PLP 139-151 (25 µg/ml). After 96 h, activated blast
cells were enriched by Ficoll centrifugation and transfected using
polybrene/DMSO-assisted gene transfer (12, 13). Cells were suspended in flat-bottomed 24-well plates at 3 × 106 cells/ml in prewarmed transfection media consisting of 10 µg/ml IL-2Prom
IL-10cDNA transgene, 1.0 µg/ml of pSV2neo plasmid (ATCC no.
37149), and 20 µg/ml polybrene (Sigma, St. Louis, MO) in
DMEM (GIBCO BRL, Gaithersburg, MD). After 6 h, the cells
were shocked with prewarmed 30% DMSO in DMEM, washed,
and cultured at 1 × 106 cells/ml in 24-well plates in a total volume
of 2.0 ml/well with 50 IU/ml mouse IL-2 (PharMingen, San Diego, CA) and 5 × 106 x-irradiated (2 × 103 rads) syngeneic splenocytes/well. After 48 h, cultures were treated with 1.0 mg/ml
geneticin (Sigma Chem. Co., St. Louis, MO; 700 µg/ml active
substance) and 50 IU/ml mouse IL-2. Cells were harvested at 6 d,
reactivated with peptide plus feeders in 24-well plates at 5 × 105
cells/well, and expanded conventionally by alternate activation/ rest cycles with antigen/IL-2. Cells were cloned at 0.3-1.0 cell/ well by limiting dilution and selection with antigen and IL-2. Proliferation assays were performed in flat-bottomed 96-well microtiter plates with 5.0 × 104 T10.11 cells/well and 5.0 × 105
x-irradiated SWXJ splenocytes/well. ELISAs were performed
with purified anti-mouse cytokine capture-detection antibody
pairs (PharMingen, San Diego, CA) on 48-h supernatants from
bulk cultures. The capture-detection antibody pairs included the
following: anti-mouse IFN-
(R4-6A2 and biotin-XMG1.2),
anti-mouse IL-4 (BVD4-1D11 and biotin-BVD6-24G2), anti-mouse TNF-
(MP6-XT22 and biotin-MP6-XT3), and anti-mouse IL-10 (JES5-2A5 and biotin-SXC-1). Standard values were plotted as absorbance at 405 nm OD versus concentrations of recombinant cytokine standards (PharMingen, San Diego,
CA). Unknown cytokine concentrations were determined as values within the linear part of the standard curve.
RNase Protection Assay (RPA).
RNA was isolated using either
guanidine isothiocyanate and CsCl as described previously or with
the TRIZOLTM reagent (GIBCO BRL, Gaithersburg, MD) following the instructions of the manufacturer (14). An Asp 700 fragment was subcloned from the IL-2Prom
IL-10cDNA transgene that included the 3
end of the IL-2 promoter region, intronic sequences, vector sequences, and the 5
end of the mouse IL-10 cDNA. This construct was linearized with EcoRI and
-[32P]UTP-labeled RNA probe synthesized using the Bluescript
T3 promoter. RPA was performed using the RNase Protection
Assay (RPA) II system (Ambion, Austin, TX). The resulting digestion products were separated on 5% Hydrolink P600 gels (J.T.
Baker, Phillipsburg, NJ), dried, and exposed to Biomax MR film
(Eastman Kodak, Rochester, NY). The RNA expression levels
were quantified using a PhosphoImager (Molecular Dynamics,
Sunnyvale, CA).
Active EAE Induction.
EAE was induced as previously described (17) by subcutaneous immunization of SWXJ mice with
the immunodominant PLP 139-151 peptide in an emulsion of
equal volumes of water and CFA (Difco, Detroit, MI). On days 0 and 3, each mouse also received intravenously 0.6 × 1010 Bordetella pertussis bacilli (Michigan Department of Public Health). All
mice were weighed and examined daily for neurologic signs in a
blinded manner.
Histologic Analysis and Quantification.
Spinal cords were fixed
in 10% phosphate-buffered formalin, and paraffin-embedded tissue
sections were cut (10 µm each) for immunostaining. Sections
were pretreated with 0.04% OsO4 and 1% H2O2 in 10% Triton
(Electron Microscopy Sciences, Fort Washington, PA) and blocked
with 5% normal goat serum (Vector, Burlingame, CA) and 5%
nonfat dehydrated milk for 60 min. Sections were treated sequentially with PLP monoclonal IgG2a antibody (Harlan) at a 1:200
dilution for 14 h at 4°C, biotinylated goat anti-mouse IgG2a (Southern Biotechnology, Birmingham, AL) at a 1:500 dilution
for 30 min at 22°C, and avidin-peroxidase complex (Vector,
Burlingame, CA) for 1 h at 1:1,000 dilution. Sections were then
treated with diaminobenzidine and 0.01% H2O2 for 8 min, 0.04%
OsO4 for 30 s and washed in PBS. Images were digitized using
the Oncor Imaging System (Gaithersburg, MD) at 640 × 480 pixel resolution. All images were normalized by adjusting background gray matter stain to the same mean intensity value using
Adobe Photoshop (Adobe Systems, Mountain View, CA). At
least 12 images per animal were analyzed. The dorsal columns
were outlined and the percentage of pixels representing the darkest 25% of stain was determined using NIH Image software.
Statistical Analysis.
The two-sided Student's t test was used
for determining differences in mean clinical scores of treated and
control EAE mice as well as differences in percent immunostaining between control and experimental spinal cords.
Results and Discussion
A transgene construct (IL-2Prom
IL-10cDNA) was designed by fusing a mouse IL-2 promoter region (
1890 to
+50) to the mouse IL-10 cDNA (Fig. 1 a). The transgene
construct also contained intron splices sites and the SV40
late polyadenylation signal region to ensure high levels of
IL-10 expression. In this way, T cells were designed so that
an IL-2 promoter region would regulate synthesis of IL-10
in an antigen-inducible, nonconstitutive manner.
Fig. 1.
The IL-2Prom
IL-10cDNA transgene and characterization of transfected T cells. (a) Schematic representation of the transgene IL-2Prom
IL-10cDNA showing IL-2 promoter region, IL-10 cDNA, and SV40 polyadenylation signals. Intron sequences are labeled and vector
sequences are indicated by solid lines. (b) Proliferative responses of T cell
clone T10.11 after peptide activation. 5 × 105 clone cells were stimulated
(25 µg/ml) with the immunodominant PLP 139-151 HSLGKWLGHPDKF, and with PLP 104-117 KTTICGKGLSATVT, a noncross-reacting
control encephalitogen for SWXJ mice (17, 26). The data show the stimulation index (cpm test/cpm background) of [3H]thymidine incorporation by
T10.11 clone cells 48 h after activation with peptide. Error bars show ± SD. (c) Cytokine levels were measured on 48-h supernatants from clone
T10.11 cells cultured without antigen (resting cells) and after activation
with PLP 139-151. Cytokine concentrations were normalized to total
cell numbers.
[View Larger Version of this Image (28K GIF file)]
T cells were prepared by in vitro activation of primed
LN cells from SWXJ mice immunized with the PLP 139-
151 peptide. In our hands, this method consistently produces encephalitogenic T cells capable of passively transferring EAE into naive animals.
Peptide-activated T cells were transfected with both the
IL-2Prom
IL-10cDNA transgene and the selectable marker
plasmid, pSV2neo. Neo-resistant T cells were expanded, and
T cell clones were isolated and analyzed. Clone T10.11 was
selected for further study because of its marked antigen
specificity (Fig. 1 b) and its ability to generate a substantial
increase in IL-10 cytokine production upon activation with
antigen (Fig. 1 c).
An RPA was used to differentiate between IL-2Prom
IL-10cDNA transgene and endogenous IL-10 gene expression. RNA from resting and antigen-activated T10.11 cells
was hybridized to an RNA probe prepared from the 5
end
of the IL-10 cDNA that incorporated transcribed-transgene vector sequences (Fig. 2 a). Two RNA transcripts were
protected from activated T10.11 cells, indicating that both
endogenous and transgene IL-10 expression were induced
after stimulation with antigen (Fig. 2 b). Transgene expression represented 40% of the total IL-10 mRNA, and increased 7.5-fold after activation with PLP 139-151 compared
with a 6.1-fold antigen-induced increase in endogenous
IL-10 mRNA. Thus, antigen-inducible expression of transgene IL-10 mRNA occurred concomitant with endogenous
IL-10 gene expression.
Fig. 2.
Detection of transgene IL-10 mRNA in T10.11 clone cells.
An RNase protection assay (RPA) was used for distinguishing endogenous and transgene mRNA. (a) The DNA construct used to generate the RNA probe for measuring IL-10 expression. The RNA probe is represented by an arrow and protected RNase digestion products are shown. (b) RNase-digestion products after hybridization to 32P-labeled RNA probe.
Lane 1, yeast RNA; lane 2, RNA from rested T10.11 clone cell 11 d after
activation with PLP 139-151; lane 3, RNA from activated T10.11 clone
cells 48 h after activation with PLP 139-151; lane 4, spleen RNA.
[View Larger Version of this Image (20K GIF file)]
Flow cytometry analysis showed that transfected T10.11
cells expressed a cell surface phenotype consistent with
normal memory T cells. After antigen stimulation, T10.11
cells were CD3+, CD4+, CD8dim, TCR
+, V
14+ T cells
with high level expression of the activation antigens CD44
(Pgp-1), CD49d (VLA-4), and CD25 (IL-2R) and low
level expression of CD62L (L-selectin), a marker for native
T cells (Table 1).
The development of EAE in SWXJ mice is characterized
by acute onset of paralytic disease within 3 wk after immunization with PLP 139-151. Mice typically recover and
undergo a relapsing-remitting disease course with progression to chronic disability accompanied by perivascular mononuclear infiltrates and demyelination in CNS white matter.
To evaluate the therapeutic potential of IL-2Prom
IL-10
cDNA transfected T cells, T10.11 clone cells were adoptively transferred into SWXJ mice before the anticipated
onset of PLP 139-151-induced EAE as well as after onset
of clinical disease. Transfer of T10.11 T cells was found to
be effective in inhibiting the onset of EAE (Fig. 3 a) and in
therapeutically altering the course of disease when transferred after initiation of neurologic signs (Fig. 3 b). The inhibitory effect of clone T10.11 was similarly mimicked by
transfer of PLP 139-151-specific T cell lines also transfected with the IL-2Prom
IL-10cDNA construct (Fig. 3
a). In contrast, transfer of either normal splenocytes or IL-2
Prom
IL-10cDNA-transfected T cell lines specific for the
irrelevant non-CNS antigen, KLH, produced no sustained
therapeutic effect on either EAE onset or progression.
KLH-specific transfected T cell lines showed antigen-inducible production of IL-10 in a manner similar to that
observed in transfected autoreactive T cells (data not
shown).
Fig. 3.
Inhibition and treatment of EAE
with autoreactive T cells transfected with IL-2
Prom
IL-10cDNA. (a) Inhibition of EAE onset after adoptive transfer of 1 × 107 T10.11
clone cells or IL-2Prom
IL-10cDNA-transfected T cell lines specific for PLP 139-151.
Recipient SWXJ mice were immunized for
EAE induction with PLP 139-151 18 d before
tail vein injection with antigen-activated T
cells. Mice showed no signs of EAE before transfer. No therapeutic effect occurred in mice
receiving 1 × 107 normal splenocytes or activated IL-2Prom
IL-10cDNA-transfected cells
specific for the irrelevant control antigen, keyhole limpet hemocyanin (KLH). (b) Therapeutic treatment after EAE onset by transfer with
IL-2Prom
IL-10cDNA-transfected T10.11
clone cells. 3 d after EAE onset, mice were injected intravenously with 1 × 107 activated T cells. No therapeutic effect was observed in mice transferred with nonactivated normal splenocytes or with
activated transfected T cells specific for KLH. All mice were weighed and examined daily for neurologic signs in a blinded manner according to the following criteria. Clinical scores are 0, no disease; 1, decreased tail tone or slightly clumsy gait; 2, tail atony and/or moderately clumsy gait and/or poor
righting ability; 3, limb weakness; 4, limb paralysis; 5, moribund state. Error bars show ± SEM.
[View Larger Version of this Image (18K GIF file)]
To determine the histologic effects after adoptive transfer
of transfected T cells, spinal cords from mice receiving either IL-2Prom
IL-10cDNA-transfected T cells (Fig. 4 b) or
normal splenocytes (Fig. 4 a) just before EAE onset were
stained immunocytochemically for PLP, and demyelination
was quantified by measuring the mean intensity of dorsal
column PLP staining. Adoptive transfer of IL-2Prom
IL-10
cDNA-transfected T cells just before EAE onset resulted in
a significant (P = 0.02) mean decrease of 12.2% in CNS
demyelination compared with EAE mice receiving normal
splenocytes.
Fig. 4.
Histologic analysis of CNS tissue after adoptive transfer of
transfected T cells. The extent of demyelination was measured by immunocytochemical staining for PLP. (a) Representative section showing demyelination (arrow) in the spinal cord dorsal column of SWXJ control
mouse adoptively transferred with normal splenocytes before EAE onset.
(b) Representative section showing uniform distribution of PLP immunostaining in the spinal cord dorsal column of SWXJ mouse adoptively
transferred before EAE onset with PLP 139-151-specific T cells transfected with the IL-2Prom
IL-10cDNA construct. Closed Bar, 50 µm.
[View Larger Version of this Image (81K GIF file)]
Ectopic expression of anti-inflammatory cytokines has
produced conflicting results in the treatment of immune-mediated inflammation. Allograft survival is prolonged after
retroviral-mediated transfection of murine cardiac transplants with viral IL-10 (18), and onset of collagen-induced
arthritis is delayed in DBA/1 mice injected with Chinese
hamster ovary (CHO) fibroblasts transfected with IL-4 or
IL-13 (19). In contrast, expression of transgene IL-10 in
pancreatic islet
cells actually accelerates the development of autoimmune diabetes in nonobese diabetic (NOD) mice
(20). Controversy is also apparent in reports which show
efficacy in treating ongoing EAE by transfer of traditional
Th2 T cell clones cells (5), but only modest therapeutic effect when Th2 T cells are transferred before disease induction (21). However, it is clear that proliferation of encephalitogenic Th1 T cells can be inhibited by IL-10, but not
by IL-4 secreted from Th2 T cells having identical antigen
specificity (22). It is worth noting that treatment with recombinant IL-10 has also produced conflicting outcomes resulting in either exacerbation (23) or amelioration (10) of EAE.
In a recent report related to the present study, Shaw et al.
(24) demonstrated a delay in EAE onset and a decrease in
disease severity after transfer of myelin basic protein-specific
T cell hybridomas that had been retrovirally transduced
with IL-4. However, in contrast with the present study,
expression of the IL-4 transgene was constitutive, and all of
the mice eventually died from overgrowth of the hybridoma
tumor cells.
The results of the present study show that transfected antigen-specific T cells are effective when used either to inhibit onset of EAE or to treat ongoing disease. In a broader
sense, our data indicate that T cells can be genetically altered with nonviral vectors to provide antigen-inducible
production of therapeutic transgene proteins while maintaining an otherwise normal memory T cell phenotype.
Thus, genetic modification of T cells may provide a means
for both identifying and delivering therapeutic transgene factors capable of modulating inflammation. Moreover, it
may be possible to use transgene-altered T cells for delivering appropriate growth factors for tissue repair, particularly
in light of recent experiments showing that T cells constitutively expressing nerve growth factor are less capable of
mediating experimental autoimmune neuritis (25). Insights
gained from genetic modification of T cells in the EAE animal model may provide a rational basis for treating the autoimmune demyelination widely believed to be responsible for chronic progression of MS.
Footnotes
Address correspondence to Dr. Vincent K. Tuohy, The Cleveland Clinic Foundation, Department of Immunology, FFb-1, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-445-9684; Fax: 216-444-8372;
E-mail: tuohyv{at}cesmtp.ccf.org
Received for publication 7 February 1997 and in revised form 6 May 1997.
We thank Dr. E. Rothenberg for the IL-2 promoter region and for her helpful advice.
This work was supported by National Multiple Sclerosis Society grants RG-2768 (V.K. Tuohy) and PP0483
(P.M. Mathisen) and by National Institutes of Health grant NS-36054 (V.K. Tuohy).
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