of April 2, 2018.This information is current as

AlfaAntigen-Specific Tolerance to AlglucosidaseB Regulatory Cells That Mediate Transient Low-Dose Methotrexate Generates

Richards and Alexandra M. JosephMitra-Kaushik, Lucy Phillips, Alida D'Angona, Susan M. Marguerite S. Joly, Roderick P. Martin, Shibani

ol.1303326http://www.jimmunol.org/content/early/2014/09/09/jimmun

published online 10 September 2014J Immunol 

MaterialSupplementary

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Transient Low-Dose Methotrexate Generates B RegulatoryCells That Mediate Antigen-Specific Tolerance toAlglucosidase Alfa

Marguerite S. Joly, Roderick P. Martin, Shibani Mitra-Kaushik, Lucy Phillips,

Alida D’Angona, Susan M. Richards, and Alexandra M. Joseph

Biologic drugs, including enzyme-replacement therapies, can elicit anti-drug Abs (ADA) that may interfere with drug efficacy and

impact patient safety. In an effort to control ADA, we focused on identifying regimens of immune tolerance induction that may be

readily available for clinical use. Data generated in both wild-type mice and a Pompe disease mouse model demonstrate that single-

cycle, low-dose methotrexate can be as effective as three cycles of methotrexate in providing a long-lived reduction in alglucosidase

alfa-specific ADA. In addition, we show that methotrexate induces Ag-specific tolerance as mice generate similar Ab responses to an

irrelevant Ag regardless of prior methotrexate treatment. Methotrexate-induced immune tolerance does not seem to involve cell

depletion, but rather a specific expansion of IL-10– and TGF-b–secreting B cells that express Foxp3, suggesting an induction of

regulatory B cells. The mechanism of immune tolerance induction appears to be IL-10 dependent, as methotrexate does not induce

immune tolerance in IL-10 knockout mice. Splenic B cells from animals that have been tolerized to alglucosidase alfa with

methotrexate can transfer tolerance to naive hosts. We hypothesize that methotrexate induction treatment concomitant with

initial exposure to the biotherapeutic can induce Ag-specific immune tolerance in mice through a mechanism that appears to

involve the induction of regulatory B cells. The Journal of Immunology, 2014, 193: 000–000.

Methotrexate was first described in 1948 by Farber andDiamond (1) as a folate antagonist capable of treating

acute leukemia in children. Since then, methotrexate

has been a widely used therapy for a number of diseases such as

neoplasms and autoimmunity (2). In oncology, high-dose courses of

methotrexate are used to kill proliferating, neoplastic cells (3). In

autoimmune diseases, such as rheumatoid arthritis and psoriasis,

continuous, low-dose methotrexate can successfully treat disease (4–

8). The mechanism by which continuous low-dose methotrexate can

control autoimmune disease most likely involves the killing of pro-

liferating cells through folate antagonism (6, 7, 9), although addi-

tional mechanisms may contribute. For instance, methotrexate has

been associated with neutrophil chemotaxis, apoptosis, and the in-

hibition of IL-1, IL-6, TNF-a, IL-4, IL-13, IFN-g, and GM-CSF (10–

15), which may play a therapeutic role (15). Also, methotrexate has

been shown to increase adenosine release (15, 16), a molecule sug-

gested to mediate the effects of regulatory T cells and hamper im-

mune responses (17, 18).A continuous, immunosuppressive regimen of methotrexate has

been associated with reduced titers of Abs that can develop against

therapeutic proteins (9). One example has been described with

coadministration of weekly methotrexate treatment and antitumor

necrosis factor (TNF) agents, such as infliximab, to provide additive

treatment effects (19–23). In such cases, not only are inflammatory

responses against diseased tissue reduced, but also the development

of Abs against the anti-TNF agent (9, 19–23). Anti-drug Abs (ADA)

that develop against anti-TNF agents have been shown to reduce

treatment effects in rheumatoid arthritis by decreasing the exposure

of the anti-TNF agent (9, 24). Therefore, reduced ADA by metho-

trexate can provide significant clinical benefit (25, 26).Our laboratory previously demonstrated that a low-dose in-

duction regimen of methotrexate can reduce ADA responses

against different protein therapies in mice and induce immune

tolerance in normal and disease settings (27–29). Low-dose

methotrexate induction treatment was subsequently included in

a clinical immune tolerance protocol that coadministered the

B cell–depleting agent, Rituximab, with optional IVIG (30–32).

The dose of methotrexate used in mice and in patients is con-

sidered to be low, as the human equivalent dose is 0.4 mg/kg s.c.

in infants. For a 5 kg infant, this would equate to 6 mg metho-

trexate per week. In a 70 kg adult patient, the human equivalent

oral dose of methotrexate is 7 mg, which for three doses would

equate to 21 mg methotrexate in 1 wk. These doses are within the

dosing range of rheumatoid arthritis treatment (5–25 mg/week)

and well below the doses used for oncology treatment (6). This

combination treatment successfully reduced ADA and induced

immune tolerance to recombinant human acid–a-glucosidase

(rhGAA, alglucosidase alfa) in a subset of infantile-onset Pompe

patients who experience poor clinical outcomes associated with

ADA (30–32). Importantly, this approach is associated with im-

proved clinical outcomes within this patient population (30–32).

Rituximab, however, induces a general, long-term B cell depletion

and is not globally available. Therefore, the use of methotrexate

alone to induce immune tolerance could provide a more tolerable

and readily available means of controlling ADA in patients with

a lower risk of immunogenicity effects.

Genzyme, a Sanofi company, Framingham, MA 01701

Received for publication December 13, 2013. Accepted for publication August 7,2014.

Address correspondence and reprint requests to Dr. Alexandra M. Joseph, Sanofi, 1Mountain Road, Framingham, MA 01701. E-mail address: alexandra.joseph@genzyme.com

The online version of this article contains supplemental material.

Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; ADA, anti-drugAb; AUC, area under the curve; KO, knockout; LAP, latency-associated peptide;mATG, murine antithymocyte globulin; MFI, mean fluorescence intensity; rhGAA,recombinant human acid–a-glucosidase; WT, wild-type.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303326

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The mechanism by which a low-dose induction regimen ofmethotrexate could induce immune tolerance remains to be dis-covered. Murine studies were initiated to investigate the mecha-nism of methotrexate-induced immune tolerance. The studies re-ported in this work demonstrate that low-dose induction treatmentof methotrexate can increase B cell subsets that express IL-10,TGF-b, and Foxp3. Moreover, adoptive transfer of purified B cellsfrom methotrexate-tolerized mice appears to transfer tolerance tonaive hosts. These results suggest a novel function of methotrexate ininducing regulatory B cells to help mediate immune tolerance.

Materials and MethodsAnimals, rhGAA, murine antithymocyte globulin, anti–TGF-b,and methotrexate treatment

C57BL/6J (Jackson ImmunoResearch Laboratories), E4-8GAA knockout(KO) (Supplemental Fig. 1; Genzyme, a Sanofi company), or B6.129P2-IL10 , tm1Cgn . /J (Jackson ImmunoResearch Laboratories) mice, be-tween 8 and 12 wk, were housed and maintained in accordance with theGuide for the Care and Use of Laboratory Animals, under the AmericanAssociation for Accreditation of Laboratory Animal Care accreditation.All animal procedures were approved by the Institutional Animal Care andUse Committee. rhGAA (Genzyme, a Sanofi Company), 20 mg/kg, wasgiven by tail vein injection once or weekly for up to 24 doses. All rhGAA-treated mice were treated prophylactically with 5–30 mg/kg diphenhy-dramine (West-ward Pharmaceutical, Eatontown, NJ) i.p. prior to rhGAAtreatment. Methotrexate (Calbiochem catalog 454125), 5 mg/kg, was de-livered by i.p. injection as either a single cycle or three cycles. A singlecycle of methotrexate represents three consecutive daily doses (0, 24, and48 h) of 5 mg/kg initiated within 15 min of the first rhGAA treatment at time0. Animals that received three cycles of methotrexate were administeredeach cycle with the first three weekly rhGAA treatments. Anti–TGF-b,a mouse IgG1 mAb (1D11; Genzyme, a Sanofi Company), or murine isotypecontrol Ab (13C4; Genzyme, a Sanofi Company) was administered to miceat 5 mg/kg i.p., weekly, every other day, through the end of study.

Serum anti-rhGAA–specific Ab titers

rhGAA-specific IgG was measured by ELISA. Briefly, 96-well plates(Corning, Corning, NY) were coated overnight with 5 mg/ml rhGAA insodium acetate buffer (pH 5.0). After blocking with 0.1% BSA in PBS,serial dilutions of serum were added in duplicate to rhGAA-coated platesand incubated at 37˚C for 1 h. The plates were washed, and HRP-conjugated goat anti-mouse IgG secondary Ab (Southern BiotechnologyAssociates, Birmingham, AL) was added and allowed to incubate for 1 h at37˚C. Following a final wash, 3,39,5,59-tetramethylbenzidine substrate(KPL, Gaithersburg, MD) was added and allowed to develop for 15 min atroom temperature. The reaction was stopped by the addition of 1 N HCl,and absorbance values were read at 450/650 nm on an ELISA plate reader(Molecular Devices, Sunnyvale, CA). End-point titers were defined as thereciprocal of the sample dilution resulting in an absorbance value of 0.2using Softmax software (Molecular Devices).

Serum anti–murine antithymocyte globulin Ab titers

The levels of anti-drug IgG in mouse serum were analyzed by ELISA.Murine antithymocyte globulin (mATG) is raised in rabbits; thus, Abresponses are assessed using a specific rabbit IgG ELISA. Briefly, 96-wellplates (Corning) were coated overnight with rabbit IgG and blocked withSuper Block Blocking Buffer (Thermo Scientific, Rockford, IL), and serialdilutions of serum were added in duplicate. Following incubation, the plateswere washed and signal was detected by first adding HRP-conjugated goatanti-mouse IgG secondary Ab (Southern Biotechnology Associates, Bir-mingham, AL) and, following another wash, developed with 3,39,5,59-tetramethylbenzidine substrate (BioFx, Owings Mills, MD). The reactionwas stopped by the addition of 1 N HCl, and absorbance values were readat 450/650 nm on an ELISA plate reader (Molecular Devices, Sunnyvale,CA). End-point Ab titers were defined as the lowest dilution above anabsorbance of 0.1 using Softmax software (Molecular Devices).

Cell preparation, B cell purification, and adoptive transfer

Animals were sacrificed at various times after initiation of treatment.Spleen, mesenteric, and inguinal lymph node were collected for flowcytometric analysis of T and B cell subsets, and sera were collected forELISA. Spleens were processed between glass slides, and RBC were lysedwith lysing buffer purchased from BD Biosciences (catalog 555899),

according to manufacturer’s instructions, and washed in PBS containing2% FCS. Lymph nodes were processed similarly. Cells were resuspendedin 200 mL PBS containing 4% FBS and 25 mg/ml total mouse IgG andblocked for 30 min at 4˚C. Approximately 3 million spleen cells and1 million lymph node cells were stained with different Ab cocktails, asdescribed, and analyzed with a high throughput sampler on a BD Bio-sciences CANTOII flow cytometer. A total of 75,000–100,000 live cellevents (based on 7-aminoactinomycin D [7AAD] positivity) was acquiredwithin the lymphocyte gate. For studies requiring B cell purification,spleens were prepared in single-cell suspension, as described above, andloaded onto the RoboSep (StemCell Technologies) instrument, accordingto the manufacturer’s instructions, and subjected to B cell–negative se-lection. Purified B cells ($90%; verified by flow cytometry) were seededat 500,000 cells/well in 96-well round-bottom plates (Costar catalog 3799),incubated in culture medium (RPMI 1640, 10% FCS, penicillin/strepto-mycin) for 2 d, and stained for various B cell subsets and cytokine ex-pression. For adoptive transfer, purified B cells were injected at variouscell concentrations by i.v. administration.

Flow cytometry

Anti-mouse Ab cocktails consist of PE-CD21/35 (catalog 552957), FITC-CD23 (catalog 553138), PE-CD138 (catalog 553714), PE-CD127 (catalog552543), allophycocyanin-Cy7-CD19 (catalog 557655), FITC-CD43(catalog 553270), PE-Cy7-CD4 (catalog 552775), FITC-CD3e (catalog553062), allophycocyanin-CD11b (catalog 553312), PE-Cy7-IgM (catalog552867), allophycocyanin-Cy7-CD8 (catalog 557654), allophycocyanin-CD138 (catalog 558626), PE-Cy7-CD11b (catalog 552850), PE-CD93(catalog 558039), allophycocyanin-CD69 (catalog 560689), Pe-Cy7-CD24 (catalog 560536), FITC-CD1d (catalog 553845), allophycocyanin-CD5 (catalog 550035), and PerCP-Cy5-7AAD (catalog 559925), allpurchased from BD Pharmingen. FITC-Foxp3 intracellular staining kit(catalog 11-5773) was purchased from eBioscience. Pacific Blue (PB)-CD25 (catalog 102022), PB-CD23 (catalog 101616), and PB-CD86 (cat-alog 105022) were purchased from BioLegend. Fluorochrome-matchedand isotype-matched controls were used to help define the gating strat-egy (Supplemental Fig. 3). Samples were run on a BD FACS Canto II andacquired using BD FACSDiva software. The analysis of lymphocyte sub-sets was performed with FCS express version 3 software provided by DeNovo Software. Percentages of each cell population within the live,lymphocyte gates were generated with the batch-processing option, andabsolute numbers were calculated according to the cell counts obtained.Spleen and lymph node cell counts were obtained with a Beckman CoulterVi-cell XR cell viability analyzer, according to the manufacturer’sinstructions. The cell types assessed include total and activated CD4+

T cells (CD4+CD192, CD69+), CD8+ T cells (CD8+CD192, CD69+),regulatory T cells (CD4+CD192CD25+Foxp3+, CD69+), follicular B cells(CD19+IgMlow/highCD23+CD932, CD862/+), total and activated marginalzone B cells (CD19+CD932CD21+CD232, CD862/+), total CD19+

CD1dhighCD5+ B cells, total and activated transitional 1 (CD19+CD232

IgMhighCD93+, CD862/+), transitional 2 (CD19+CD23+IgMhighCD93+,CD862/+), and transitional 3 (CD19+IgMlowCD23+CD93+), CD862/+)B cells (33, 34).

In vitro and cytokine analysis

C57BL/6 (Jackson ImmunoResearch Laboratories) or E4-8GAAKO (Gen-zyme, a Sanofi company) mice, 8–12 wk old, were given methotrexate(Calbiochem catalog 454125), 5 mg/kg, by i.p. injection for 1 cycle (3consecutive daily doses) commencing with i.v. rhGAA 20 mg/kgtreatment. Animals were sacrificed on day 6 or 7 post-rhGAA initiation,depending on the mouse strain. Spleens were prepared in single-cell sus-pension and loaded onto the RoboSep (StemCell Technologies) instrument,according to the manufacturer’s instructions, and subjected to B cell–negative selection. Purified B cells were seeded at 500,000 cells/well in 96-well round-bottom plates (Costar catalog 3799) and incubated with nostimulation. Cells were allowed to incubate for at least 48 h at 37˚C.Samples were transferred to V-bottom wells (USA Scientific catalog651201) and spun at 1200 rpm for 5 min at 4˚C. Cells were resuspended in200 mL PBS containing 4% FBS and 25 mg/ml total mouse IgG andblocked for 30 min at 4˚C. Plates were spun again and resuspended in90 mL PBS with 2% FCS with addition of 10 ml Ab mixture, as describedabove, and incubated for 20 min at 4˚C with addition of 5 mL 7AAD forthe last 10 min of the staining procedure. Addition of 100 mL buffer to thesamples with subsequent spin sufficed as a wash; samples could beresuspended in buffer for surface analysis of protein and immediate ac-quisition or resuspended in Fix/Perm (eBioscience catalog 11-5773) forintracellular staining of IL-10 (BioLegend catalog 505008), TGF-b(latency-associated peptide [LAP]/TGF-b1; BioLegend catalog 141404),

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and Foxp3 (eBioscience catalog 11-5773-82), according to the manu-facturer’s instructions. Calculation of cell number was obtained with theaddition of 50 mL CountBright absolute counting beads (Molecular Probescatalog C36950). All samples were acquired and analyzed, as describedabove.

ResultsSingle cycle and three cycles of methotrexate similarly reduceanti-rhGAA IgG titers

To investigate whether ADA titers against rhGAA could be re-duced with a brief course of methotrexate in Pompe mice, anti-rhGAA–specific IgG titers were assessed after single or threecycles of methotrexate were given with 12 weekly i.v. treatments of20 mg/kg rhGAA in E4-8GAAKO mice (a model of Pompe dis-ease; Supplemental Fig. 1). A single cycle of methotrexate rep-resents three consecutive daily doses of 5 mg/kg (at 0, 24, and 48 h)initiated within 15 min of the first rhGAA treatment at time 0.Animals that received three cycles of methotrexate were adminis-tered each cycle along with the first three weekly rhGAA treat-ments. Both induction regimens similarly reduced anti-rhGAA IgGtiter (Fig. 1). Subsequently, rhGAA treatment was halted for 4 wk,and the animals were then rechallenged. Anti-rhGAA IgG responseswere not increased in methotrexate-treated mice as they werein rhGAA-alone–treated mice. The overall reduction of rhGAA-specific IgG by single- and three-cycle methotrexate comparedwith rhGAA alone was 78% (p = 0.014) and 71% (p = 0.025),respectively, as measured by area under the curve (AUC) (Fig. 1).No statistical difference was observed between the two differentregimens of methotrexate.

Methotrexate-induced immune tolerance appears to be Agspecific

The ability of a single cycle of methotrexate to induce Ag-specifictolerance to rhGAA was investigated. Wild-type (WT), C57BL/6mice were treated weekly with 20 mg/kg i.v. rhGAA for 26 wk.A subset of animals was dosed with a single cycle of 5 mg/kgmethotrexate. Starting at week 12, each group of animals wasalso treated monthly with mATG (5 mg/kg, i.p.). Monthly treat-

ments of mATG in C57BL/6 mice generate robust Ab responses(29), and thus enabled our investigation of the Ag-specificnature of methotrexate-induced immune tolerance. Methotrexate re-duced anti-rhGAA–specific titers by 78% as measured by AUC(Fig. 2A). Irrespective of methotrexate treatment, both groups ofanimals generated similar anti-mATG IgG responses (Fig. 2B).These data suggest that animals tolerized to one Ag with a singlecycle of methotrexate remain fully responsive to subsequentchallenge with other Ags.

B cell subpopulations are enriched in methotrexate-treatedmice

To investigate how the induction regimen of methotrexate generatesimmune tolerance, the absolute numbers of various lymphocyte sub-sets from C57BL/6 WT and E4-8GAAKO mouse spleen and lymphnodes were assessed by flow cytometry. Included in the subset anal-ysis were total and activated CD4+ T cells (CD4+CD692/+), total andactivated CD8+ T cells (CD8+CD692/+), total and activated naturalT regulatory cells (CD4+CD25+Foxp3+ CD692/+), and total and ac-tivated B cells (CD19+CD862/+). No significant, reproduciblechanges were observed in the numbers of total or activated CD4+,CD8+, or T regulatory cells from the lymph node and spleen days 4–8following rhGAA treatment on day 1 between animals treated withand without methotrexate (data not shown; Supplemental Fig. 2). Ondays 7 and 8, significant increases were observed in the absolutenumbers of activated, splenic B cells isolated from WT animalstreated with rhGAA plus methotrexate when compared with B cellsisolated from animals treated with rhGAA alone (Fig. 3A). Similarly,the numbers of activated B cells in rhGAA plus methotrexate-treatedE4-8GAAKO mice were significantly increased by ∼60% whencompared with mice treated with rhGAA alone (Fig. 3B). In both WTand E4-8GAAKO mice, activated B cells were not expanded in thelymph node (data not shown). In contrast, no significant differenceswere observed between rhGAA-treated and saline-treated mice orbetween saline- and methotrexate alone–treated mice (Fig. 3B).

FIGURE 1. Low-dose methotrexate induction treatment specifically

reduces Ab responses that develop against rhGAA. Six E4-8GAAKO

mice/group were treated weekly for the first 12 wk with 20 mg/kg i.v.

rhGAA alone or in combination with either single-cycle methotrexate

(three consecutive, daily i.p. treatments of 5 mg/kg starting within 15 min

of rhGAA treatment) or three-cycle methotrexate (three consecutive, daily

i.p. treatments of 5 mg/kg given alongside the 3 wk of rhGAA treatment).

After 12 wk, animals were rested for 3 wk and then rechallenged with

rhGAA. Anti-rhGAA titers were assessed weeks 2, 4, 8, 10, 13, 15, 17, and

20. The overall reduction of rhGAA-specific IgG by single- and three-

cycle methotrexate compared with rhGAA alone was 78% (p = 0.014) and

71% (p = 0.025), respectively. These data are representative of three

separate experiments. Statistical comparison of AUC was performed by

building an ANOVA model with repeated measurement nested with study

mice, using treatments, weeks, and their interaction as the exploratory

variables in the model.

FIGURE 2. Low-dose methotrexate induces Ag-specific immune toler-

ance to rhGAA. Twelve C57BL/6 mice/group were treated weekly for

22 wk with 20 mg/kg rhGAA i.v. in the presence or absence of single-

cycle methotrexate, as described above. Starting at 12 wk, both groups

were treated with 5 mg/kg, i.p., mATG every 4 wk through week 20. Anti-

rhGAA–specific IgG titers were assessed by ELISA every other week

throughout the course of the study (A), and anti-mATG IgG titers were

evaluated by ELISA every 2 wk starting at week 12 (B). These data are

representative of two separate experiments.

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Additional B cell subsets were then investigated that includedfollicular, marginal zone, transitional 1, transitional 2, transitional3, and B10-like (CD1dhighCD5+) B cells (Table I). The activationstatus of most of these subsets was assessed by CD86 expression.

The B10-like B cells, activated transitional 2 B cells, and activatedfollicular B cell subpopulations isolated from the spleens of ani-mals cotreated with rhGAA plus methotrexate were consistentlyand significantly increased in cell number by an average of 50, 70,

FIGURE 3. Methotrexate appears to increase the absolute numbers of activated splenic B cells. Four to five C57BL/6 mice/group were treated with one dose of

20 mg/kg rhGAA, i.v., in the presence or absence of single-cycle methotrexate, as described above. On days 4–8, spleens were harvested and processed into single-

cell suspensions. Activated B cells were analyzed by flow cytometry, as described in Materials and Methods, and the average number of activated B cells per

group over time is presented (A). The four to five E4-8GAA KO mice/group were similarly treated with rhGAA and a single cycle of methotrexate. Experimental

control groups of mice treated with methotrexate alone and saline alone were included. Spleens were harvested on day 6 from each animal/group, processed

individually, and analyzed by flow cytometry. The average numbers of total B cells and activated B cells from each group are presented (B). Splenocytes were

prepared from four to five E4-8GAAKOmice treated with one dose of rhGAA in the presence or absence of single-cycle methotrexate, as described above, or with

either saline or methotrexate alone. Six days following treatment, multiple splenic B cell subsets, including CD19+CD1dhighCD5+ B cells, activated transitional 2

(CD19+CD23+IgMhighCD93+, CD86+), and activated follicular B cells (CD19+IgMlow/highCD23+CD932, CD86+), were assessed for each animal within each

group. The gating strategy for each subset is shown (C). The average total number of cells within each subset is presented (D). Student t tests were performed, and

significance is noted as *#0.05, **#0.01. These data are representative of three separate experiments.

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and 70%, respectively, when compared with animals treated withrhGAA alone (Fig. 3D). No increases were observed betweensaline-treated and rhGAA alone–treated mice or between saline-treated and methotrexate only–treated mice in any of the pop-ulations examined (Fig. 3D).

Methotrexate treatment increases the numbers of CD1dhigh

CD5+B cells that are IL-10, TGF-b, and Foxp3 positive

IL-10 expression was assessed in CD1dhighCD5+ B cells isolatedfrom mice treated with rhGAA plus methotrexate or rhGAA alonein three independent experiments to determine whether metho-trexate induces the expansion of B10 B cells. B10 B cells areregulatory IL-10–secreting CD1dhighCD5+ B cells (35, 36). Spe-cifically, splenic B cells were purified from WT animals 7 d aftertreatment, cultured without any stimulation for 2 d, and then an-alyzed by flow cytometry. A subset of CD1dhighCD5+ B cells fromanimals treated with rhGAA alone appears to express IL-10. The

numbers of IL10+CD1dhighCD5+ B cells isolated from rhGAAplus methotrexate–treated mice were significantly increased by anaverage of 70% over those isolated from animals treated withrhGAA alone (Fig. 4A) across three experiments in WT mice. Inaddition, the total mean fluorescence intensity (MFI) of IL-10 inCD1dhighCD5+ B cells obtained from methotrexate-treated micewas also significantly increased by an average of 33% across threeseparate experiments (Fig. 4D).TGF-b and Foxp3 are additional proteins that have been associ-

ated with immunosuppressive effects of regulatory B cells (37–39).The expression of these proteins in CD1dhighCD5+ B cells was in-vestigated by flow cytometry, as described above. CD1dhighCD5+

B cells isolated from rhGAA-treated mice appear to show TGF-b,specifically the propeptide LAP/TGF-b1, and Foxp3 expression ina small subset of the population. In contrast, mice treated withrhGAA plus methotrexate appear to consistently exhibit an average70% increase in numbers of both TGF-b+CD1dhighCD5+ and Foxp3+

CD1dhighCD5+ B cells (Fig. 4B, 4C). Moreover, the overall expres-sion levels of TGF-b and Foxp3, as measured by MFI, withinCD1dhighCD5+ B cells were significantly increased by 42 and 33%,respectively, in CD1dhighCD5+ B cells cultured from rhGAA plusmethotrexate–treated mice as compared with rhGAA alone–treatedmice within each and among all three experiments (Fig. 4E, 4F).

Methotrexate treatment increases the numbers of transitional 2and follicular B cells that are IL-10, TGF-b, and Foxp3positive

Because methotrexate treatment appeared to influence the ex-pression of IL-10, TGF-b, and Foxp3 in CD1dhighCD5+ B cells,we investigated whether methotrexate induced similar effects in

Table I. B cell subsets assessed by flow cytometry

B Cell Subset Name Surface Phenotype Assessed

Folliculara CD19+CD932IgM+/2CD23+

Marginal zonea CD19+CD932CD21highCD232/low

Transitional 1a CD19+CD93+IgMhighCD232

Transitional 2a CD19+CD93+IgMhighCD23+

Transitional 3a CD19+CD93+IgMlowCD23+

B10-likeb CD19+CD5+CD1dhigh

aActivation status of these cell types was assessed by CD86 expression.bThese cells are termed B10-like because initially we did not assess IL-10 ex-

pression.

FIGURE 4. Methotrexate treatment increases the numbers of CD1dhighCD5+ B cells that are IL-10, TGF-b, and Foxp3 positive. B cells were purified

from splenocytes, isolated, and processed from C57BL/6 mice treated with either rhGAA alone or in combination with single- cycle methotrexate, as

described above. Five animals were treated per group. Cells isolated from each treatment group were pooled and cultured in replicates of six without

stimulation for 2 d. Flow cytometry was then performed on each replicate to assess the presence of IL-10, TGF-b, and Foxp3 in CD1dhighCD5+ B cells, as

described in Materials and Methods. The average total number of IL-10+ CD1dhighCD5+, TGF-b+ CD1dhighCD5+, and Foxp3+ CD1dhighCD5+ B cells

obtained in three independent experiments is graphed, with each character representing the average number for each experiment (A–C). Representative

histograms IL-10, TGF-b, and Foxp3 in total CD1dhighCD5+ B cells are depicted; the average MFI observed in three independent experiments are graphed

(D–F). In addition to the p values noted on the figure, significance is represented as *#0.05, **#0.01.

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transitional 2 and follicular B cells. The number of IL-10+ tran-sitional 2 B cells increased significantly and consistently by anaverage of 45% in mice treated with rhGAA plus methotrexatewhen compared with animals treated with rhGAA alone in threeindividual experiments (Fig. 5A). The total MFI of IL-10 expressionin transitional 2 B cells appears to be enhanced in methotrexate-treated mice (by 43%), although the level of increase is variablebetween experiments (Fig. 5D). Analogous increases in cell num-bers were observed with TGF-b+ and Foxp3+ transitional 2 B cells(40 and 50%, respectively), and these increases were significantacross three independent experiments (Fig. 5B, 5C). The averageMFI of TGF-b+ and Foxp3+ also was increased (by 46 and 52%,respectively; Fig. 5E, 5F). These increases were generally signifi-cant in each individual experiment, but the level of increase variedamong the three experiments. Moreover, the increase in totalTGF-b expression in transitional 2 B cells was less consistent, asit was significant in only two of the three studies.In three separate experiments, IL-10, TGF-b, and Foxp3 were

observed in ∼1% of follicular B cells obtained from rhGAA-treatedmice. The numbers of IL-10+ follicular B cells, TGF-b+ follicularB cells, and Foxp3+ follicular B cells were significantly expandedwith methotrexate treatment by an average of 55, 57, and 72%,respectively, across three experiments (Fig. 6A–C). The most vari-ability across experiments was observed with increases in the num-bers of Foxp3+ follicular B cells (Fig. 6C). A significant shift inMFI for these proteins was not observed within the total follicular

B cell population. However, when activated CD86+ follicular B cellswere analyzed, we observed slight but significant increases in theMFI of IL-10, TGF-b, and Foxp3 in cells obtained from mice treatedwith rhGAA plus methotrexate compared to animals treated withrhGAA alone. Specifically, the MFI of IL-10, TGF-b, and Foxp3 wasincreased by 32, 34, and 40% respectively (Fig. 6D–F).

IL-10, but not active, secreted TGF-b, appears to be necessaryfor methotrexate-induced immune tolerance

Because IL-10 has consistently been described as a critical me-diator of B regulatory cell function (35), we assessed whether IL-10 was required for methotrexate-induced immune tolerance torhGAA. Both WT C57BL/6 mice and IL-10KO mice of the samegenetic background were treated weekly with 20 mg/kg rhGAA,i.v., with and without single cycle methotrexate. Anti-rhGAA–specific IgG titers were assessed biweekly by ELISA on weeks 4,6, and 8. The C57BL/6 WT mice responded as expected with an80% reduction in AUC of titers over time in rhGAA plus meth-otrexate–treated mice compared with rhGAA alone–treated mice(Fig. 7A). In contrast, no reduction in anti-rhGAA–specific titerswas observed in IL-10KO mice treated with rhGAA plus metho-trexate when compared with those treated with rhGAA alone(Fig. 7B). Expectedly, the anti-rhGAA titer values were lower inthe IL-10KO mice than in the WT mice. IL-10 is also known topromote plasma cell differentiation and Ab secretion (40, 41).Therefore, these studies were initiated with the expectation that

FIGURE 5. Methotrexate treatment increases the numbers of transitional 2 B cells that are IL-10, TGF-b, and Foxp3 positive. B cells were purified from

splenocytes isolated and processed from C57BL/6 animals treated with either rhGAA alone or in combination with single-cycle methotrexate, as described

above. B cells isolated from each treatment group were pooled and cultured in replicates of six without stimulation for 2 d. Flow cytometry was then

performed on each replicate to assess the presence of IL-10, TGF-b, and Foxp3 in transitional 2 B cells, as described in Materials and Methods. Rep-

resentative dot plots are shown for each population assessed from rhGAA and rhGAA plus methotrexate–treated animals. The average total number of

IL-10+ transitional 2, TGF-b+ transitional 2, and Foxp3+ transitional 2 B cells obtained in three independent experiments is graphed, with each character

representing the average number for each experiment (A–C). Representative histograms IL-10, TGF-b, and Foxp3 in total transitional 2 B cells are depicted,

and the average MFI observed in three independent experiments are graphed (D–F). In addition to the p values noted on the figure, significance is rep-

resented as *#0.05, **#0.01.

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anti-rhGAA–specific IgG titers would be lower in the IL-10KOmice, but not too low to evaluate the effect of methotrexate. Asdemonstrated in Fig. 7D, single-cycle methotrexate can success-fully reduce titers when maximal titer values in a study are in therange of 2–5 3 104, which is within the range observed in the IL-10KO mice treated with rhGAA.After observing that methotrexate could not control titers in IL-

10KO mice, we evaluated the numbers of splenic IL-10+ B10 B cells,TGF-b+ B10 B cells, and Foxp3+ B10 B cells in WT C57BL/6 miceand IL-10KO mice that were treated with rhGAA alone or rhGAAplus methotrexate. Not surprisingly, the numbers of IL-10+ B10B cells in the IL-10KO mice were not increased with methotrexatetreatment as compared with WT mice (Fig. 7C). However, the num-bers of TGF-b+ B10 B cells and Foxp3+ B10 B cells were similarlyincreased in IL-10KO mice and C57BL/6 WT mice (Fig. 7C).The impact of TGF-b inhibition on anti-rhGAA–specific IgG

titer by methotrexate-induced immune tolerance was assessed byadministering a TGF-b–neutralizing murine IgG1 mAb, 1D11.The 1D11 neutralizes all active, secreted forms of TGF-b. The1D11 or its isotype control Ab (13C4) was administered threetimes per week throughout the course of the study. The 1D11treatment had no impact on the ability of methotrexate to induceimmune tolerance to rhGAA (Fig. 7D).

Splenic B cells isolated from mice tolerized to rhGAA bymethotrexate transferred tolerance to naive animals

Adoptive transfer studies were performed to assess whether totalsplenic B cells, which include B10, activated transitional 2, and

activated follicular B cells, could transfer tolerance to naive hosts(Fig. 8). Total splenic B cells were therefore purified from donormice, 7 d after treatment with either rhGAA alone or rhGAA plusmethotrexate. Following negative B cell separation, donor B cellswere 94–95% pure (Fig. 8B). Five million B cells from rhGAAalone–treated mice were transferred to naive recipient mice.Recipients of B cells purified from rhGAA plus methotrexate–treated mice were administered between 500,000 and 5 millionB cells. Following transfer, the recipient mice were treated weeklywith 20 mg/kg rhGAA i.v. Anti-rhGAA IgG titers were thenassessed biweekly through 16 wk. The highest average titer wasobserved in animals that received B cells from rhGAA alone–treated donor mice. These titers were similar in range to thosereported through week 16 for naive C57BL/6 mice treated withweekly rhGAA (Fig. 2A). Recipient mice of B cells from rhGAAplus methotrexate–treated donor mice exhibited the lowest anti-rhGAA–specific IgG titers with a percent reduction in AUCranging from 88 to 99% (Fig. 8C). Recipients of B cells frommethotrexate-treated mice also exhibited little variability in titeramong the animals within each of the four recipient groups(Fig. 8C). Of note, two of the eight animals that received splenicB cells from rhGAA alone–treated mice did not mount an Abresponse to rhGAA, and one animal exhibited a very low anti-rhGAA IgG titer. The reason for this lack of response is unclear.These animals were not included in the data presented in Fig. 8C.Statistical significance was conservatively assessed by removingthe two animals with no response and comparing the AUC dif-ference between the recipients of 5 million nontolerized cells and

FIGURE 6. Methotrexate treatment increases the numbers of follicular B cells that are IL-10, TGF-b, and Foxp3 positive. B cells were purified from

splenocytes isolated and processed from C57BL/6 mice treated with either rhGAA alone or in combination with single-cycle methotrexate, as described

above. Cells isolated from each treatment group were pooled and cultured in replicates of six without stimulation for 2 d. Flow cytometry was then

performed on each replicate to assess the presence of IL-10, TGF-b, and Foxp3 in follicular B cells, as described in Materials and Methods. Representative

dot plots are shown for each population assessed from rhGAA and rhGAA plus methotrexate–treated animals. The average total number of IL-10+ follicular,

TGF-b+ follicular, and Foxp3+ follicular B cells obtained in three independent experiments is graphed, with each character representing the average number

for each experiment (A–C). Representative histograms IL-10, TGF-b, and Foxp3 in activated (CD86+) follicular B cells are depicted, and the average MFI

observed in three independent experiments are graphed (D–F). In addition to the p values noted on the figure, significance is represented as *#0.05.

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recipients of 5 million tolerized cells (p = 0.02), recipients of2 million tolerized cells (p = 0.008), recipients of 1 milliontolerized cells (p = 0.01), and recipients of 500,000 tolerized cells(p = 0.01). When all eight recipient mice of rhGAA alone–treatedB cells were included in the statistical comparison, the p valuerange was 0.052–0.1, in which the comparison between the re-cipient mice of 5 million nontolerized and 2 million tolerizedmice was the closest to reaching statistical significance.A schematic representation of the proposed effects of low-dose

methotrexate induction treatment on antibody responses is shownin Fig. 9.

DiscussionThese studies expand our understanding of the function of metho-trexate. Methotrexate has classically been described as a dihy-drofolate reductase antagonist (2). Folate inhibition by methotrexateinterferes with purine metabolism and DNA synthesis, and thusinduces death in proliferating cells (2). Through this mechanism,methotrexate has served as a successful oncology therapy for manyyears. Continuous, low-dose, weekly administration of methotrexate

has been associated with a similar proposed mechanism of actionin the treatment of rheumatoid arthritis (7, 9), although othermechanisms of methotrexate could also contribute in this setting(14, 15). Cytokine inhibition, apoptosis, adenosine release, andchemotaxis may be additional means by which methotrexatemay modulate inflammation (10–13, 15, 16).Recently, low-dose, methotrexate induction treatment has

been shown to induce immune tolerance to protein therapies inseveral different mouse models (27–29). In the context ofmurine-specific antithymocyte globulin (mATG), a single cycleof three daily doses of methotrexate at the initiation of monthlymATG treatment more effectively reduced ADA and enhancedmATG pharmacodynamic activity than three consecutive cycles ofmethotrexate (29). Our present investigation of single- versus three-cycle methotrexate in rhGAA tolerance demonstrated equivalentADA reduction by both regimens. Importantly, reduced Ab titerswere similarly maintained through rhGAA rest and rechallenge,suggesting durable immune tolerance with methotrexate inductiontreatment. In addition, regardless of whether rhGAA-treated micereceived methotrexate or not, both sets of animals were able to

FIGURE 7. IL-10, but not active,

secreted TGF-b, appears to be neces-

sary for methotrexate-induced immune

tolerance. IL-10KO mice or C57BL/6

mice were treated weekly for 8 wk with

rhGAA in the presence and absence of

single-cycle methotrexate, as described

above. Anti-rhGAA–specific IgG titers

were assessed by ELISA at weeks 4, 6,

and 8, as described in Materials and

Methods. Two studies were conducted;

one study consisted of 12 animals per

group, and the other consisted of 8

animals per group. Data from the two

individual experiments were graphed

together (A and B). C57BL/6 mice

and IL-10KO mice were treated

with rhGAA alone or rhGAA plus sin-

gle-cycle methotrexate. Each group

contained six animals. Seven days

following treatment, the absolute cell

numbers of splenic IL10+ B10 B cells,

TGF-b+ B10 B cells, and Foxp3+ B10 B

cells were assessed, as described above

(C). These data are representative of

two independent studies. Four groups of

10 WT C57BL/6 mice each were treated

weekly with 20 mg/kg i.v. rhGAA.

Three of the rhGAA-treated groups

were additionally given single-cycle

methotrexate in the presence of either

the murine IgG1, anti–TGF-b mAb,

1D11, or the isotype control Ab, 13C4, at

5 mg/kg i.p., weekly, every other day

through the end of the study, as described

above. Anti-rhGAA–specific IgG titers

were assessed biweekly throughout the

course of the study (D). These data are

representative of two separate experi-

ments. Significance is represented as

*p , 0.05, ***p , 0.005.

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produce comparable responses against a different Ag, mATG.Therefore, methotrexate can induce Ag-specific tolerance when ad-ministered as a single cycle.Although these results support the utility of methotrexate in

immune tolerance induction, they do not explain the mechanismby which methotrexate exerts such effects. Because methotrexatetreatment has been associated with the death of activated, prolif-erating cells, as well as with apoptosis induction (15), we inves-tigated whether cell numbers were altered following methotrexatetreatment. Specifically, T and B cell subsets within spleen andlymph nodes were assessed as these populations would be acti-vated upon rhGAA treatment to enable B cell differentiation intoAb-secreting plasma cells. In addition, T regulatory cell numberswere investigated because methotrexate has been described toinduce adenosine, and adenosine has been associated with Tregulatory cell function (16–18). No differences were observed inthe total numbers of Th, T cytotoxic, T regulatory, or B cells

between mice treated with rhGAA alone or with rhGAA plusmethotrexate in either tissue. These results were expected becauseonly a small percentage of total T and B cells should be responsiveto rhGAA, and minor shifts in cell numbers could be easilymasked within assessments of the total populations. Becausemethotrexate specifically targets activated cells rather than rest-ing cells (15), the numbers of activated Th, T cytotoxic, T regu-latory, as measured by CD69 expression, and activated B cells, asmeasured by CD86 expression, were investigated. No changes inthe cell numbers of these populations were observed in the lymphnodes. Interestingly, rhGAA plus methotrexate induced signifi-cant increases in the absolute numbers of activated splenic B cells,whereas no changes were observed in any activated T cell pop-ulation. The increases observed in the numbers of activated B cellswere consistent among both WT and E4-8GAAKO mice, and thetiming of the increase was a bit faster in the E4-8GAAKO mouse.There were no significant differences between saline-treated miceand rhGAA alone–treated mice, nor between the control groups,saline-treated mice, and methotrexate only–treated mice.To further understand the effect of methotrexate on B cells,

different splenic B cell subsets were examined. These includedfollicular, marginal zone, transitional 1, transitional 2, transitional3, and B10-like B cells. These peripheral subsets represent fullymature B cells, B cells transitioning from immaturity to maturity, aswell as regulatory B cells (35, 42). Of these subsets, significantincreases in the cell numbers of activated follicular, activatedtransitional 2, and B10-like B cells were reproducibly observed inanimals treated with rhGAA plus methotrexate when comparedwith animals treated with rhGAA alone. CD1dhighCD5+ B cellsthat secrete IL-10 have been described in both mouse and humansas B10 regulatory B cells (35, 36). B10 B cells have been im-plicated in controlling immune responses in murine disease set-tings that include, but are not limited to experimental autoimmuneencephalomyelitis (43), sclerodermatous graft-versus-host disease(44), and chronic intestinal inflammation (45). In addition, B10B cells have been shown to reduce IgG responses in mice (46).IL-10 secretion has been identified as a critical mediator of B10B cell regulatory function (35). Therefore, we investigatedwhether the CD1dhighCD5+ B10-like B cell subset that is in-creased upon methotrexate treatment expresses IL-10. We ob-served IL-10+ CD1dhighCD5+ cells and consistently foundsignificant increases in the numbers of IL-10+ CD1dhighCD5+ cellsin rhGAA plus methotrexate–treated mice when compared withthose isolated from rhGAA alone–treated animals. Moreover, theoverall expression level of IL-10, as measured by MFI, within theCD1dhighCD5+ population was increased in methotrexate-tolerizedmice. Cytokine expression by regulatory B cells is typically mea-sured following in vitro culture with stimulation by LPS or PMAand ionomycin along with monensin treatment (47). Although theincreases in IL-10 MFI are ∼2-fold, our assessments of IL-10 areconducted in primary B cells cultured for 2 d in the absence of anystimulation, suggesting that rhGAA plus methotrexate is a potentinducer of this response. These data suggest that methotrexatewhen administered with rhGAA can induce the expansion of B10B cells, and that B10 B cells may be involved in mediating theimmune-tolerizing effects of methotrexate.In addition to B10 B cells, several other regulatory B cell subsets

have been described in mice (48, 49). Some of these subsets appearto express TGF-b, which is linked with their immunosuppressiveproperties (48, 50–53). Although TGF-b expression has not yetbeen described for B10 B cells, we evaluated whether TGF-bexpression is influenced by methotrexate treatment in B10 B cells.There are three isoforms of TGF-b, as follows: TGF-b1, TGF-b2,and TGF-b3 (54). The predominant form of TGF-b in immune

FIGURE 8. Splenic B cells isolated from mice tolerized to rhGAA by

methotrexate appear to transfer tolerance to naive animals. Donor C57BL/6

mice were treated with rhGAA alone or rhGAA plus methotrexate (A), as

described above. Seven days following treatment, spleens were harvested

and B cells were purified and pooled from the donor animals (A). B cells

isolated from rhGAA alone–treated mice were 95% pure, and B cells iso-

lated from rhGAA plus methotrexate–treated mice were 94% pure (B). Five

million B cells from rhGAA alone–treated mice (nontolerized) and 5, 2.5, 1,

and 0.5 million B cells from rhGAA plus methotrexate–treated mice

(tolerized) were i.v. injected into naive, syngeneic recipient mice (A). The

recipients were then treated weekly for 15 wk with 20 mg/kg i.v. rhGAA.

Anti-rhGAA–specific IgG were assessed every other week by ELISA, as

described in Materials and Methods, throughout the 16-wk study (C). These

data are representative of two separate experiments.

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cells is TGF-b1, and it is synthesized as an inactive proproteincalled LAP (54). LAP/TGF-b interacts with latent TGF-b–bindingprotein, and the entire complex is secreted from cells (54). TGF-bis then activated upon additional stimulation such as binding tocell surface proteins (54). We used an anti–LAP/TGF-b1 Ab tomeasure TGF-b in the experiments described above. In threeseparate experiments, animals that were treated with rhGAA plusmethotrexate exhibited increases in not only the numbers of TGF-b+CD1dhighCD5+ cells, but in the MFI of TGF-b within thesecells when compared with animals treated with rhGAA alone.Finally, although Foxp3 expression has been widely demonstrated

in T regulatory cells, there is an example in which Foxp3 has beenobserved in human CD5+CD19+ B cells (55). Moreover, in T cells,TGF-b can induce Foxp3 expression and promote the developmentof T regulatory cells (56). In addition, T regulatory cells can expressFoxp3, IL-10, and TGF-b, and these proteins are associated withimmunosuppression (57). We investigated whether we could observeFoxp3 expression in methotrexate-induced CD1dhighCD5+ cells. In-terestingly, mice treated with rhGAA plus methotrexate exhibitedsignificant increases in the numbers of Foxp3+CD1dhighCD5+ B cells.In addition, the expression level of Foxp3 in this cell subset wasincreased in methotrexate-treated mice.Because rhGAA plus methotrexate treatment appeared to ex-

pand the absolute numbers of two other B cell subtypes that have alsobeen associated with immune regulation, transitional 2 B cells andfollicular B cells (58, 59), we decided to evaluate whether thesepopulations expressed the immunosuppressive proteins IL-10,TGF-b, and Foxp3 and whether these subsets were enhanced byrhGAA plus methotrexate treatment. Transitional 2 and follicularB cells isolated from rhGAA plus methotrexate–treated mice wereIL-10+, TGF-b+, and Foxp3+. The numbers of these cell subsetswere significantly increased in mice treated with rhGAA plusmethotrexate when compared with those isolated from animalstreated with rhGAA alone. Generally, the expression of theseproteins in transitional 2 B cells was increased with methotrexatetreatment, although the MFI of TGF-b was only significantly in-creased in two of the three experiments. In contrast, because sucha small percentage of total follicular B cells was expanded withmethotrexate treatment, shifts in the MFI of IL-10, TGF-b, andFoxp3 were only observed in activated CD86+ follicular B cells.One open question was whether IL-10 is a critical mediator of

tolerance to rhGAA in this treatment paradigm, as has beenconsistently reported in both human and mouse for B regulatorycell–mediated suppression (35, 36, 60). To assess this, we com-pared the ability of methotrexate to reduce rhGAA-specific IgG inC57BL/6 WT mice and C57BL/6-derived IL-10KO mice. An 80%

reduction in titer was observed in C57BL/6 WT mice, whereas noreduction in rhGAA-specific IgG by methotrexate was observedin IL-10KO mice. These data suggest that IL-10 is a critical medi-ator of methotrexate-induced tolerance. Upon further evaluation, thenumbers of IL-10+ B10 B cells in IL-10KO mice were not increasedwith rhGAA plus methotrexate treatment as observed in C57BL/6WT mice. In contrast, the numbers of TGF-b+ B10 B cells andFoxp3+ B10 B cells were similarly increased in both murine strains.These data suggest that TGF-b and Foxp3 expression in B10 B cellscannot, independently of IL-10, enable methotrexate-induced im-mune tolerance.The role of TGF-b in methotrexate-induced immune tolerance

to rhGAA was investigated further. A neutralizing murine IgG1anti–TGF-b mAb (1D11) that binds to active TGF-b1, TGF-b2,and TGF-b3 was used. Alongside rhGAA alone or rhGAA plusmethotrexate treatment, 1D11 was given three times per week at5 mg/kg in a regimen that has demonstrated TGF-b neutralizationin mice (61). Two independent studies demonstrated that the in-hibition of active TGF-b did not interfere with the ability of metho-trexate to control anti-rhGAA–specific IgG, indicating thatsecreted, active TGF-b may not be an essential mediator of thisprocess.To investigate whether B cells isolated from methotrexate-

tolerized mice can transfer immune tolerance to naive hosts,purified splenic B cells from animals treated with either rhGAAalone or rhGAA plus methotrexate were transferred into naiveanimals that began weekly rhGAA treatment the same day as celltransfer. Because small populations of B10, follicular, and tran-sitional 2 B cells appeared to be responding to rhGAA plusmethotrexate treatment, we decided to assess the effects of all ofthem by transferring total splenic B cells. Methotrexate-inducedchanges in B10, transitional 2, and follicular B cells were con-sistently observed 7 d following rhGAA plus methotrexate treat-ment in C57BL/6 WT mice; thus, this time point was chosen forharvest and adoptive transfer. In the recipient mice, anti-rhGAA–specific IgG was evaluated every other week for 16 wk. Recipientsof 5 million cells isolated from nontolerized mice treated withrhGAA alone displayed the highest average titer. In contrast,recipients of either 5, 2, 1, or 0.5 million cells purified from micetolerized to rhGAA by methotrexate exhibited lower titers of anti-rhGAA IgG and overall reductions in area under the effect curveranging from 88 to 99%. The reduction in Ab responses remainedthroughout the 16-wk study. Moreover, whereas the variability intiter among the individual animals that received B cells fromrhGAA alone–treated mice was large, the variability exhibitedamong animals within each of the four groups that received B cells

FIGURE 9. Proposed schematic of the effects of

low-dose methotrexate induction treatment on Ab

responses.

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from methotrexate-treated mice was low. The data from thesestudies collectively suggest that B cells represent a key mediatorof methotrexate-induced Ag-specific immune tolerance.In conclusion, the studies reported in this work identify a novel

mode of methotrexate function. Particularly, low-dose inductiontreatment of methotrexate given together with Ag stimulationexpands IL-10+, TGF-b+, and Foxp3+ B cell populations thatappear to control Ab responses and contribute to immune toler-ance induction in mice (Fig. 9). Regulatory B cells with similarprotein expression profiles have been observed in autoimmunepatients, tolerized human transplant patients, and allergy (39, 58,62–64). Thus, it is conceivable that methotrexate may success-fully induce tolerance in humans through a similar mechanism.Moreover, methotrexate treatment in rheumatoid arthritis pa-tients may exert similar effects that may help control disease.Currently, high-risk, infantile Pompe patients who may not re-spond to enzyme-replacement therapy due to ADA interferenceare receiving an immune tolerance regimen that incorporatesa low-dose methotrexate induction treatment along with Ritux-imab and optional i.v. IG (30–32). This regimen has successfullytolerized a number of these high-risk patients and has enabledeffective response to therapy (30–32). The use of Rituximab canbe associated with infection risk and is not available worldwide.The availability of a methotrexate-only tolerance strategy mayfurther enable treatment responsiveness in Pompe patients, in-cluding high-risk patients unable to receive Rituximab.

AcknowledgmentsWe thank Michael Phipps and Adam Palermo for help characterizing the

E4-8GAAKO mouse, the Department of Comparative Medicine for help

executing the animal studies, Patrick DeCourcy for help with ELISA,

Wenfei Zhang and Yuefeng Lu for performing some of the statistical assess-

ments, and Alison Schroeer for work finalizing the figures.

DisclosuresAll of the authors were employed by Genzyme, a Sanofi company, and held

some equity in the company while this work was being conducted.

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