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Division of Immunology and Rheumatology, Departments of Medicine and
Pediatrics, Stanford University School of Medicine, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production in developing and effector Th1 populations, indicating its
potential to modulate Th1-promoted inflammation. We have previously
demonstrated that transduction of myelin basic protein-specific
CD4+ T cells with pGC retroviral vectors can result in
efficient and stable transgene expression. Therefore, we adoptively
transferred myelin basic protein-specific CD4+ T cells
transduced to express IL-12 p40 into mice immunized to develop
experimental autoimmune encephalomyelitis and demonstrated a
significant reduction in clinical disease. In vivo tracking of
bioluminescent lymphocytes, transduced to express luciferase, using
low-light imaging cameras demonstrated that transduced CD4+
T cells trafficked to the central nervous system, where histological
analysis confirmed long-term transgene expression. These studies have
demonstrated that retrovirally transduced autoantigen-specific
CD4+ T cells inhibited inflammation and promoted
immunotherapy of autoimmune disorders. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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has
been shown to regulate autoimmune inflammation (4, 5, 6, 7, 8).
However, systemic cytokine delivery can also lead to increased risk of
infections and malignancies (9, 10). Therefore, recent
approaches have explored local delivery of regulatory proteins as an
alternative to systemic therapeutic regimens (11, 12, 13).
Although adenoviruses have been a common tool for gene delivery, these
viruses allow only transient expression in infected cells
and have been associated with lethal events
in recent clinical trials (14, 15, 16). In contrast,
retrovirally mediated gene transfer safely provides stable integration
of transgenes (17) with resultant long-term expression.
Furthermore, retroviral gene delivery has been used to treat human
disease (18) and has avoided the viral protein
immunogenicity that occurs with the use of adenoviral gene
delivery. Previously, we have shown that retroviral transduction can preferentially target Ag-specific CD4+ T cells (19). Due to the intrinsic tissue-specific homing properties of autoantigen-specific CD4+ T cells, they should serve as ideal vehicles for site-specific transgene delivery. Indeed, we have demonstrated that autoantigen-specific T cell hybridomas retrovirally transduced to express a regulatory cytokine, IL-4, were capable of trafficking to autoimmune lesions in the CNS to ameliorate experimental autoimmune encephalomyelitis (EAE)4 (20). Disease could be abrogated or exacerbated depending on the cytokine delivered (21). Other studies have used transduced T cell clones instead of hybridomas to deliver immunosuppressive proteins to lesions of EAE (22, 23, 24). However, characteristics of uncontrolled growth and abnormal homing properties render hybridomas and T cell lines a poor option for the therapy of human disease. Therefore, the work presented here explores the use of short-term ex vivo retroviral transduction of primary autoreactive CD4+ T cells for therapeutic transgene delivery.
Th1-type CD4+ T cells are considered to be a
predominant contributor to the initiation and persistence of
autoimmunity; however, there is conflicting evidence regarding the
influence of proinflammatory cytokine expression on autoimmune disease.
Mice genetically deficient in proinflammatory mediators such as
IFN-, TNF, and lymphotoxin are as susceptible to EAE as
wild-type mice (25, 26, 27). Furthermore, in some
cases, administration of Th1 cytokines can provide protection from
autoimmune disease (28, 29, 30). In contrast, IL-12
administration has been shown to promote autoimmune inflammation. IL-12
both accelerated diabetes onset and decreased IL-4 production by
islet-infiltrating cells when administered to prediabetic nonobese
diabetic mice (31). Ab neutralization of IL-12 was
protective for both EAE (32) and collagen-induced
arthritis (CIA) in IFN-R-/- mice
(33), and IL-12-/- mice were
resistant to EAE or CIA induction (34, 35). Collectively,
these studies demonstrate the significant contribution of IL-12 to
Th1-directed autoimmune inflammation. Indeed, IL-12 is the predominant
cytokine that promotes differentiation of Th1 effector cells
(36). Produced by macrophages, dendritic cells, and B
cells, IL-12 stimulates IFN- production from NK cells and
CD4+ and CD8+ T cells.
IL-12 exists as a 70- to 75-kDa heterodimer consisting of
disulfide-bonded 40-kDa (p40) and 35-kDa (p35) subunits, and both p40
and p35 subunits must be present for biological activity.
Interestingly, the IL-12 p40 subunit can bind with high affinity to the
IL-12R (37) and has been reported to act as an IL-12R
antagonist (38). Antagonism of the IL-12R in vivo by IL-12
p40 may inhibit the development of Th1 effector cells, thus providing
an interesting approach to therapy of autoimmune diseases. Thus, rather
than administering cytokines that could incidentally promote tissue
pathogenesis (39), we chose to regulate IL-12 via
CNS-specific delivery of an IL-12R antagonist, the IL-12 p40 subunit.
The studies presented here investigate local IL-12 p40 delivery to
provide adoptive immunotherapy of EAE using myelin basic protein
(MBP)-specific CD4+ T cells retrovirally
transduced to express IL-12 p40.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PL/J (H-2u) and B10.PL
(H-2u) mice were purchased from The Jackson
Laboratory (Bar Harbor, ME). B10.PL mice transgenic (Tg) for a
V
4/V8.2 TCR specific for MBP N-acetylated
(NAc)1–11 have been described elsewhere
(40) and were obtained from Dr. C. C. Whitacre (Ohio
State University, Columbus, OH). Mice were used between 6 and 8 wk of
age and were maintained according to institutional guidelines under
approved protocols in the Stanford Medical Center’s Department of
Comparative Medicine (Stanford, CA).
Induction of EAE
MBP peptide NAc1–11 (NAc-ASQKRPSQRHG) was synthesized and HPLC purified at the Protein and Nucleic Acid Facility (Beckman Center, Stanford University). T cell medium (complete RPMI, RPMI-C) consisted of RPMI 1640 (Life Technologies, Gaithersburg MD) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), nonessential amino acids (1%), sodium pyruvate (1 mM), HEPES buffer (10 mM), 2-ME (50 µM), and FCS (10%) (HyClone Laboratories, Logan UT). For passive EAE, splenocytes from MBP TCR-Tg mice (5 x 106 cells/ml) were cultured in RPMI-C with NAc1–11 (10 µg/ml) and 100 U/ml IL-12 (BD PharMingen, San Diego CA) and restimulated with NAc1–11, IL-12, and irradiated APCs on days 4 and 8. Cells were maintained at 37°C with 6% CO2. On day 11, 1 x 106 CD4+ MACS-isolated (Miltenyi Biotec, Auburn CA) cells were transferred i.v. to B10.PL recipients. Mice also received 200 ng pertussis toxin (List Biological Laboratories, Campbell, CA) i.p. in 0.2 ml PBS at the time of cell transfer and 48 h later.
Active EAE was induced as previously described (41). Mice were injected s.c. over four sites on the back with a total of 100 µl CFA (containing 200 µg Mycobacterium tuberculosis Jamaica strain) combined with 200 µg guinea pig MBP (Sigma, St. Louis, MO) for B10.PL mice and 100 µg guinea pig MBP for PL/J mice. Pertussis toxin was administered as described above. Animals demonstrated clinical signs (cs) within 7–14 days and were scored as follows: 1, limp tail or waddling gait with tail tonicity; 2, waddling gait with limp tail (ataxia); 2.5, ataxia with partial limb paralysis; 3, full paralysis of one limb; 3.5, full paralysis of one limb with partial paralysis of second limb; 4, full paralysis of two limbs; 4.5, moribundity; and 5, death. Daily cs are averaged for the group and mean incidence; cumulative cs, cs per day, day of onset, highest cs, and the number of relapses ± SEM are described. Mean cumulative cs was calculated by averaging the sum of daily clinical scores for individual animals. The cs per day value was calculated by dividing the cumulative cs by the number of days the animal remained in the study. To determine the mean day of onset, animals not developing EAE were not included in the analysis. To determine the mean highest cs, mice not developing EAE were assigned a value of 0 and included in the analysis. Relapses were defined by a drop in clinical score sustained for at least 2 days followed by an increase in clinical score sustained for at least 2 days.
Vectors
pGCy (6700 bp) was derived from the previously described Moloney murine leukemia virus-based retroviral vector, pGCIRES, which contains a SrfI polylinker and an encephalomyocarditis internal ribosome entry site (19). The enhanced yellow variant of green fluorescent protein (GFP) (713 bp) was PCR amplified from pEYFP-C1 (CLONTECH Laboratories, Palo Alto, CA) with oligonucleotide primers (sense, 5'-TCG CCA CCA TGG TGA GCA AGG GCG-3'; and antisense, 5'-TCC TCC GGA TCA TTA CTT GTA CAG CTC GTC CAT-3') and subcloned into pGCIRES at NcoI and BstEI sites, replacing GFP. The murine IL-12 p40 cDNA was obtained from Riken Gene Bank (Tsukuba, Japan) with permission from Dr. H. Hamada (Cancer Chemotherapy Center, Cancer Institute, Tokyo, Japan). The IL-12 p40 gene (1000 bp) was PCR amplified from pMFGmIL-12 p40 with oligonucleotide primers (sense, 5'-GGG TGC ATG CAT GTG TCC TCA GAA GCT AAC C-3'; and antisense, 5'-GCT GCC ATG GCT AGG ATC GGA CCC TGC AGG G-3') and subcloned into pGCy using SrfI restriction ligation and termed pGCy.p40 (7691 bp). For bioluminescent cell tracking, the GFP/yellow-green luciferase gene fusion (gfp/luc) (42) was isolated from pJW.GFP-yLuc.1 by restriction digestion with HpaI and BglII and was then subcloned into the Moloney murine leukemia virus-based retroviral vector pGCpgk at the SalI and BamHI restriction sites and termed pGCgfp/luc (9200 bp).
Cell lines
The Phoenix retroviral packaging cell lines are derived from a
293 T cell line and have been described elsewhere (43).
Ecotropic packaging cells (Phoenix-E) were cultured in complete DMEM
(DMEM-C), which contained DMEM (Life Technologies) supplemented with
FCS (10%), glutamine (2 mM), penicillin (100 U/ml), and streptomycin
(100 µg/ml). Phoenix-E cells were maintained in DMEM-C with
diphtheria toxin (1 µg/ml) (Calbiochem, La Jolla, CA) for selection
of the ecotropic envelope (env) gene and hygromycin B (140
µg/ml) (Boehringer Mannheim, Indianapolis, IN) for selection of the
virion assembly genes (gag-pol), and cells were removed from
selection before transfection. The NIH3T3 cell line (ATCC CCL92)
(American Type Culture Collection, Manassas, VA) was cultured in DMEM
supplemented with bovine serum (10%) (JRH Biosciences, Lenexa KS),
L-glutamine (2 mM), penicillin (100 U/ml), and
streptomycin (100 µg/ml). MBP and collagen type II (CII)-specific T
cell hybridomas were produced as previously described
(44). Briefly, spleen cells from either CII-specific or
MBP-specific TCR-Tg mice were stimulated with specific peptide Ags (40
µg/ml for CII and 30 µg/ml for MBP NAc1-11).
Forty-eight hours after stimulation, CD4+ T cells
were purified by MACS using anti-CD4 MicroBeads (Miltenyi Biotec).
These cells (1 x 106) were then fused with
the BW5147 TCR--negative T cell line (1 x
106) using 40% polyethylene glycol. Fusions were
selected in hypoxanthine-aminopterin-thymidine-supplemented medium for
3 wk, hypoxanthine-thymidine-supplemented medium for 2 wk, and
were then cultured in RPMI-C and further selected by FACS sorting for
V8.2 TCR-positive cells, yielding 
98% purity. All cells were
maintained at 37°C in 6% CO2.
Transfection assay
A total of 2 x 106 Phoenix-E packaging cells were cultured in 12 ml DMEM-C in 100-mm tissue culture dishes (Nalge Nunc International, Roskilde, Denmark). Following overnight incubation, cells were transfected with 10 µg retroviral plasmid DNA (Qiagen, Valencia, CA) or no plasmid DNA (mock) using a modified version of the calcium phosphate precipitation protocol, which is described elsewhere (43). At 8–12 h after transfection, calcium phosphate-containing medium was replaced with DMEM-C, and cultures were maintained at 37°C in 6% CO2 for 24–48 h and then at 32°C in 6% CO2 for 16–24 h. Viral supernatant from transfected cultures was harvested and filtered using a 0.45-µm filter (Nalge Nunc International) and was then stored at -80°C. Virus titers were determined using NIH3T3 lines as previously described (45), and virus stocks with titers >4 x 106 were used.
Transduction assay
Splenocytes from MBP TCR-Tg mice (5 x 106 cells/ml) were cultured in RPMI-C with NAc1–11 (10 µg/ml) for 24 h. Then, 2–5 x 106 activated splenocytes or unactivated NIH3T3 cells were cultured in six-well plates (0.5 ml/well) and overlaid with 2–3 ml thawed recombinant retroviral supernatant supplemented with protamine sulfate (8 µg/ml; Sigma). Plates were centrifuged at 2500 rpm at 32°C for 2 h and transferred to 32°C in 6% CO2 for 16 h. Media was exchanged with RPMI-C medium supplemented with 10 U/ml murine rIL-2 (R&D Systems, Minneapolis, MN) and transferred to 37°C in 6% CO2 for an additional 24 h.
FACS analysis
Cells were stained with rat anti-mouse CD4-PE (Caltag Laboratories, San Francisco CA), and dead cells were excluded by propidium iodide (PI) (Sigma). Yellow fluorescent protein (YFP) reporter expression was analyzed using the FITC channel and isolation of YFP+CD4+ cells was conducted using a FACStar flow cytometer (Stanford FACS Facility, Stanford University). Sorted samples were then reanalyzed to confirm purity (>95%). Cell surface analysis was performed on a FACScan cytometer (BD Biosciences, Mountain View CA). All data were analyzed using FlowJo (version 3.1.1) flow cytometry software (Tree Star, San Carlos, CA).
ELISA analysis
Protein production was measured by a standard sandwich ELISA
protocol. Cells were Ag stimulated in RPMI-C or X-Vivo 20 serum-free
medium (TGF-) (BioWhittaker, Walkersville MD), and supernatants were
harvested at 48 (IL-2, IL-4, IL-5, and IFN-) and 72 h (IL-12
p40, IL-10, and TGF-). Capture Abs were incubated in 96-well
Maxisorp ELISA plates (Nalge Nunc International) at 4°C overnight
with 1 µg/ml of anti-IL-2, IL-4, or IL-5, 4 µg/ml of
anti-IFN-, 5 µg/ml of anti-IL-10, 6 µg/ml of
anti-IL-12 p40 (C15.6) (BD PharMingen), or 1.5 µg/ml of chicken
anti-TGF- (R&D Systems). After washing and a 1-h room
temperature incubation with 5% FCS in PBS or 0.5% enzyme
immunoassay-grade gelatin (TGF-) (Bio-Rad, Hercules, CA), 50
µl sample or standard dilution of murine rIL-2, murine rIL-4, murine
IL-5, murine rIFN-, murine rIL-10, murine rIL-12 p40 (BD
PharMingen), or human rTGF- (R&D Systems) was incubated overnight at
4°C. TGF- samples (100 µl) were acidified for 15 min with 4 µl
1N HCl and were neutralized with 4 µl NaOH before analysis.
Biotinylated anti-IL-2 (0.5 µg/ml), IL-4 (0.5 µg/ml), IL-5 (1
µg/ml), IFN- (0.5 µg/ml), IL-10 (1 µg/ml), or IL-12 p40
(C17.8, 1 µg/ml) (BD PharMingen) was added for 3 h at 4°C. For
detection of TGF-, 0.5 µg/ml mouse anti-TGF- 1, 2, 3
(Genzyme, Cambridge MA) was added for 2 h, followed by washing,
then a 1-h incubation with 2 µg/ml of biotinylated horse
anti-mouse IgG (Vector Laboratories, Burlingame CA). For all
cytokines, biotinylated Abs were removed, plates were washed, and
extravidin (1:1000) (Sigma) was added for 45 min. Wells were developed
with 3,3',5,5'-tetramethyl-benzidine substrate (Sigma), and the
reaction was stopped with 1N HCl. Plates were read at 450 nm on a
microtiter plate reader (Wallac, Gaithersburg, MD). Cytokine
concentrations (nanograms per milliliter) were determined by comparing
the OD of samples to the standard curve.
Proliferation assay
Splenocytes from MBP TCR-Tg mice were cultured in round-bottom 96-well plates (4 x 105 cells/well) with or without NAc1–11 (10 µg/ml) in RPMI-C for 72 h. Wells were pulsed with 1 µCi [3H]thymidine 18 h after culture. Cultures were harvested onto glass-fiber filter mats using a Skatron harvester (Molecular Devices, Sunnyvale, CA) and counted by liquid scintillation on a Wallac Betaplate (Wallac). The means of replicate wells were determined, and results are expressed as mean stimulation index, which was calculated as follows: (mean cpm of cultures with Ag/mean cpm of cultures with medium alone) ± SEM.
Bioluminscent imaging
Bioluminescent imaging was performed as previously described
(46). Briefly, 1 x 106 MBP
TCR-Tg splenocytes tranduced with pGCgfp/luc were transferred i.v. to
PL/J recipients. Luciferin substrate (126 mg/kg) (Biosynth
Technologies, Naperville IL) was injected i.p., and then mice were
anesthetized with 250 mg/kg Avertin (Aldrich, Milwaukee WI). Imaging
was conducted using an IVIS imaging system equipped with a cooled
charge-coupled device camera (Xenogen, Alameda CA) and a Navitar f 0.9
lens (Navitar, Rochester, NY). Mice were imaged in dorsal, ventral,
then lateral positions by collecting two images, a grayscale body
surface reference image collected under weak illumination and an image
of light emission from the animals. The emission images were collected
with 5-min integration times, and pseudocolor representation of light
intensity (red being the most intense and blue being the least intense)
was superimposed over the grayscale body surface reference image. Data
acquisition and analyses were performed using the LivingImage (Xenogen)
software overlay on the IgorPro image analysis package (WaveMetrics,
Seattle, WA). Animals recovered from the anesthetic after 
30 min
under close supervision and were imaged again at the indicated time
points.
Histology
Tissue samples were harvested and fixed in 4% buffered
paraformaldehyde for 8 h and were then removed to 30% buffered
sucrose for 4 h. Frozen samples embedded into OCT compound 4583
(Tissue-Tek, Torrence CA) were then cut into 6-µm sections. Sections
were overlaid with PI mounting medium (Vectashield; Vector
Laboratories, Burlingame CA), air-dried for 30 min, then examined using
fluorescent microscopy for YFP-positive cells (FITC excitation) and
PI-positive cells (PE excitation) (Khavari Laboratory, Stanford
University).
Statistical analysis
Experiments with two groups were analyzed by nonparametric ANOVA with Mann Whitney U analysis. Differences in experiments with more than two groups were determined by one-way ANOVA with Tukey-Kramer multiple comparison analysis. All analyses was performed using InStat 2.01 software (GraphPad, San Diego, CA), and values were considered significantly different at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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production in
transduced MBP-specific T cells
We have recently established an efficient method of transduction
of murine CD4+ T cells using a retroviral vector
that allowed bicistronic expression of both the therapeutic transgene
and a GFP marker (19). GFP has since been further
mutagenized to form a YFP (47, 48). YFP has GFP excitation
with red-shifted emission, thus providing a brighter marker protein
also detected using the FITC channel. Therefore, YFP was used in the
therapeutic retroviral vector used for these studies (Fig. 1
A). Splenocytes from MBP
TCR-Tg mice were infected with recombinant retrovirus containing the
IL-12 p40 transgene, and FACS analysis demonstrated that 60% of
CD4+ cells were transduced (i.e., YFP positive;
Fig. 1B). Cells transduced with empty vector had similar
transduction efficiencies, whereas mock-transduced (no vector) cells
had no YFP expression (data not shown). Transduced cells were sorted by
FACS into low and high YFP-expressing populations and restimulated with
Ag in vitro. Supernatants were analyzed for IL-12 p40 content by ELISA,
and data presented in Fig. 1C demonstrate that IL-12 p40
protein levels correlated with YFP protein expression and are
significantly higher in "p40 high"-transduced populations.
Supernatants were also analyzed for T cell cytokines. There was no
observable change in IL-4, IL-10, IL-5, TGF- (data not shown), or
IL-2 production (Fig. 1D, 
). However, IFN- levels were
markedly reduced with increased levels of IL-12 p40 expression (Fig. 1D, 
). These data suggest that IL-12 p40 can inhibit Th1
differentiation (i.e., IFN- production) of transduced T cells
without hindering T cell viability (i.e., IL-2 production) or promoting
Th2 development. However, for therapeutic application,
transgene-derived IL-12 p40 must also regulate existing Th1 cells
within the autoimmune lesion.
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production in developing and effector
CD4+ T cell populations
To assess the regulatory effects of IL-12 p40 on Th1 cells,
transgene-derived IL-12 p40 was overlaid onto Th1 cell cultures. For
robust production of transgene-derived IL-12 p40, NIH3T3 cells were
transduced with the IL-12 p40 retroviral vector. Transduction in NIH3T3
cells yielded 98% efficiency, and supernatants containing
transgene-derived IL-12 p40 were collected and quantified by ELISA.
Immunoprecipitation using supernatants from NIH3T3 cells transduced for
expression of IL-12 p40 confirmed the presence of both monomeric and
dimeric IL-12 p40 species (data not shown). Splenocytes from MBP TCR-Tg
mice were then cultured with the immunodominant
NAc1–11 MBP epitope and increasing
concentrations of transgene-derived IL-12 p40. ELISA analysis of
supernatants demonstrated that without IL-12 p40 (0 ng/ml),
MBP-specific T cells underwent Th1 differentiation (i.e., IFN-
production) upon Ag stimulation in vitro (Fig. 2A, ). There were no
observable differences in IL-2, IL-4, or IL-10 production (data not
shown) or in proliferative responses to Ag following IL-12 p40 addition
(Fig. 2A, ). However, IFN- production was markedly
reduced. These data demonstrate the ability of IL-12 p40 to inhibit Th1
differentiation in developing CD4+ T cells
without altering proliferative responses or promoting Th2 cytokine
production.
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B ()
support what is typically observed with MBP-specific Tg cells, in that
effector T cells proliferate less robustly than after primary Ag
stimulation (Fig. 2A). However, it is important to note that
proliferation responses are not altered by IL-12 p40 supplementation.
In contrast, T cells that produced high amounts of IFN- after
primary Ag stimulation produced significantly less IFN- with the
addition of IL-12 p40 at secondary Ag exposure (). Subsequent
experiments have shown that CD4+ T cells must be
exposed to IL-12 p40 within 12 h of Ag exposure to inhibit IFN-
production (data not shown). These observations demonstrate that IL-12
p40 has a significant inhibitory effect on Th1 effector cells, which
suggests that the encephalitogenic potential of Th1 cells may also be
suppressed by IL-12 p40. IL-12 increases encephalitogenicity of TCR-Tg MBP-specific T cells
To demonstrate the role of bioactive IL-12 on encephalitogenicity
of MBP-specific Tg cells, splenocytes from MBP TCR-Tg mice were Ag
stimulated in vitro with or without IL-12 supplementation. Passive EAE
was then initiated by transferring activated cells to syngeneic non-Tg
recipients. As shown by data presented in Fig. 3A, the addition of IL-12 to
cultures significantly enhanced the encephalitogenicity of MBP-specific
cells. Mean disease incidence (3 of 6 vs 4 of 4) and cumulative
clinical score ± SEM (6.8 ± 3.1 vs 24.9 ± 5.4*)
increased in mice receiving IL-12-treated cells. In addition, the day
of disease onset was earlier (12.3 ± 0.2 vs 10.0 ± 1.2),
and the highest clinical score achieved (1.0 ± 0.4 vs 2.8 ±
0.5) was greater upon transfer of 1 x 106
IL-12-treated cells. Of the mice receiving IL-12-treated cells, 3 of 4
reached peak clinical scores (2.5, 3, and 4) by day 12 after transfer,
whereas all mice receiving cells not treated with IL-12 demonstrated a
very mild EAE clinical course (maximum disease score of 2). It is
likely that IL-12 enhanced Th1 differentiation of the Ag-primed cells,
because supernatant from IL-12-treated cultures demonstrated a 5-fold
increase in IFN- production over non-IL-12-treated cells (data not
shown). These studies establish the importance of bioactive IL-12 in
promoting the encephalitogenic response and suggest that local
targeting of the IL-12 pathway could provide protection from
EAE.
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For CNS-targeted delivery of an IL-12R antagonist, MBP-specific T
cell hybridomas were transduced to express IL-12 p40 and were
adoptively transferred to syngeneic mice immunized to develop EAE. Data
presented in Fig. 3B demonstrate that IL-12 p40-transduced
MBP-specific hybridomas significantly suppress disease. Interestingly,
adoptive transfer of T cell hybridomas specific for CII and producing
equivalent amounts of IL-12 p40 had no effect on EAE in similarly
immunized mice but were therapeutic when transferred to mice with CIA
(49). The mean cumulative clinical scores ±
SEM (12.6 ± 5.4 vs 34.1 ± 4.2 and 34.3 ± 9.1),
clinical score per day (0.4 ± 0.2 vs 1.1 ± 0.1 and 1.1
± 0.3), highest clinical score (1.2 ± 0.5* vs 3.0 ± 0.3
and 3.0 ± 0.4), and the number of relapses (0.0 vs 1.0 ±
0.3 and 0.4 ± 0.2) were also reduced in IL-12 p40-treated mice
when compared with nontreated or CII-specific hybridoma-treated mice,
respectively. These data suggest that the site-specific TCR expression
is required for therapy, presumably providing transduced cell retention
by specific Ag recognition in the inflammatory lesion.
IL-12 p40-transduced primary T cells can suppress EAE
Due to the intrinsic hazards of administration of transformed
(hybridoma) cells in vivo, it was preferable to establish an IL-12 p40
adoptive immunotherapy protocol using untransformed T cells. Therefore,
similar experiments were performed using primary T cells from MBP
TCR-Tg mice. MBP-specific CD4+ T cells were
transduced to express IL-12 p40 and adoptively transferred to mice
immunized for EAE. Following encephalitogenic challenge, B10.PL mice
exhibited an initial acute disease followed by intermittent relapses
and/or chronic EAE. To determine whether IL-12 p40 could provide
therapeutic benefit in vivo, 1 x 106 IL-12
p40-transduced MBP-specific CD4+ T cells were
adoptively transferred just before disease onset (day 10 after MBP
immunization). Because the disease course is unique for each animal,
the mean daily clinical score for each group is shown. Disease was
significantly reduced in mice receiving IL-12 p40-transduced cells
(Fig. 3C). Mean cumulative clinical score ± SEM
(49.5 ± 9.3 vs 32.8 ± 8.7), clinical score per day
(1.4 ± 0.3 vs 0.9 ± 0.2), highest clinical score (3.3
± 0.4 vs 2.2 ± 0.4), and the number of relapses (0.5 ± 0.2
vs 0.2 ± 0.2) from the day of treatment through 35 days after
transfer were also reduced in IL-12 p40-treated mice. It is important
to note that the transfer of vector-only-transduced MBP-specific cells
did not alter disease when compared with nontreated control mice (data
not shown). Furthermore, the amount of IL-12 p40 produced by the 1
x 106 transferred IL-12 p40-transduced T cells
(40 ng/ml; Fig. 1C) was approximately equal to levels
that inhibited the activation of Th1 cells (50 ng/ml; Fig. 2), as
demonstrated by in vitro analysis. These studies have demonstrated the
therapeutic potential of IL-12 p40 delivery. However, even though
analysis of serum samples 4 and 12 days after cell transfer did not
demonstrate detectable systemic levels of IL-12 p40 protein (data not
shown), these data alone do not demonstrate that the therapeutic IL-12
p40 transgene is delivered locally to sites of inflammation.
MBP-specific transduced CD4+ T cells traffic to the CNS
To assess the trafficking kinetics of transduced MBP-specific
CD4+ T cells, we used in vivo bioluminescent
imaging, which allows for sensitive detection of labeled cells in
rodent models of human disease (46). This technique used T
cell expression of a firefly luciferase gene to track T cell
trafficking patterns in vivo. The enzymatic reaction between luciferase
and its substrate, luciferin, causes photon emission that can be
detected by a cooled charge-coupled device camera and represented as a
pseudocolor image of light intensity overlaid onto a grayscale image of
an anesthetized mouse. Recipients with white coats minimize light
absorption; therefore, syngeneic PL/J mice were used for these studies.
Although this mouse strain is susceptible to EAE, under our
MBP/CFA/pertussis toxin immunization protocol, PL/J mice typically
demonstrated a mild EAE clinical course with late onset and no
relapses, which is in contrast to the robust relapsing/chronic EAE in
B10.PL mice used to study the therapeutic efficacy of IL-12 p40 (Fig. 3, B and C). MBP-specific
CD4+ T cells were transduced with a retroviral
vector containing a GFP-luciferase gene fusion (pGCgfp/luc; Fig. 1A) and were then analyzed and sorted using FACS. Cells
expressing the GFP/luc marker protein were transferred (i.v.) to naive
or MBP-immunized recipients before the onset of EAE. Upon peritoneal
administration of luciferin, luciferase-expressing cells could be
detected in the lungs within 5 min of transfer (Fig. 4A). One day after transfer,
mice were reanesthetized, and image analyses demonstrated
luciferase-positive cells within the peripheral lymph nodes, spleen,
and at the sites of immunization. Luciferase-positive cells were
retained only in MBP-immunized mice and were demonstrable in regions
consistent with the lumbar and thoracic regions of the spinal cord at
approximately the time of disease onset. Interestingly, the time
required for CD4+ T cells to traffic to
CNS-related sites correlated with the kinetics of therapeutic effects
after transfer of IL-12 p40-transduced cells (Fig. 3C).
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8 days in naive mice (n = 2). However,
in MBP/CFA-immunized mice, luciferase-positive cells were present in
sites of immunization, and five of six mice with EAE demonstrated
luciferase-positive cells in the brain within 3 days of transfer (Fig. 4B). No luciferase-positive cells were detected in the CNS
of naive control mice at any time, and the administration of pertussis
toxin alone did not alter transduced cell trafficking when compared
with naive control mice (data not shown). These observations suggest
that T cell trafficking was accelerated following the breech of the
blood-brain barrier. Further analysis demonstrated that
luciferase-positive cells were detected as long as 50 days after
transfer (data not shown). Therefore, these studies suggest that
retrovirally transduced CD4+ T cells traffic to
sites of autoimmune inflammation and are capable of long-term
persistence. Retrovirally transduced T cells provide long-term local transgene expression
To confirm that therapeutic transgenes are delivered and expressed
locally, histological evaluation was performed on brain and spinal cord
sections from EAE mice that were treated with IL-12 p40-transduced
cells. Approximately 30 days after adoptive transfer,
YFP+ cells were detected in both brain (Fig. 5) and spinal cord sections (data not
shown). There was minimal fluorescence emission in organs from mice
receiving nontransduced MBP-specific cells (Fig. 5). However,
YFP+ cells were detected in sections from spleen
and cervical lymph nodes of MBP-immunized mice receiving vector or
IL-12 p40-transduced MBP-specific cells (data not shown). It is
important to note that vector-only as well as IL-12 p40-transduced cell
recipients demonstrated YFP+ cells in the CNS
upon histopathological analysis. Therefore, both populations of
retrovirally transduced CD4+ T cells are capable
of trafficking to the CNS, but only IL-12 p40-transduced cells provided
therapeutic effects (Fig. 3C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Analysis in vitro demonstrated IL-12 p40
regulation of IFN- production (Figs. 1 and 2). Th1 cytokine
production from developing and effector Th1 cells was inhibited without
altering T cell viability or proliferative responses. These data
suggest that IL-12 p40 can act therapeutically by interrupting
Th1-mediated inflammatory autoimmune responses.
The differentiation of CD4+ T cells into Th1
cells is greatly influenced by the presence of bioactive IL-12 during
Ag recognition (50, 51). Therefore, the expression of the
IL-12R plays a critical role in determining the Th1-Th2 balance during
the course of an immune response. Unlike the IL-12R 1 chain, the
IL-12R 2-chain is not expressed on naive or resting T cells, but is
induced after TCR engagement with Ag/MHC class II on APCs
(52). As schematically represented in Fig. 6, the T cell up-regulates CD40 ligand
(CD40L) surface expression upon activation, which binds to CD40 on the
activated APC. Interestingly, CD40-mediated signaling is thought to be
more effective in DC activation than engagement of MHC class II
molecules (53). This CD40L-CD40 interaction triggers the
APC to secrete preformed stores of bioactive IL-12 (p35/p40). IL-12
binding to the high affinity IL-12R, consisting of both the 1 and
2 chains, activates STAT1, STAT3, and STAT4 and results in the
production of IFN- and Th1 differentiation (54). The
p40 subunit of IL-12 has been proposed to bind to the high affinity
IL-12R and block engagement of bioactive IL-12 (37), thus
inhibiting Th1 commitment. It is known that
IFN--/- mice are susceptible to EAE
(25); therefore, it is likely that other cytokines also
influence Th1 differentiation. IL-4 can regulate the development of Th1
cells by inhibiting expression of the IL-12R 2 chain. However, this
inhibition can be overcome by high levels of IFN-, even in cells
that have begun to differentiate along the Th2 pathway (52, 55). Thus, Th1 differentiation is critically dependent on the
cytokine microenvironment during T cell priming, and IL-12 p40
expression may inhibit such pathways. Interestingly, the IL-12 p40
subunit can also associate with a p19 subunit produced by dendritic
cells to form the newly characterized cytokine, IL-23
(56). It has been suggested that IL-23 may have a similar
biological function to IL-12. Although p19 expression within the CNS
has not yet been described, it is known that astrocyte production of
IL-12 will promote the development of type 1 T cell cytokine responses
and NK cellular immunity (57). Therefore, it remains
possible that the therapeutic effects of IL-12 p40 may be attributable
to inhibition of both IL-12- and IL-23-mediated signaling during CNS
inflammatory responses.
|
A), in which luciferase
production was no longer detected in naive mice once transduced T cells
returned to a quiescent state (9 days after transfer). Interestingly,
we observed that MBP-specific hybridomas were more efficient in
reducing EAE clinical signs when compared with primary-transduced T
cells (Fig. 3, B and C), even though each can
produce comparable levels of IL-12 p40. It is well established that T
cell hybridomas are greatly dysregulated in their growth and cytokine
production. Therefore, transduced hybridomas will produce transgene and
proliferate at a constant rate. In contrast, transduced primary T cells
require Ag presentation for activation, proliferation, and transgene
production. Therefore, not only is more time required to establish a
"therapeutic" population of transduced primary T cells, but it is
also highly likely that primary T cells are more subject to in vivo
regulatory processes when compared with T cell hybridomas. Thus,
transduced primary T cells offer an effective yet more regulated method
for Ag-specific in vivo immunotherapy.
In vitro analysis demonstrated the ability of IL-12 p40 to inhibit both
developing and effector Th1 populations (Fig. 2). These observations
were also evidenced in vivo by the ability of IL-12 p40 to suppress EAE
clinical signs when cells were administered 10 days after MBP
immunization (Fig. 3C), at which time effector Th1
populations most likely already exist within the CNS. Furthermore, T
cell expression of IL-12 p40 provided long-term disease suppression,
even though recipient mice were actively immunized, which most likely
results in the continual repopulation of encephalitogenic T cells.
Interestingly, the amelioration of the relapsing/chronic phase of EAE
suggests that responses to other CNS Ags could also be suppressed by
IL-12 p40 expression. To inhibit T cell IFN- production in vitro, we
observed that IL-12 p40 must be present in sufficient quantities within
12 h of Ag exposure. Therefore, the encephalitogenicity of T cells
stimulated by Ag before sufficient local expression of IL-12 p40 will
not be inhibited. Thus, the observed delay between cell transfer and
reduced cs may be attributed to the time required for site-specific
transduced T cell trafficking, replacement of the proinflammatory
environment with IL-12 p40-mediated suppression, and resolution of
established inflammation and restoration of motor ability. Preliminary
studies suggest that the administration of IL-12 p40-transduced cells
during established disease may also suppress EAE; however, further
investigation is required (G. L. Costa and J. M. Benson,
unpublished observations).
These studies demonstrate that IL-12 p40 did not alter T cell viability
or proliferation (Figs. 1 and 2); therefore, adoptively transferred
IL-12 p40-transduced CD4+ T cells were expected
to retain the ability to traffic to autoimmune sites. This was
confirmed by in vivo bioluminescent studies that demonstrated
trafficking of luciferase-transduced T cells to the CNS of mice
immunized for EAE (Fig. 4). In addition, transduced
CD4+ T cells were also present in secondary
lymphoid tissue. Yet, if expression of the IL-12 p40 transgene in
secondary lymphoid tissue was sufficient for CNS therapy, it would be
expected that adoptive transfer of non-antigen-specific
CD4+ T cells tranduced for IL-12 p40 expression
would also provide therapeutic effect. In contrast, we found that
collagen-reactive T cell hybridomas transduced to express IL-12 p40
were not protective when adoptively transferred into recipients
immunized for EAE (Fig. 3B). The lack of protection is most
likely due to inadequate retention in sites of autoimmune lesions
because subsequent bioluminescent cell trafficking studies have
demonstrated that collagen-reactive T cell hybridomas traffic to and
persist in peripheral lymph nodes in patterns similar to MBP-specific T
cell hybridomas (49). Thus, even though it cannot be
conclusively determined whether peripheral expression of the IL-12 p40
transgene influenced disease suppression, it is likely that local
expression is required for amelioration of EAE. These experiments
support previous studies that describe the requirement of site-specific
TCR for adoptive immunotherapy (23, 59) and provide
definitive evidence that the IL-12 p40 subunit can effectively block
CNS autoimmune Th1 responses in vivo.
Local delivery of an IL-12R antagonist represents a therapeutic
strategy for the treatment of organ-specific autoimmune diseases that
may be preferable to direct cytokine delivery. Data presented here
supports previous observations of IL-12 p40-mediated suppression of
diabetes in nonobese diabetic mice (60, 61). However, we
have shown that site-specific expression of the IL-12 p40 subunit can
be used to target and ablate autoimmune lesions without demonstrable
side effects that are associated with elevated levels of regulatory
cytokines (21, 39). Although it was not anticipated that
high concentrations of receptor antagonist would exacerbate disease,
the YFP marker protein allowed cells to be sorted for low or high
transgene expression to assess regulatory effects. We demonstrated that
IL-12 p40 exhibited therapeutic benefit at levels as low as 40 ng/ml
and had no detrimental or toxic effects on T cell populations even when
expressed at high levels (100 ng/ml) (
Figs. 1–3). Obtaining
autoantigen-specific CD4+ T cells from patients
with autoimmune diseases would be technically demanding; however, the
work presented here demonstrates the efficacy of adoptive gene therapy
using very low numbers of transduced cells. These data suggest that
stable expression of antagonist molecules, such as IL-12 p40, using
autoreactive CD4+ T cells that traffic
preferentially to inflammatory lesions may provide an effective
adoptive immunotherapy for organ-specific autoimmune diseases.
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Footnotes |
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2 G.L.C. and J.M.B. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. C. Garrison Fathman, Division of Immunology and Rheumatology, Department of Medicine, Stanford University School of Medicine, Room 2225, Center for Clinical Science Research Building, 300 Pasteur Drive, Stanford, CA 94305-5166. E-mail address: cfathman{at}stanford.edu
4 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; CIA, collagen-induced arthritis; MBP, myelin basic protein; RPMI-C, complete RPMI medium; cs, clinical signs; GFP, green fluorescent protein; YFP, yellow fluorescent protein; Tg, transgenic; DMEM-C, complete DMEM; CII, collagen type II; PI, propidium iodide; CD40L, CD40 ligand; NAc, N-acetylated.
Received for publication February 28, 2001. Accepted for publication June 11, 2001.
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