Demyelinating Disease Regulatory T Cells During Autoimmune ...

Pre-Emptive Targeting of the Epitope Spreading Cascade with Genetically Modified Regulatory T Cells During Autoimmune Demyelinating Disease1 Ling Yin,2 Min Yu, Andrea E. Edling, Julie A. Kawczak, Peter M. Mathisen,3 Tania Nanavati, Justin M. Johnson, and Vincent K. Tuohy4 Epitope spreading or endogenous self-priming has been implicated in mediating the progression of autoimmune disease. In the present study we created an immune-deviated, epitope spreading response in SWXJ mice after the onset of experimental autoimmune encephalomyelitis, a prototypic autoimmune animal model widely used in multiple sclerosis research. We established an immunoregulatory spreading repertoire by transferring T cells genetically modified to produce high levels of IL-10 in response to a dominant epitope spreading determinant. Installation of a Th2/Tr1-like spreading repertoire resulted in a marked and prolonged inhibition of disease progression and demyelination characterized by 1) bystander inhibition of the recall response to the priming immunogen, and 2) a Th13 Tr1 immune-deviated spreading response involving a shift in the source of IL-10 production from the transferred regulatory population to the host-derived, endogenously primed repertoire. Thus, our data provide a rationale for cell-based therapeutic intervention in multiple sclerosis by showing that pre-emptive targeting of the epitope spreading cascade with regulatory T cells effectively induces an immune-deviated spreading response capable of inhibiting ongoing inflammatory autoreactivity and disease progression. The Journal of Immunology, 2001, 167: 6105– 6112.

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ultiple sclerosis (MS),5 and its related animal model, experimental autoimmune encephalomyelitis (EAE), are often characterized by a relapsing-remitting clinical course that eventually leads to progression of symptoms and chronic debilitation (1–3). Although there is no consensus regarding a single underlying mechanism that may account for chronicity in autoimmune demyelination, there is considerable evidence that progression of EAE may be due in part to immunoregulatory failures that allow reactivation of memory T cells specific for the priming determinant used to initiate disease (4 –9). In addition, recent studies have implicated acquired neoautoreactivity or epitope spreading in chronic progression of autoimmune disease (10 –12). We have shown that in SWXJ mice primed with the immunodominant encephalitogenic determinant of myelin proteolipid protein (PLP)139 –151, progression to chronicity is invariably accompanied by the sequential accumulation of Th1 neoautoreactivity directed against the immunodominant determinant of myelin basic protein (MBP)87–99 as well as other encephalitogenic pep-

Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195 Received for publication January 2, 2001. Accepted for publication September 19, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants NS-36054 and NS-37476 and National Multiple Sclerosis Society Grant RG-3070. 2 Current address: Department of Neurology, General Hospital of the People’s Liberation Army #301, Beijing, People’s Republic of China. 3

Current address: Aventis Pharmaceuticals, Bridgewater, NJ 08807.

4

Address correspondence and reprint requests to Dr. Vincent K. Tuohy, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, NB30, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: [email protected] 5 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; PLP, myelin proteolipid protein.

Copyright © 2001 by The American Association of Immunologists

tides of PLP, including PLP178 –191. Thus, progression to chronicity in the SWXJ mouse model is distinguished by the development of the defined epitope spreading cascade PLP139 –1513 MBP87–993 PLP178 –191 in mice initially primed to PLP139 –151 (12). EAE progression may be inhibited when Th2- or Tr1-like T cells specific for the PLP139 –151 priming determinant are adoptively transferred after the onset of disease (13, 14) or when tolerance to spreading determinants is induced after disease onset (11, 12). However, there are presently no studies showing that spreading determinants may serve as effective targets for adoptive immunotherapy. Therefore, in the current study we examined the efficacy of pre-emptive targeting of the epitope spreading cascade by adoptive transfer of regulatory T cells after onset of disease. We hypothesized that transfer of regulatory T cells specific for components of the epitope spreading cascade may pre-emptively establish an immune-deviated bias in the development of neoautoreactivity. Our experimental approach involved the generation of a panel of Th2/Tr1-like T cell lines specific for the immunodominant MBP87–99 spreading determinant and for control Ags, including PLP104 –117, an immunodominant determinant not substantially involved in the early epitope spreading cascade in SWXJ or SJL/J mice (11, 12). We generated Th2/Tr1-like T cell lines by transfecting primed lymph node cells with a transgene construct designed to produce mouse IL-10 regulated by a mouse IL-2 promoter (IL-2Prom3 IL10cDNA). This method provides a reliable and efficient way to generate stable Ag-specific Th2-like T cells (14) that show many of the features of high IL-10-producing Tr1 regulatory T cells (15). We found that after induction of EAE with PLP139 –151, adoptive transfer of MBP87–99-specific Th2/Tr1-like T cells resulted in a marked and prolonged inhibition of disease progression accompanied by a significant decrease in spinal cord demyelination as determined by quantitative immunocytochemistry. Transfer of Th2/ Tr1-like T cell lines specific for the PLP104 –117 control determinant or for the irrelevant BSA Ag had no comparable effect 0022-1767/01/$02.00

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PRE-EMPTIVE TARGETING OF EPITOPE SPREADING IN EAE

on disease progression. The observed therapeutic effect was accompanied by two concurrent immunoregulatory events: 1) bystander inhibition of the recall response to the priming immunogen characterized by decreased production of IFN-␥, and 2) a Th13 Tr1 immune deviation of the response to the MBP87–99 spreading determinant characterized by enhanced production of IL-10 and decreased production of IFN-␥, IL-2, and IL-12. Moreover, we observed a shift in the source of IL-10 production from the transferred regulatory T cells to native T cells endogenously primed to the MBP87–99 spreading determinant. Thus, our data indicate that the transferred regulatory T cells actively induced a host-derived, immune-deviated spreading response capable of inhibiting ongoing inflammatory autoreactivity and disease progression.

2Prom3 IL-10cDNA transgene, 1.0 ␮g/ml pSV2neo plasmid (ATCC 37149), and 20 ␮g/ml hexadimethrine bromide (Sigma-Aldrich) in DMEM (Life Technologies) supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 2 mM fresh L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 30 mM HEPES buffer (Life Technologies). 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 (BD PharMingen, San Diego, CA) and 5 ⫻ 106 x-irradiated (2 ⫻ 103 rad) syngeneic splenocytes per well. After 48 h, cultures were treated with 1.0 mg/ml Geneticin (Sigma-Aldrich; 700 ␮g/ml active substance) and 50 IU/ml mouse IL-2. Cells were harvested at 6 days, reactivated with Ag plus splenocyte feeders in 24-well plates at 5 ⫻ 105 cells/well, and expanded conventionally with at least three activation/ rest cycles involving, respectively, Ag/IL-2 treatments. Before transfer into EAE mice, the Ag specificities and cytokine profiles of the generated T cell lines were confirmed by proliferation and ELISA analyses, respectively, as described below.

Materials and Methods

Proliferation assays

Mice

On day 35 after EAE onset (day 29 after transfer of Th2/Tr1-like T cells), spleens were excised and T cells were partially purified by centrifugation on a Ficoll/sodium metrizoate gradient as described above. Cells collected from the interface were washed in HBSS and resuspended in DMEM supplemented as described above. Cells were cultured in triplicate test and control wells at 2 ⫻ 105 cells/well in a total volume of 200 ␮l in flat-bottom 96-well microtiter Falcon plates (BD Biosciences, Lincoln Park, NJ). Test wells contained various doses of MBP87–99, PLP139 –151, PLP104 –117, or BSA. Positive control wells contained 10 ␮g/ml anti-mouse CD3 (BD PharMingen), 10 –100 U/ml tuberculin purified protein derivative (Medeva, Surrey, U.K.), or 20 ␮g/ml M. tuberculosis H37RA (Difco), while negative control wells contained no peptide. In each experiment dose responses (0.1–100 ␮g/ml) were also tested to whole human PLP prepared from a washed total lipid extract of human brain white matter (16) that was purified (17) and converted to the aqueous form as previously described (18). After 72 h of culture, wells were pulsed with [methyl-3H]thymidine (l.0 ␮Ci/well; sp. act., 6.7 Ci/mmol; New England Nuclear, Boston, MA). Sixteen hours after pulsing, cultures were harvested by aspiration onto glass-fiber filters. Levels of incorporated radioactivity were determined by scintillation spectrometry. Results are expressed as the mean cpm of triplicate experimental cultures with Ag divided by the mean cpm of cultures without Ag (stimulation index).

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Female SWXJ.Thy-1b/b (H-2 ) mice were bred at Lerner Research Institute (Cleveland, OH) by mating SWR/J.Thy-1b (H-2q) females with SJL/ J.Thy-1b (H-2s) males purchased from The Jackson Laboratory (Bar Harbor, ME). In addition, female SWXJ.Thy-1a/b mice were generated by mating conventional SWR/J.Thy-1b females with SJL/J.Thy-1a males provided by Dr. H. Tse (Wayne State University, Detroit, MI). All protocols were approved by the institutional animal care and use committee in compliance with the Public Health Service policy on humane care and use of laboratory animals.

Peptides PLP139 –151 (HSLGKWLGHPDKF; serine for cysteine at residue 140), PLP104 –117 (KTTICGKGLSATVT), and MBP87–99 (VHFFKNIVTPRTP) were synthesized at the Molecular Biotechnology Core Facility of Lerner Research Institute with standard solid phase methodology using 9-fluorenylmethoxycarbonyl side chain-protected amino acids. Peptides were purified ⬎90% by reverse phase HPLC, and their identities were confirmed by mass spectrometry.

EAE induction and clinical evaluation EAE was induced by s.c. injection in the abdominal flanks with 100 nmol PLP139 –151 (154 ␮g) and 400 ␮g of Mycobacteria tuberculosis H37RA (Difco, Detroit, MI) in 200 ␮l of an emulsion of equal volumes of water and IFA (Difco). Each mouse was also injected i.v. on days 0 and 3 with 0.60 ⫻ 1010 Bordetella pertussis bacilli (Michigan Department of Public Health, Lansing, MI). After immunization all mice were weighed and examined daily for neurologic signs according to the following criteria: 0, no disease; l, 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; and 5, moribund state.

The IL-2Prom3 IL-10cDNA transgene The plasmid construct IL-2Prom3 IL-10cDNA was designed so that a mouse IL-2 promoter would regulate the expression of mouse IL-10 cDNA. The IL-2Prom3 IL-10cDNA construct was generated by subcloning a mouse IL-2 promoter region (⫺1890 to ⫹50; a gift from Dr. E. Rothenberg) and mouse IL-10 cDNA (pcD(SR␣)-F115; ATCC 68027, American Type Culture Collection, Manassas, VA) into a derivative of the pSI expression vector (Promega, Madison, WI) as previously described (14).

Generation of stable Ag-specific Th2/Tr1-like T cell lines Stable transfection of autoreactive T cells with the IL-2Prom3 IL10cDNA plasmid serves as an effective way to generate stable Ag-specific autoreactive Th2/Tr1-like T cells (14). Th2/Tr1-like T cell lines were prepared by transfection of primed T cells with IL-2Prom3 IL-10cDNA using Polybrene/DMSO-assisted gene transfer as previously described (14). Briefly, 7–10 days after immunization of SWXJ mice with 100 ␮g of Ag, primed LN cells were reactivated in vitro with Ag at 25 ␮g/ml. After 96 h, activated blast cells were enriched by centrifugation on a mixture of 14% Ficoll (Sigma-Aldrich, St. Louis, MO) and 32% sodium metrizoate (Accurate Chemical and Scientific, Westbury, NY) at a ratio of 12:5 (v/v) for 20 min at 2500 rpm. Cells collected from the interface were washed three times with HBSS (Life Technologies, Grand Island, NY) and suspended in flat-bottom 24-well plates (BD Labware, Franklin Lakes, NJ) at 3 ⫻ 106 cells/ml in prewarmed transfection medium consisting of 10 ␮g/ml IL-

ELISAs Purified capture/detection Ab pairs and recombinant cytokines were obtained commercially (BD PharMingen) and used to measure 48-h supernatant cytokine concentrations according to the manufacturer’s specifications. The capture/detection Ab pairs used in the present study included anti-mouse IFN-␥ (R4-6A2 and biotin-XMG1.2), anti-mouse IL-2 (JES61A12 and biotin-JES6-5H4), anti-mouse IL-4 (BVD4-1D11 and biotinBVD6-24G2), anti-mouse IL-5 (TRFK5 and biotin-TRFK4), anti-mouse IL-10 (JES5-2A5 and biotin-SXC-1), and anti-mouse IL-12 p35/p70 (RedT/G297-289 and biotin-C17.8). The Red-T/G297-289 Ab reacts with the p35 subunit of mouse IL-12 p70 and thereby measures biologically active IL-12 p70. After ELISA, plates were processed and absorbance at 405 nm was measured using a model 550 ELISA microplate reader (Bio-Rad, Hercules, CA). Standard values were plotted as absorbance (OD) vs cytokine concentration, and sample cytokine concentrations were determined as values within the linear part of the standard curve established using known concentrations of each cytokine.

Adoptive transfer of Th2/Tr1-like T cells and clinical evaluation As mice developed EAE, they were incorporated on a rotational basis into each experimental group. On day 6 after EAE onset, mice were injected i.v. with 1 ⫻ 107 activated IL-2Prom3 IL-10cDNA-transfected Th2/Tr1-like T cells specific for MBP87–99, PLP104 –117, or BSA (Sigma-Aldrich). Control mice received 1 ⫻ 107 normal splenocytes or PBS vehicle. All experiments were performed in a blinded manner in such a way that the investigator evaluating the mice remained unaware of the treatments used on each mouse. Relapse was assessed when mice showed an increase of at least one clinical score unit of neurologic disability typically accompanied by an abrupt substantial weight loss.

Isolation and purification of transferred Thy-1a⫹ T cells In some experiments, MBP87–99-specific Th2/Tr1-like T cells were generated from SWXJ.Thy-1a/b female mice and transferred into conventional SWXJ.Thy-1b/b females. In this way, transferred T cells expressing the

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FIGURE 1. Characterization of Ag-specific Th2/Tr1-like T cell lines. Ag-specific T cell lines were generated by transfecting primed SWXJ lymph node cells with the IL-2Prom3 IL-10cDNA construct. a, Proliferation assays showed that lines earmarked for adoptive transfer expressed appropriate Ag specificities against the immunodominant spreading determinant MBP87–99, the nonspreading control determinant PLP104 –117, or the irrelevant non-self Ag BSA. b, All adoptively transferred T cell lines expressed the hybrid cytokine phenotype of both Th2 and Tr1 T cells with Th2-like high production of IL-4 and IL-5 and Tr1-like enhanced production of IL-10 (IL-4high/IL-5high/IL-10veryhigh). All data in Fig. 1 are from representative T cell lines.

uncommon Thy-1a cell surface isoform may be distinguished by flow cytometry from host cells expressing the common Thy-1b isoform (19, 20). Four weeks after transfer, Thy-1a⫹ T cells were purified from whole splenocytes for functional analysis. Purification of the transferred Thy-1a⫹ T cells involved sequential negative and positive selection. Non-T cells were substantially removed by incubating whole splenocytes for 15 min at 4°C with a mixture of microbeads coated with anti-mouse CD11b and anti-mouse CD45R or B220 (Miltenyi Biotec, Auburn, CA). T cells were then negatively selected by magnetic bead separation on a MidiMACS cell separator (Miltenyi Biotec). The negatively selected cells were then treated with PE-labeled anti-mouse Thy-1a (BD PharMingen) at a 1/100 dilution for 30 min at 4°C, and the Thy-1a⫹ T cells were positively selected (⬎98%) using a FACSVantage cell sorter (BD Biosciences, San Jose, CA). The purified Thy-1a⫹ T cells were then cultured at 5 ⫻ 104 cells/well in 200 ␮l of DMEM supplemented as described above in 96-well flat-bottom microtiter Falcon plates (BD Biosciences) previously coated overnight at 37°C with 10 ␮g/ml anti-mouse CD3 (BD PharMingen). Supernatants were collected at 48 h for measuring cytokine concentrations by ELISA.

Immunohistochemistry Spinal cords were fixed in 10% phosphate-buffered formalin and cut into multiple pieces, and paraffin-embedded tissue sections were cut (30 ␮m each) for immunostaining as described previously (21). Sections were pre-

treated with 0.04% OsO4 and 1% H2O2 in 10% Triton X-100 (Electron Microscopy Sciences, Fort Washington, PA) and blocked with 5% normal goat serum (Vector Laboratories, Burlingame, CA) and 5% nonfat dehydrated milk for 60 min. Sections were treated sequentially with a PLP mouse monoclonal IgG2a Ab (Serotec, Oxford, U.K.) at a 1/200 dilution for 18 h at 4°C, biotinylated goat anti-mouse IgG2a (Southern Biotechnology Associates, Birmingham, AL) at a 1/500 dilution for 30 min at 22°C, and avidin-peroxidase complex (Vector Laboratories) for 1 h at a 1/1000 dilution. Sections were then treated with diaminobenzidine and 0.01% H2O2 for 1– 8 min and with 0.04% OsO4 for 30 s and washed in PBS. To accommodate the staining variability that occurs from one procedure to another, care was taken to couple untreated, control, and test spinal cord tissue samples in a side-by-side manner during each staining procedure.

Quantitative digitized image analysis Demyelination was quantified by analysis of digitized images of PLP-immunostained spinal cord sections as previously described (21). Briefly, digital images of PLP-immunostained spinal cord sections were captured at 640 ⫻ 480 pixel resolution and ⫻11.67 magnification using the AlphaImager 2000 System (Alpha Innotech, San Leandro, CA). Gray matter backgrounds were uniformly normalized using Adobe Photoshop (Adobe Systems, Mountain View, CA), and the mean intensity of myelin immunostaining was assessed using National Institutes of Health image software (National Institutes of Health, Bethesda, MD) to measure mean pixel blackness of outlined dorsal columns. At least 10 images from the cervical, thoracic, lumbar, and sacral regions of the spinal cord were analyzed per animal by a person blinded to the treatment regimen. The mean pixel blackness measured in untreated age- and sex-matched control mice was considered to represent 100% of the maximum myelin immunostaining and was used as a standard for comparing relative immunostaining in experimental EAE tissue.

Statistical analysis Clinical scores, relapse rates, time to first relapse, proliferative responses, and cytokine responses of treatment groups were compared using repeated measures ANOVA. Outcome differences for overall significance were determined by the Tukey-Kramer multiple comparison procedure. Differences in PLP immunostaining of spinal cord sections were determined by the unpaired Student t test.

Results Characterization of Ag-specific Th2/Tr1-like T cell lines FIGURE 2. Th2/Tr1 adoptive immunotherapy. Ag-activated Th2/Tr1like T cells (1 ⫻ 107) were adoptively transferred into SWXJ mice 6 days after the onset of EAE induced by immunization with PLP139 –151. Mice that received Th2/Tr1-like T cells specific for the immunodominant MBP87–99 spreading determinant showed a significantly improved clinical outcome (p ⫽ 0.02) compared with control mice that received PBS, normal splenocytes, or Th2/Tr1-like T cells specific for the irrelevant Ag BSA. No significant therapeutic effect was observed in mice that received Th2/Tr1like T cells specific for the nonspreading PLP104 –117 determinant.

Primed SWXJ LN cells were activated in vitro with Ag and transfected with the IL-2Prom3 IL-10cDNA construct. After selection by neomycin resistance, T cell lines were conventionally expanded through three additional stimulation/rest cycles by alternate Ag/ IL-2 treatments. All transferred T cell lines showed appropriate Ag specificity (Fig. 1a) and produced cytokines consistent with the phenotype of Th2-like T cells (16, 17) with high level production of IL-4, IL-5, and IL-10 and minimal production of IL-2 and

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Table I. Therapeutic effect of Th2/Tr1 adoptive immunotherapy on EAE

Treatment

No. of Mice per Group

Total Relapses per Group

Mean Time to First Relapse (⫾SE)a

No. of Mice with Multiple Relapses

No. of Mice not Relapsing

Mean Clinical Score (⫾SE)b

MBP 87–99 Th2/Tr1 T cells PLP 104–117 Th2/Tr1 T cells BSA Th2/Tr1 T cells Normal splenocytes PBS

8 8 8 8 8

8c 15 14 15 14

18.1 ⫾ 1.3d 16.5 ⫾ 1.2 15.5 ⫾ 1.8 16.4 ⫾ 1.2 15.3 ⫾ 1.3

2/8 6/8 5/8 6/8 5/8

2/8 0/8 0/8 0.8 0.8

1.7 ⫾ 0.32e 2.9 ⫾ 0.27 3.8 ⫾ 0.41 3.9 ⫾ 0.42 3.9 ⫾ 0.38

Mean number of days from time of EAE onset to time of first relapse ⫾ SE. Mean clinical score for each group at the end of the experimental period ⫾ SE. c SD, p ⫽ 0.02. d SD, p ⬍ 0.05. e SD, p ⫽ 0.02. a b

IFN-␥ (Fig. 1b). All lines produced IL-10 levels consistently higher than their IL-4 or IL-5 concentrations, thereby indicating that IL-10-transduced T cell lines shared features with high IL-10producing human regulatory Tr1 T cells generated in vitro in the presence of exogenous IL-10 (15). Thus, the IL-10-transfected T cell lines expressed the hybrid cytokine phenotype of both Th2 and Tr1 T cells, with Th2-like high production of IL-4 and IL-5 and Tr1like enhanced production of IL-10 (IL-4high/IL-5high/IL-10veryhigh). Th2/Tr1 adoptive immunotherapy EAE was induced by immunization of SWXJ mice with PLP139 –151. Ag-specific Th2/Tr1-like T cells were adoptively transferred into SWXJ EAE mice 6 days after onset of disease. Mice were weighed and clinically evaluated daily in a blinded manner during the 4-wk period following transfer. Mice that received Th2/Tr1-like T cells specific for the immunodominant MBP87–99 spreading determinant had a significantly improved clinical outcome ( p ⫽ 0.02) compared with control mice that received PBS, normal splenocytes, or Th2/Tr1-like T cells specific for the irrelevant Ag BSA (Fig. 2 and Table I). The improved clinical score at the end of the experimental period was also accompanied by a significant reduction in the relapse rate ( p ⫽ 0.02) and a significant delay in the mean time to onset of first relapse ( p ⬍ 0.05; Table I). In addition, the therapeutic group had the fewest mice with multiple relapses and was the only group with mice that did not relapse (Table I). A modest therapeutic effect appeared to be evident in mice that received Th2/Tr1-like T cells specific for PLP104 –117, an encephalitogenic determinant not in-

volved in early epitope spreading (11, 12). However, this apparent effect (Fig. 2) was not found to be significant and was not accompanied by a decreased relapse rate (Table I). The improved clinical outcome achieved by adoptive transfer of Th2/Tr1-like T cells specific for MBP87–99 was accompanied by a corresponding histologic improvement (Fig. 3). Digitized image analysis of PLP-immunostained spinal cord sections showed that mice receiving Th2/Tr1-like T cells specific for MBP87–99 had detectable PLP representing 94.37 ⫾ 0.52% (⫾ SE) of that found in normal spinal cord, while PBS-treated mice had only 91.94 ⫾ 0.50% of normal PLP levels (Fig. 4). The 2.43% increased detectable PLP found in spinal cords from the therapeutic treatment group was highly significant ( p ⬍ 0.001) and represented a 30% decrease in myelin loss compared with PBS-treated mice.

Recall proliferative responses to self after Th2/Tr1 adoptive immunotherapy Splenocytes from each mouse were tested for proliferative responses to self-determinants 4 wk after adoptive transfer (Fig. 5). Overall comparison of all treatment groups by ANOVA showed no significant differences in response to any recall Ag. However, comparison between mice receiving MBP87–99-specific Th2/Tr1-like T cells (n ⫽ 8) and the mean of all other treatment groups in their responses to the priming PLP139 –151 immunogen (n ⫽ 32) showed a tendency toward significance ( p ⫽ 0.07), suggesting that adoptive immunotherapy with MBP87–99-specific Th2/Tr1-like T cells

FIGURE 3. PLP immunostained spinal cord sections from SWXJ EAE mice 5 wk after onset of disease. Spinal cord sections were immunostained for detection of PLP, and digital images were captured at ⫻11.67 magnification. a, Normal-appearing PLP-immunostained dorsal columns from normal mice without EAE. b, Severe demyelination appearing as unstained areas in dorsal columns of EAE control mice treated with PBS. c, Marked inhibition of demyelination in dorsal columns of EAE mice adoptively transferred with Th2/Tr1-like T cells specific for the immunodominant MBP87–99 spreading determinant. Scale bar ⫽ 50 ␮m.

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FIGURE 4. Quantitative immunohistochemistry. Demyelination was quantified by digitized image analysis of PLP-immunostained dorsal column sections from eight mice per treatment group, including normal untreated control mice without EAE, PBS-treated control mice with EAE, and EAE mice adoptively transferred with Th2/Tr1-like T cells specific for the immunodominant MBP87–99 spreading determinant. EAE mice receiving Th2/Tr1-like T cells specific for MBP87–99 showed a highly significant (p ⬍ 0.001) 30% decreased myelin loss compared with PBS-treated mice. Error bars show ⫾ SE.

may marginally inhibit proliferative responses directed against the priming determinant used to initiate disease. Cytokine phenotypes of recall responses to self after Th2/Tr1 adoptive immunotherapy In contrast to the marginal impact on proliferative responses to self-determinants, adoptive transfer of MBP87–99-specific Th2/ Tr1-like T cells significantly inhibited production of proinflammatory Th1 cytokines in recall responses to both the PLP139 –151 priming immunogen (Fig. 6a) and the MBP87–99 spreading determinant (Fig. 6b). Responses to PLP139 –151 showed significant inhibition of IFN-␥ production ( p ⫽ 0.02), while responses to MBP87–99 showed significantly inhibited production of both IFN-␥ ( p ⫽ 0.03) and IL-2 ( p ⫽ 0.002). However, the most significant difference in cytokine production was evident in the enhanced production of IL-10 observed in the recall response to MBP87–99 ( p ⬍ 0.001). This enhanced IL-10 response to MBP87–99 was accompanied by a complementary significant decrease in supernatant IL-12 p70 concentrations ( p ⫽ 0.01), a reciprocal relationship associated with resistance to autoimmune disease (22). We did not observe any significant differences in the production of IL-4 or IL-5 between any of the experimental groups, nor did we observe significant differences in the production of any cytokine by mice receiving Th2/Tr1-like T cells specific for either PLP104 –117 or BSA. Thus, adoptive transfer of Th2/Tr1-like T cells targeted to an immunodominant epitope spreading determinant established a high IL-10 Tr1-like, immune-deviated spreading response sufficient to

FIGURE 5. Recall proliferative responses to self after Th2/Tr1 adoptive immunotherapy. Splenocyte proliferative responses to self-determinants were measured ex vivo 4 wk after adoptive immunotherapy. No significant differences were observed in recall proliferative responses between any of the treatment groups. The apparent inhibition of the proliferative response to the PLP139–151 priming determinant in mice adoptively transferred with MBP87–99-specific Th2/Tr1like T cells was not statistically significant. Error bars show ⫾ SE.

The enhanced production of IL-10 in the recall response to MBP87–99 appeared to mimic the characteristic high IL-10-producing Tr1 feature of the hybrid Th2/Tr1-like transferred T cells (15) (Fig. 1b). To determine whether the transferred population served as the source of the enhanced IL-10 production, MBP87–99-specific Th2/Tr1-like T cells were generated from SWXJ.Thy-1a/b female mice and transferred into conventional SWXJ.Thy-1b/b females 6 days after EAE onset. Four weeks after transfer, Thy-1a⫹ and Thy1a⫺ T cells were purified ⬎98% from whole splenocytes by magnetic bead separation and cell sorting and cultured in anti-CD3coated microtiter wells. ELISA analysis of 48-h supernatants showed that the predominant source of IL-10 was host-derived Thy-1a⫺ T cells rather than the transferred Thy-1a⫹ population (Fig. 7). These surprising results indicated that the transferred regulatory T cells eventually failed to produce IL-10, but only after they actively induced a high IL-10 Tr1-like, host-derived spreading repertoire.

Discussion Prior studies have shown that a therapeutic outcome may be achieved when Th2- or Tr1-like T cells specific for the priming encephalitogenic determinant are adoptively transferred into EAE mice after the onset of clinical symptoms (13, 14). The current study extends this work by showing that a similar therapeutic effect may be achieved by transfer of Th2/Tr1-like T cells specific for secondary epitope spreading determinants. Moreover, the present study shows that pre-emptive installation of an immune-deviated Th2/Tr1-like epitope spreading repertoire mediates bystander inhibition of the established autoreactive T cell repertoire. Th2/Tr1 adoptive immunotherapy targeted against the immunodominant MBP 87–99 spreading determinant effectively manufactures an immune-deviated spreading response that inhibits the production of proinflammatory Th1 cytokines not only from T cells responding to the immunodominant MBP87–99 spreading determinant, but also from T cells responding to the PLP139 –151 priming determinant. Thus, the pre-emptive orchestration of an anti-inflammatory epitope spreading phenotype directly alters the secondary spreading response and also mediates bystander inhibition of ongoing autoreactivity directed against the disease-initiating determinant. Before transfer, IL-10-transduced T cells expressed the hybrid cytokine phenotype of both Th2 and Tr1 T cells, with Th2-like

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FIGURE 6. Cytokine phenotypes of recall responses to self after Th2/ Tr1 adoptive immunotherapy. Four weeks after adoptive immunotherapy, splenocytes were stimulated ex vivo with either the PLP139 –151 priming determinant (a) or the MBP87–99 spreading determinant (b). At 48 h supernatant cytokine concentrations were determined by ELISAs. Adoptive transfer of MBP87–99-specific Th2/Tr1-like T cells resulted in 1) a significant bystander inhibition of IFN-␥ production (p ⫽ 0.02) in the recall response to the PLP139 –151 priming immunogen, 2) a significant inhibition of both IFN-␥ (p ⫽ 0.03) and IL-2 (p ⫽ 0.002) in response to the MBP87–99 spreading determinant, and 3) a significant increased production of IL-10 (p ⬍ 0.001) and a reciprocal inhibition of IL-12 p70 (p ⫽ 0.01) in response to the MBP87–99 spreading determinant. Error bars show ⫾ SE.

high production of IL-4 and IL-5 and Tr1-like enhanced production of IL-10 (IL-4high/IL-5high/IL-10veryhigh). However, 4 wk after transfer, ex vivo analysis showed that the final response to the MBP87–99 spreading determinant was skewed significantly toward the Tr1 phenotype with low production of IL-4 and IL-5 and enhanced production of IL-10 (IL-4low/IL-5low/IL-10veryhigh). Moreover, the source of the IL-10 was found to be native rather than transferred T cells. This unexpected and rather surprising finding indicates that transferred regulatory T cells may not be the ultimate effector population regulating autoimmunity but, instead, may induce native T cells to provide ultimate long-term inhibition of autoimmunity. Thus, our observations are consistent with the view that the high IL-10-producing transferred T cells induced a

FIGURE 7. IL-10 production by native T cells rather than transferred T cells after Th2/Tr1 adoptive immunotherapy. MBP87–99-specific Th2/Tr1like T cells generated from SWXJ.Thy-1a/b female mice were transferred into conventional SWXJ.Thy-1b/b females. Four weeks after transfer, Thy1a⫹ T cells were purified ⬎98% from whole splenocytes by magnetic bead separation and cell sorting. The purified T cells were cultured in flat-bottom microtiter wells coated with anti-mouse CD3, and the 48-h supernatants were assayed by ELISA for IL-10. By 4 wk after adoptive immunotherapy, the transferred Thy-1a⫹ T cells no longer produced substantial levels of IL-10. The predominant source of IL-10 at this time was host-derived Thy-1a⫺ T cells. Data show the combined results of two similar experiments. Error bars show ⫾ SE.

host-derived immune-deviated Tr1 spreading response capable of inhibiting the production of IFN-␥ by T cells responding to the PLP139 –151 priming determinant. Tr1 self-priming may have occurred preferentially as a consequence of IL-10-mediated inhibition of IL-12-dependent Th1 priming (22–24). Thus, the transferred high IL-10-producing regulatory T cells may have simply initiated a self-sustainable Tr1 self-priming milieu. This proposed regulatory mechanism does not preclude the possibility that the transferred Th2/Tr1 repertoire may have become physically compartmentalized into functionally discrete populations, as has been recently described in the memory response to OVA (25). Such a scenario implies that some transferred T cells may still function as high IL-10-producing memory T cells but do not reside in traditional lymphoid compartments such as the spleen. The present study indicates that the selection of targeted Ag is critical for achieving optimum adoptive immunotherapy and suggests that there may be a clear therapeutic determinant hierarchy. However, the basis for this therapeutic hierarchy is not completely resolved. Recent studies have shown that the immunodominance of the PLP139 –151 encephalitogenic determinant in SJL/J mice may be due to the increased frequency of high-affinity T cell clones capable of responding to the peptide (26, 27). Since transfected Th2/Tr1-like T cell lines showed a remarkably consistent adherence to the MBP87–99 ⬎ PLP104 –117 ⬎ BSA reactivity profile (Fig. 1a), our data suggest that the basis for the therapeutic determinant hierarchy may also be related directly to the availability of high-affinity clones capable of responding to the determinant. Thus, the ability to generate Th2/Tr1 T cell lines may be directly related to the availability and frequency of high-affinity clones in the native repertoire capable of responding to each determinant. By extrapolation, the predominance of the role that a given epitope plays in the spreading cascade may also be due to its ability to elicit a high-affinity clonal response, thereby directly linking prominence in the epitope spreading cascade with relative therapeutic

The Journal of Immunology effectiveness in adoptive immunotherapy. In this regard, regulatory T cells specific for the nonspreading PLP104 –117 determinant may have failed to mediate a profound therapeutic effect because they lacked the potent high-affinity clones required for inducing immune deviation of the host autoreactive repertoire. Our current data may have important clinical implications in choosing target autoantigens for Th2/Tr1 adoptive immunotherapy in human autoimmune disease. Selecting immunodominant spreading determinants as therapeutic targets provides the advantage of expanding a naive T cell population that has not undergone any prior extensive in vivo expansion. Thus, naive or partially expanded spreading repertoires have not undergone chronic selfstimulation to the point of T cell exhaustion. As a result, spreading repertoires by their nature have not endured any extensive anergy, suppression, or partial deletion that may occur over time within established autoreactive repertoires (8, 28). Thus, spreading repertoires provide a fresh source of vibrant autoreactivity relatively unaffected by immunoregulatory forces acting in varying degrees upon established chronically activated autoreactive T cell repertoires. Cell-based adoptive immunotherapy provides distinct advantages over the use of Ag-based therapy in treating human autoimmune disease. This is particularly evident from the results of recently terminated clinical trials in which treatment with an MBP83–99-altered peptide ligand was poorly tolerated by patients with relapsing-remitting MS (29, 30) and in some cases may have contributed to disease exacerbation (29). While a specific altered peptide ligand may serve as an antagonist or partial agonist for some T cell clones, it may concurrently serve as a superagonist for another subset of autoreactive T cells or may enhance responsiveness to native cognate self in vivo as has been recently suggested (31–33). Adoptive immunotherapy eliminates such possibilities by providing well-characterized T cell populations with specific antigenicities and defined cytokine phenotypes. For the treatment of human autoimmune disease, autoreactive Th2/Tr1-like T cells may be genetically modified to express the gene for herpes simplex viral thymidine kinase. Thus, if unexpected adverse effects occur, the transferred cells may be eliminated in vivo by treating patients with ganciclovir, thereby halting any undesirable effects and preventing unanticipated disease exacerbation (34, 35). However, in light of the observed inability of regulatory T cells to produce transgene IL-10 4 wk after transfer, such anticipated adverse effects may be inherently self-limiting and of minimal concern. The present study shows that therapy for ongoing autoimmune demyelinating disease may be achieved by adoptive transfer of Th2/Tr1-like T cells specific for components of the epitope spreading cascade. Our data provide a rational basis for developing Ag-specific cell-based therapies for treating MS. In addition, our results indicate that spreading determinants may serve to generate vibrant autoreactive T cell repertoires that may be genetically modified to optimize their anti-inflammatory activities and perhaps their ability to remyelinate and provide protection against the marked axonal damage associated with progression of MS (36). Such enhanced T cell protective autoimmunity (37, 38) may prove to be most useful in inhibiting the inflammation and tissue damage responsible for chronic debilitation in MS.

Acknowledgments We thank Dr. Ellen Rothenberg for the IL-2 promoter region and for her helpful advice. We also thank Dr. Paul Elson for performing the statistical analysis.

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