IL-10-Producing CD4+CD25+ Regulatory T Cells Play a Critical Role in Granulocyte-Macrophage Colony-Stimulating Factor-Induced This information is current as Suppression of Experimental Autoimmune of September 29, 2021. Thyroiditis Eryn Gangi, Chenthamarakshan Vasu, Donald Cheatem and Bellur S. Prabhakar Downloaded from J Immunol 2005; 174:7006-7013; ; doi: 10.4049/jimmunol.174.11.7006 http://www.jimmunol.org/content/174/11/7006 http://www.jimmunol.org/ References This article cites 51 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/174/11/7006.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-10-Producing CD4؉CD25؉ Regulatory T Cells Play a Critical Role in Granulocyte-Macrophage Colony-Stimulating Factor-Induced Suppression of Experimental Autoimmune Thyroiditis1

Eryn Gangi,* Chenthamarakshan Vasu,† Donald Cheatem,* and Bellur S. Prabhakar2*

Our earlier study showed that GM-CSF has the potential not only to prevent, but also to suppress, experimental autoimmune thyroiditis (EAT). GM-CSF-induced EAT suppression in mice was accompanied by an increase in the frequency of CD4؉CD25؉ regulatory T cells that could suppress mouse thyroglobulin (mTg)-specific responses in vitro, but the underlying mechanism of this suppression was not elucidated. In this study we show that GM-CSF can induce dendritic cells (DCs) with a semimature Downloaded from phenotype, an important characteristic of DCs, which are known to play a critical role in the induction and maintenance of regulatory T cells. Adoptive transfer of CD4؉CD25؉ T cells from GM-CSF-treated and mTg-primed donors into untreated, but mTg-primed, recipients resulted in decreased mTg-specific T cell responses. Furthermore, lymphocytes obtained from these donors and recipients after adoptive transfer produced significantly higher levels of IL-10 compared with mTg-primed, untreated, control mice. Administration of anti-IL-10R Ab into GM-CSF-treated mice abrogated GM-CSF-induced suppression of EAT, as indicated by increased mTg-specific T cell responses, thyroid lymphocyte infiltration, and follicular destruction. Interestingly, in http://www.jimmunol.org/ -vivo blockade of IL-10R did not affect GM-CSF-induced expansion of CD4؉CD25؉ T cells. However, IL-10-induced immuno suppression was due to its direct effects on mTg-specific effector T cells. Taken together, these results indicated that IL-10, -produced by CD4؉CD25؉ T cells that were probably induced by semimature DCs, is essential for disease suppression in GM CSF-treated mice. The Journal of Immunology, 2005, 174: 7006–7013.

xperimental autoimmune thyroiditis (EAT)3 is a well-es- response. Specifically, injection of CD8aϩ DCs triggers the devel- tablished mouse model for Hashimoto’s thyroiditis (HT). opment of Th1 cells, whereas CD8aϪ DCs induce Th2-type re- HT is an organ-specific autoimmune disease character- sponses to soluble Ags (15–17). Therefore, targeted expansion of

E by guest on September 29, 2021 ized by lymphocyte infiltration of the thyroid that eventually leads a particular DC subset might be used to shift an immune response to follicular destruction. In HT, thyroglobulin (Tg)-specific T cells from one type to another and thereby prevent autoimmune disease are generated, and they migrate to the thyroid. These cells produce development. In addition, DC maturation can be modulated using IFN-␥, which induces the expression of MHC class II on thyro- different cytokines to induce either regulatory T cells (Treg) or cytes and results in further expansion and accumulation of activated effector T cells (18–22). mouse Tg (mTg)-specific T cells (1–5). The mechanism(s) of thyroid Neither CD8aϩ nor CD8aϪ DCs can induce optimal T cell re- destruction, although not completely understood, appears to involve sponses when they are immature, but they become potent activa- cytokine production by thyroid-infiltrating T cells that can facilitate tors of T cells when they are mature (15–17). Although immature apoptosis of thyrocytes through caspase activation (6–9). DCs, characterized by the expression of low levels of costimula- Although dendritic cells (DCs) are essential for the induction of tory molecules and proinflammatory cytokines, can promote an- an effective immune response against foreign Ags, they can also ergy; semimature DCs that express significant levels of MHC class play a critical role in promoting and maintaining tolerance to self- II and costimulatory molecules, but low levels of proinflammatory Ags (10–14). Modulation of DC phenotype and maturation status cytokines, compared with mature DCs can induce Treg (10, 21, in vitro and in vivo can have a profound effect on T cell activation 23). These observations clearly illustrate that modulation of func- and differentiation and may skew the immune response. Different tional properties of DCs can be an effective therapeutic approach subsets of DCs can preferentially influence a Th1- or a Th2-type for autoimmune conditions. Our earlier studies (24, 25) showed that administration of GM- CSF or Flt3 ligand, potent DC growth factors, resulted in suppres- Departments of *Microbiology and Immunology and †Surgery, University of Illinois, Chicago, IL 60612 sion or augmentation of EAT, respectively. Treatment with GM- Ϫ Received for publication October 22, 2004. Accepted for publication March 23, 2005. CSF induced CD8a DCs and caused a shift in the immune response against Tg from a Th1 response to a Th2 response, as 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 seen by increased IL-4 production with a concomitant decrease in with 18 U.S.C. Section 1734 solely to indicate this fact. IFN-␥ production. However, GM-CSF-induced suppression of 1 This work was supported by National Institutes of Health Grant R21DK066634. EAT was associated not only with mere Th2 skewing but also with ϩ ϩ 2 Address correspondence and reprint requests to Dr. Bellur S. Prabhakar, Department a selective expansion of CD4 CD25 Treg that could suppress of Microbiology and Immunology (M/C 790), Room E-709, Building 935, 835 South mTg-specific responses in vitro (24). CD4ϩCD25ϩ Treg play a Wolcott Avenue, Chicago, IL 60612. E-mail address: [email protected] critical role in the suppression of autoimmunity. Depletion or ab- 3 Abbreviations used in this paper: EAT, experimental autoimmune thyroiditis; DC, ϩ ϩ ; FasL, Fas ligand; HT, Hashimoto’s thyroiditis; mTg, mouse Tg; Tg, sence of CD4 CD25 Treg has been shown to result in the de- thyroglobulin; Tr1, type 1 ; Treg, regulatory T cell. velopment of autoimmune disease (26, 27). Although how

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 7007

ϩ ϩ CD4 CD25 Treg suppress autoimmunity is not fully understood, samples and were used for PCR. Cytokine transcript levels for IL-10, IL-6, suppressor cytokines, such as IL-10, have been implicated (28– TNF-␣, IL-1, and IL-12 were determined using a multiplex PCR method 33). In GM-CSF-treated mice, there was a considerable increase in according to the manufacturer’s guidelines (Maxim Biotech). the levels of IL-10, and neutralization of IL-10 in lymphocyte cul- Adoptive transfer of CD4ϩCD25ϩ T cells tures derived from GM-CSF-treated mice restored mTg-specific T cell responses. Furthermore, lymphocytes from GM-CSF-treated CFA control, mTg control, GM-CSF/mTg, and CD4ϩCD25ϩ T cell recip- mice that were depleted of CD4ϩCD25ϩ T cells showed enhanced ient groups were included in this experiment. Mice from GM-CSF/mTg group were used as donors and were killed on day 34; CD4ϩCD25ϩ T cells mTg-specific proliferation, with a concomitant decrease in the lev- were isolated from splenocytes and lymph nodes using a CD4ϩCD25ϩ els of IL-10 in vitro, suggesting that these cells were the source of isolation kit following the manufacturer’s instructions (Miltenyi Biotec). IL-10 (24). These data implied a role for IL-10 in GM-CSF-in- The isolated population was Ͼ90% pure. One set of mTg-immunized mice ϩ ϩ duced suppression of EAT. was adoptively transferred with donor CD4 CD25 T cells (1 ϫ 106/ In the current study we investigated the direct role of mouse) by i.v. injection on day 28 after initiation of immunization. Ani- mals were killed on day 45 (day 18 posttransfer), and lymph nodes, CD4ϩCD25ϩ T cells and IL-10 in GM-CSF-induced suppression ϩ ϩ spleens, thyroids, and sera were collected for analyzing the mTg-specific of EAT. We show that adoptive transfer of CD4 CD25 T cells immune response. from GM-CSF-treated mice into mTg-primed mice can suppress mTg-specific proliferation, and cells from recipient mice can pro- CD4ϩCD25ϩ T cell coculture duce higher levels of IL-10. Furthermore, in vivo blockade of IL- The mTg control and GM-CSF mTg mouse groups were used in this study. 10R can abrogate GM-CSF-induced suppression and restore mTg- Mice were treated with GM-CSF and immunized with mTg as described specific T cell responses, resulting in the development of EAT. above. They were killed on day 35, spleen and lymph node cells were Downloaded from ϩ ϩ Moreover, we observed an increase in DCs with a semimature collected, and CD4 CD25 T cell were isolated from mice in the GM- phenotype in GM-CSF-treated mice, which suggested a putative CSF/mTg group as described above. Effector T cells were isolated from spleen and lymph node cells from mice in the mTg control group using mechanism for the induction of Treg. These data show the critical ϩ ϩ magnetic cell sorting (Miltenyi Biotec) and were stained with CFSE at a role that CD4 CD25 T cells and IL-10 play in GM-CSF-induced concentration of 1 ␮M for 10 min at 37°C. Cells were washed three times suppression of EAT. and plated into 96-well, flat-bottom plates at 0.5 ϫ 106 cells/well. Isolated CD4ϩCD25ϩ T cells from group 3 were added to cultures at a 5:1 effector: http://www.jimmunol.org/ Treg ratio. T cell-depleted spleen cells (0.5 ϫ 106 cells/well) or enriched Materials and Methods DCs (0.1 ϫ 106 cells/well) from naive mice (both accomplished by mag- Mice netic cell sorting; Miltenyi Biotec) were used as feeder cells in this study. Cells were harvested after 7 days in culture and were tested for CFSE Six- to 8-wk-old female CBA/J were purchased from The Jackson Labo- dilution using a FACS analyzer (BD Biosciences). ratory. Mice were housed at the Biological Resources laboratory facility at University of Illinois and were provided food and water ad libitum. Ani- mals were cared for in accordance with the guidelines set forth by the Anti-IL-10R Ab treatment University of Illinois animal care and use committee. All mice were used Six groups (group 1, CFA controls; group 2, mTg controls; group 3, GM- at 8–10 wk of age. CSF/isotype controls; group 4, GM-CSF/anti-IL-10R no. 1; group 5, GM- GM-CSF, Abs, and mTg CSF/anti-IL-10R no. 2; and group 6, GM-CSF/anti-IL-10R no. 3) were by guest on September 29, 2021 included in this set of experiments, with four or five mice per group. Recombinant mouse GM-CSF was purchased from Cell Sciences. FITC- Groups 1–3 correspond with those mentioned above, except that animals in conjugated anti-CD11c and PE-conjugated anti-I-Ak (MHC class II), anti- group 3 received i.p. injections of rat IgG isotype control Ab (0.5 mg/ CD8a, anti-CD80, anti-CD86, and anti-CD40 (BD Pharmingen) and PE- mouse) on days 6, 11, 20, 25, and 32. Animals in groups 4–6 were treated conjugated anti-CD4, anti-CD8a, and anti-CD25 (Caltag Laboratories) Abs with GM-CSF and immunized with mTg as described above and received were used in flow cytometry. Neutralizing rat mAb to mouse IL-10R (clone i.p. injections of anti-IL-10R (0.5 mg/mouse) on days 6, 11, 20, 25, and 32; 1B1.3a) was provided by K. Moore (DNAX, Palo Alto, CA). Purified rat days 6 and 20; and days 11, 25, and 32, respectively. All animals were IgG was used for isotype control (Fitzgerald). T cells, CD4ϩCD25ϩ T killed on day 45, and lymph nodes, spleen, thyroids, and serum were col- cells, and DC magnetic bead isolation kits were obtained from Miltenyi lected to evaluate thyroiditis. Biotec. Normal mouse thyroids were obtained from BiochemMed, and Tg was prepared as described previously (24). Effects of GM-CSF treatment on thyroid microenvironment GM-CSF treatment and immunization with mTg CFA control, mTg control, and GM-CSF/mTg mice were used in this study. Three mice from each group were killed on day 21. Thyroids were Three groups of mice (group 1, CFA controls; group 2, mTg controls; and collected, pooled within groups, and digested with collagenase D (0.5 mg/ group 3, GM-CSF/mTg) were used throughout the study unless otherwise ml)for1hat37°C to prepare single cell suspensions. Cells were washed specified. Mice in groups 1 and 2 were injected with PBS, and mice in with PBS supplemented with 2% FBS and blocked with anti-CD16/CD32 group 3 were injected with 2 ␮g of GM-CSF/mouse/day from days 1–5 and Fc-Block (BD Pharmingen) on ice for 30 min. Cells were stained with from days 15–19. Mice in groups 2 and 3 were immunized s.c. with mTg FITC-conjugated anti-mouse CD4 along with PE-conjugated anti-mouse (100 ␮g/mouse) emulsified in CFA on days 6 and 20. Mice in group 1 CD25 mAbs on ice for 15 min, washed, and analyzed using a FACS an- (CFA control) received PBS emulsified in CFA on days 6 and 20. Mice alyzer (BD Biosciences) and CellQuest software. At least 10,000 cells/ from these three groups were killed at different time points for different sample were analyzed. To determine cytokine/chemokine production, thy- experiments, as described below, to study the various aspects of GM-CSF- roid cell suspensions were maintained in RPMI 1640 medium containing mediated immunomodulation. 2% normal mouse serum for 36 h. Cell-free supernatants were collected Effects of GM-CSF treatment on DC maturation from these cultures, and spontaneous cytokine (IL-4, IL-10, and IFN-␥) and chemokine (MCP-1 and RANTES) productions were detected by a The above-mentioned mouse groups 2 and 3 (i.e., mTg controls and GM- multiplex cytokine/chemokine assay kit using Luminex technology at the CSF/mTg, respectively) were used in this study. Animals were killed be- Luminex core facility of University of Pittsburgh Cancer Center. The sug- fore (days 6 and 20) and after (days 8 and 22) each mTg immunization. The gested lowest detection levels using this kit are 5 pg/ml for IL-4, 1 pg/ml immunization schedule was staggered in such a way that all animals were for IFN-␥, 15 pg/ml for IL-10, 5 pg/ml for MCP-1, and 5 pg/ml for killed at the same time. Spleen cells from these mice were stained with RANTES. FITC-conjugated anti-mouse CD11c in combination with PE-conjugated To assess apoptotic molecule expression on thyrocytes, thyrocytes were anti-mouse MHC class II, B7.1, B7.2, or CD40 Abs and analyzed in a separated from other resident cells, and mRNA was isolated using an FACS analyzer (BD Biosciences). DCs were isolated from splenocytes mRNA isolation kit, following the manufacturer’s instructions (Miltenyi using a CD11c isolation kit, and mRNA was extracted using an mRNA Bitoec). RT-PCR was conducted using mRNA and gene-specific primers isolation kit (Miltenyi Biotec). All procedures were conducted according to for Fas, Fas ligand (FasL), and caspase-8 (Maxim Biotech). ␤-Actin was the manufacturer’s instructions. The cDNAs were synthesized from mRNA used as a control to ensure equivalent amounts of RNA in the assay. 7008 IL-10-PRODUCING CD4ϩCD25ϩ CELLS IN PREVENTING EAT mTg-specific T cell proliferation Statistical analysis Mouse splenocytes or lymph node cells (5 ϫ 105 cells/well) were plated in Mean, SD, and statistical significance were calculated using an SPSS ap- 96-well, flat-bottom tissue culture plates in triplicate in RPMI 1640 con- plication. Statistical significance was determined using the nonparametric taining 2% normal mouse serum at a final volume of 0.25 ml/well. The Wilcoxon signed test. In most cases, values of individual treated and im- mTg was added at a concentration of 20 ␮g/ml. Con A (1 ␮g/ml) was used munized groups were compared with those of untreated but immunized Յ as a positive control. Cells were incubated for 72 h at 37°C in a CO2 groups. A value of p 0.05 was considered significant. incubator. Cells were pulsed with 1 ␮Ci of [3H]thymidine/well for the last 16 h of culture, transferred into 96-well, U-bottom tissue culture plates, washed twice with PBS, lysed in water, and dried overnight at 37°C. Scin- tillation fluid was added to these wells (50 ␮l/well) and counted using a Results 96-well plate (Microbeta counter; PerkinElmer Wallac). To evaluate and to GM-CSF-induced DCs maintain semimature phenotype test the T cell pattern of proliferation, cells were stained with CFSE as described above, plated in 96-well, flat-bottom tissue culture plates in the To determine the effects of GM-CSF treatment on the maturation presence or the absence of mTg (20 ␮g/ml) in RPMI 1640 containing 2% of DCs, we analyzed the expression of MHC class II and costimu- normal mouse serum at a final volume of 0.25 ml/well, maintained for 7 latory molecules as well as the production of proinflammatory cy- days, harvested, and tested for CFSE dilution using FACS (BD tokines from DCs isolated from GM-CSF-treated and untreated Biosciences). mice before and after mTg immunization. Spleens from mice ϩ Measurement of cytokine production treated with GM-CSF showed increased numbers of CD11c cells (7.51%) compared with untreated controls (3.61%; Fig. 1A). De- Spleen or lymph node cells (5 ϫ 106 cells/well; 12-well plate) were incu- bated in the presence or the absence of mTg (20 ␮g/ml) in 1.5 ml of RPMI spite an increase in the number of DCs, expression levels of MHC 1640 medium supplemented with 2% normal mouse serum for 36 h. Cell- class II, B7.1, B7.2, and CD40 were comparable in GM-CSF- Downloaded from free culture supernatants were collected after 36 h by centrifugation. Cy- treated and untreated mice after immunization with mTg (Fig. 1B). tokine levels in cell-free supernatants were assayed by ELISA, using paired However, levels of proinflammatory cytokines, such as TNF-␣, ␥ Abs for detection of IL-2, IL-10, IL-4, and IFN- , following the manu- IL-12, and IL-1␤, evaluated by RT-PCR, were significantly higher facturer’s instructions (eBioscience), and the OD450 was recorded using a Microplate reader (Bio-Rad). The amount of cytokine was determined us- in DCs from untreated, mTg-immunized mice than in DCs from ing corresponding cytokine standards. The suggested lowest detection lev- GM-CSF-treated, mTg-immunized mice (Fig. 1C). These data sug- ␥ els using this kit are 2 pg/ml for IL-2, 4 pg/ml for IL-4, 15 pg/ml for IFN- , gest that DCs from GM-CSF-treated, but not untreated, mice main- http://www.jimmunol.org/ and 15 pg/ml for IL-10. tain a semimature status after mTg immunization. Evaluation of EAT Thyroids collected from mice at the time of death were fixed in formalin, CD4ϩCD25ϩ T cells from GM-CSF-treated mice suppress embedded in paraffin, sectioned, and stained with H&E. Thyroids were anti-mTg response in vivo scored for the extent of thyroid lymphocytic infiltration, as a marker of disease severity, using a scale of 1ϩ to 5ϩ. An infiltrate of at least 125 To determine whether CD4ϩCD25ϩ T cells from GM-CSF-treated cells in one or several foci was scored 1ϩ, 10–20 foci of cellular infiltra- mice can suppress mTg-specific autoimmune responses in vivo, tion involving up to 25% of the gland was scored 2ϩ, an infiltration in- ϩ ϩ purified CD4 CD25 T cells from GM-CSF-treated and mTg- volving up to 25–50% of the gland was scored 3ϩ, destruction of Ͼ50% by guest on September 29, 2021 of the gland was scored 4ϩ, and near-complete destruction of the gland, primed mice were adoptively transferred to untreated mice that with few or no remaining follicles, was scored 5ϩ. were primed with mTg. As shown in Fig. 2A, mice receiving

FIGURE 1. Effects of GM-CSF treatment on DC maturation. CBA/J mice were left untreated or were treated with GM-CSF for 5 consecutive days starting on days 1 and 15 as described in Materials and Methods.In addition, mice were immunized with mTg emulsified in CFA on days 6 and 20. Mice were killed before (days 6 and 20) and after (days 8 and 22) first and second mTg immunizations to obtain spleens. A, Splenocytes iso- lated from mice killed on day 8 were stained with FITC- anti-CD11c and with either PE-anti-MHC class II or PE- anti-CD8a and analyzed by FACS. B, The same cells were stained with FITC-anti-CD11c and with PE-anti- B7.1, PE-anti-B7.2, or PE-anti-CD40 and analyzed by FACS. Black lines indicate the isotype control, dark gray lines indicate the mTg control, and light gray lines indicate GM-CSF-treated mice. C, DCs were isolated from spleens before or after mTg immunization, using magnetic column separation. mRNA was isolated and used in a multiplex RT-PCR assay to detect cytokine transcripts. The results shown are representative of two independent experiments using two mice per group. The Journal of Immunology 7009

suggested that IL-10 produced by CD4ϩCD25ϩ T cells is required to suppress mTg-specific proliferation.

Treatment with anti-IL-10R mAb abolishes GM-CSF-induced suppression of EAT Next, we investigated the role of IL-10 in GM-CSF-induced sup- pression of EAT. The effects of IL-10 were blocked by the admin- istration of saturating concentrations of anti-IL-10R mAb to GM- CSF-treated mice at various times during disease induction. Regardless of the time of administration, almost all animals that received anti-IL-10R mAb, with the exception of some mice treated with anti-IL-10R mAb immediately after GM-CSF treat- ment (i.e., GM-CSF/anti-IL-10R no. 2), showed increased mTg- specific proliferation compared with mice that received GM-CSF and isotype control mAb. A significant increase in proliferation was seen in mice that received anti-IL-10R mAb 5 days after GM- CSF treatment (i.e., GM-CSF/anti-IL-10R no. 3) or throughout the

course of the disease (i.e., GM-CSF/anti-IL-10R no. 1; p ϭ 0.001 Downloaded from and p ϭ 0.005, respectively; Fig. 4A). Interestingly, we observed an increase in the frequency of CD4ϩCD25ϩ T cells in all GM- CSF-treated mice regardless of the time of administration of anti- FIGURE 2. Adoptive transfer of CD4ϩCD25ϩ T cells from GM-CSF- IL-10R mAb (Fig. 4B), suggesting that blocking IL-10 had no treated mice. CD4ϩCD25ϩ T cells were purified from mice treated with effect on the expansion of these cells by GM-CSF-induced DCs.

GM-CSF and immunized with mTg as described in Materials and Meth- As shown in Fig. 4C and Table I, thyroids from anti-IL-10R http://www.jimmunol.org/ ϩ ϩ ods. Purified CD4 CD25 T cells were adoptively transferred into mTg- mAb groups of mice, with the exception of some mice treated with 6 immunized mice (1 ϫ 10 cells/mouse), and mice were killed on day 18 anti-IL-10R mAb immediately after GM-CSF treatment (i.e., GM- after transfer to assess the immune response to mTg compared with con- CSF/anti-IL-10R no. 2), showed more severe lymphocytic infil- trols. A, Splenocytes were cultured in the presence or the absence of mTg. tration compared with thyroids from GM-CSF/isotype control The proliferative response to mTg was measured by 3H incorporation as- mice. Taken together, these results indicated that IL-10 is the pri- say. Values for ⌬cpm (mTg-stimulated cpm Ϫ (nonstimulated) background cpm) are plotted. Background cpm were Ͻ200 in this assay. Spent medium mary mediator of GM-CSF-induced disease suppression. collected at 36 h from the above cultures was tested for IFN-␥ (B), IL-4 (C), and IL-10 (D) by ELISA. Results are expressed as the mean Ϯ SD of

Effect of GM-CSF treatment on thyroid microenvironment by guest on September 29, 2021 ,ء .values obtained from triplicate determinations in three individual mice Statistically significant difference vs mTg control mice. To test the effects of GM-CSF on the target organ, we investigated the cell type and cytokine production in the thyroids of treated mice. GM-CSF treatment resulted in the expansion of CD8aϪ DCs in the periphery (Fig. 1A); however, this expansion was not re- ϩ ϩ CD4 CD25 T cells from GM-CSF-treated mice showed signif- flected within the thyroid (data not shown). In contrast, there was icantly lower mTg-specific proliferation compared with mTg con- an increase in the percentage of CD4ϩCD25ϩ T cells in the thy- trol mice ( p ϭ 0.021). Analysis of mTg-induced cytokine produc- roids of GM-CSF-treated mice relative to untreated mice (24.57 tion by spleen cells from different groups of mice showed similar and 20.06%, respectively; Fig. 5A). Previous studies had shown ϩ ϩ levels of IFN-␥ in both CD4 CD25 T cell recipient and nonre- that MCP-1 preferentially attracts CD4ϩCD25ϩ T cells to the thy- cipient mTg control mice (Fig. 2B). In contrast, cells from recip- roid, whereas RANTES preferentially attracts CD4ϩ effector T ient mice produced significantly higher amounts of IL-4 ( p ϭ cells (34). Therefore, we tested for the levels of these two chemo- 0.045) and IL-10 ( p ϭ 0.035) than nonrecipient, mTg-primed mice kines. MCP-1 production was comparable among all experimental (Fig. 2, C and D, respectively). groups, whereas RANTES was undetectable (data not shown),

ϩ ϩ suggesting that these chemokines could not account for the ob- IL-10 produced by CD4 CD25 T cells is important for served increase in CD4ϩCD25ϩ T cell frequency in GM-CSF- suppressing mTg-specific T cell response treated thyroids. Next, to determine whether IL-10 produced by GM-CSF-induced Next, we quantified cytokine production by thyrocytes and thy- CD4ϩCD25ϩ T cells was responsible for suppressing mTg-spe- roid-resident lymphocytes. Although a slight increase in IL-10 pro- cific T cell responses, T cells from untreated, mTg-primed mice duction with a very small decrease in IFN-␥ production were ob- were cocultured with CD4ϩCD25ϩ T cells from mTg-primed and served in GM-CSF-treated mice compared with mTg control mice GM-CSF-treated mice in the presence of anti-IL-10R mAbs or (Fig. 5B), these differences were not significant. isotype control. T cell-depleted spleen cells (Fig. 3A) or isolated Several studies have suggested that thyrocyte destruction in HT DCs (Fig. 3B) from naive mice were used as feeder cells. As is due to Fas-mediated apoptosis through increased caspase ex- shown in Fig. 3, mTg-primed T cells cultured with CD4ϩCD25ϩ pression. Therefore, we tested for the expression levels of Fas, T cells from mTg-primed and GM-CSF-treated mice in the pres- FasL, and caspase 8 on thyrocytes by RT-PCR. Although we ob- ence of isotype control Ab showed reduced mTg-specific T cell served a slight increase in Fas expression in GM-CSF-treated mice proliferation relative to controls, as indicated by reduced CFSE compared with CFA and mTg control mice, there was no detect- dilution (0.72 and 8.18% vs 2.42 and 11.46%, respectively). How- able FasL expression in any of the groups. Furthermore, there was ever, the response was restored to the control levels or higher in the no substantial difference in the expression levels of caspase 8 presence of anti-IL-10R mAb (i.e., 2.15 and 16.62%). These data among the groups of mice (Fig. 5C). 7010 IL-10-PRODUCING CD4ϩCD25ϩ CELLS IN PREVENTING EAT Downloaded from

FIGURE 3. Role of IL-10 in CD4ϩCD25ϩ T cell-induced suppression of mTg-specific T cell proliferation. CBA/J mice were treated with or without GM-CSF for 5 consecutive days starting on days 1 and 15 and were immunized with mTg emulsified in CFA on days 6 and 20 as described in Materials and Methods. Mice were killed on day 35 to obtain lymph node and spleen cells. CD4ϩCD25ϩ T cells from GM-CSF-treated mice and T cells (effector cells) from untreated mice were purified from pooled spleen and lymph node cells using the magnetic separation method. Effector T cells were stained with http://www.jimmunol.org/ CFSE, cocultured with isolated CD4ϩCD25ϩ T cells (5:1 effector:Treg ratio), and stimulated with mTg in the presence of saturating concentrations of anti-IL-10R or isotype control mAb. Either T cell-depleted spleen cells (A) or enriched DCs (B) from naive mice were used as APCs. The proliferative response to mTg was assessed by CFSE dilution, as determined by FACS on day 7. Histograms shown are gated on the CD4ϩ T cell population. Results Statistically significant difference compared with the isotype control ,ء .shown are representative of two independent experiments conducted in triplicate mice shown in the middle panel.

Discussion ability to induce generation of IL-10-producing type 1 Treg (Tr1) In this study we investigated mechanisms by which GM-CSF treat- that do not express significant levels of CD25 unless they are ac- by guest on September 29, 2021 ment can cause suppression of EAT. Our results showed that GM- tivated (38, 39). However, other studies have clearly shown that CSF can expand DCs and maintain them in a semimatured status immature and other tolerogenic DCs can help expand IL-10-pro- in vivo, promote expansion of CD4ϩCD25ϩ T cells, and induce ducing CD4ϩCD25ϩ Treg (36, 37, 40), which may play an im- higher levels of IL-10 production required for EAT suppression. portant role in the induction and differentiation of Tr1 These results further extend our earlier studies (24) in which we cells (28, 41, 42). Ϫ showed that GM-CSF treatment can expand CD8a DCs and Although several types of Treg have been described, each with ϩ ϩ CD4 CD25 Treg and suppress EAT. a specific surface phenotype and a cytokine profile, naturally oc- Although DC function is traditionally associated with the induc- curring CD4ϩCD25ϩ Treg, which constitute 5–10% of peripheral tion of primary T cell responses, there is increasing evidence that CD4ϩ T cells, are the predominant suppressors of autoreactive T they play a critical role in peripheral tolerance (10–14). DCs pass cells that escape central tolerance (43–45). Previously we (24) through several stages of maturation (10), and earlier studies have demonstrated that CD4ϩCD25ϩ T cells from GM-CSF-treated shown that semimatured DCs play a critical role in the induction mice could suppress the mTg-specific proliferative response of ef- and expansion of Treg (10, 18–23). Because GM-CSF treatment fector T cells in vitro. However, CD4ϩCD25ϩ T cells from un- led to an increase in the frequency of CD4ϩCD25ϩ T cells with treated, but mTg-primed, mice failed to show similar suppression regulatory properties (24), we asked whether GM-CSF exerted its of mTg-specific responses. More interestingly, depletion of effects by affecting DC maturation. We found that DCs from GM- ϩ ϩ CD4 CD25 T cells from in vitro cultures of lymphocytes from CSF-treated mice displayed a semimature phenotype, as indicated GM-CSF-treated mice restored mTg-specific proliferation (24). by high levels of expression of MHC class II and B7 molecules, This showed that effector T cells were generated in GM-CSF- but low levels of expression of proinflammatory cytokines com- treated mice as they were in untreated, mTg-primed mice, but their pared with untreated mTg control mice. This suggested that GM- ϩ ϩ CSF treatment most likely induced and/or promoted tolerance function was suppressed by CD4 CD25 T cells that were in- duced/expanded in GM-CSF-treated mice. In this study, adoptive through the expansion of semimature DCs, which are known to aid ϩ ϩ in the generation of Treg (10, 18–23). transfer of CD4 CD25 T cells from GM-CSF-treated mice into In fact, an earlier study showed that DCs generated by culturing mTg-primed mice resulted in a significant suppression of mTg- bone marrow precursor cells in low concentrations of GM-CSF are specific proliferation compared with mTg-primed nonrecipients. maturation resistant, and inoculation of these DCs pulsed with al- Although we cannot rule out the possibility that the transferred ϩ ϩ lopeptides could prolong allograft survival in vivo (35). Genera- CD4 CD25 T cell population contained some activated effector tion of tolerogenic DCs capable of preventing autoimmune dis- T cells, the suppressive property observed suggested that the pop- eases and allotransplant rejections have been reported extensively ulation was primarily composed of CD4ϩCD25ϩ Treg. Further- (10–14, 36, 37). One of the major properties of such DCs is their more, lymphocytes from recipient mice, upon in vitro stimulation The Journal of Immunology 7011

Table 1. Effect of anti-IL-10R Ab on GM-CSF-induced EAT suppressiona

01ϩ 2ϩ 3ϩ 4ϩ p Value

Group CFA control 4 0000 mTg control 0 1310 GM-CSF/isotype control 0 5000 GM-CSF/anti-IL-10R no. 1 0 04100.002 GM-CSF/anti-IL-10R no. 2 0 22000.09 GM-CSF/anti-IL-10R no. 3 0 03110.008

a Mice were treated with GM-CSF, immunized with mTg, and further treated with anti-IL-10R mAb or isotype control Ab as described in Materials and Methods. These mice were sacrificed along with control mice on day 45, thyroids were collected from mice, at the time of sacrifice, were fixed in formalin, embedded in paraffin, and sectioned for histological H&E staining. Thyroiditis cellular infiltration index was determined as described in Materials and Methods. Values of p were calculated by comparing anti-IL-10R mAb-treated mice with isotype control mice.

with mTg, produced higher levels of IL-10 and IL-4 than mTg- Downloaded from primed controls. This indicated that adoptively transferred CD4ϩCD25ϩ T cells exerted suppressive effects on recipient ef- fector T cells, as seen in GM-CSF-treated donor mice. To explore the mechanism of suppression of mTg-specific re- sponses by GM-CSF-induced CD4ϩCD25ϩ T cells, we conducted

additional studies. Because in an earlier study we had ruled out a http://www.jimmunol.org/ critical role for IL-4 in EAT suppression (24), and IL-10 is a crit- ical mediator of Treg-induced suppression of effector T cell func- tion (28–33), we tested the role of IL-10 in both the expansion and the function of CD4ϩCD25ϩ T cells in GM-CSF-treated mice. Blockade of IL-10 function in vivo using anti-IL-10R Ab reversed the suppressive effects of CD4ϩCD25ϩ T cells from GM-CSF- treated mice on mTg-specific T cell responses in vitro and sug- gested a critical role for this cytokine in GM-CSF-induced sup- pression of EAT. Furthermore, we showed that blockade of IL-10 by guest on September 29, 2021 function in vivo completely abolished the disease-suppressive ef- fects of GM-CSF and allowed development of EAT. Initiation of treatment with anti-IL-10R Ab at different time points during dis- ease development allowed us to address two major questions; namely, whether IL-10 is required for the induction and/or expan- sion of CD4ϩCD25ϩ T cells in vivo, and whether it is required for merely suppressing autoreactive effector T cell function, resulting in consequent suppression of EAT. Our results showed that re- gardless of the time of treatment, blocking IL-10 abolished the EAT-suppressive capacity in a majority of mice. Interestingly, the number of CD4ϩCD25ϩ T cells was higher in all GM-CSF-treated mice, compared with untreated mice regardless of anti-IL-10R Ab treatment. Consistent with previous reports (46, 47), our results showed that IL-10 is not essential for the expansion of CD4ϩCD25ϩ T cells. However, IL-10 produced by these Treg is critical for the suppression of effector T cells. IL-10 is a key regulator of inflammation, and it can inhibit both Th1- and Th2-type immune responses through the suppression of FIGURE 4. In vivo effects of anti-IL-10R Ab on GM-CSF-induced sup- proinflammatory cytokines and T cell proliferative responses (48). pression of EAT. GM-CSF-treated mice were treated with anti-IL-10R One of the major mechanisms of IL-10-mediated suppression of T mAb as described in Materials and Methods and killed on day 45 along cells is through selective inhibition of the CD28 costimulatory with control mice to obtain lymph nodes and spleen cells. A, Splenocytes pathway (46). However, in thyroiditis, alternative mechanisms of were stained with CFSE and stimulated with mTg for 7 days. The prolif- action of IL-10 have been proposed (7, 9, 49–52). Injection of erative response to mTg was assessed by CFSE dilution, as determined by cDNA expression vectors encoding IL-10 into the thyroid can sig- FACS. Histograms shown are gated on CD4ϩ T cell population. B, Spleno- nificantly inhibit lymphocyte infiltration and development of EAT cytes were stained with FITC-labeled anti-mouse CD4 and PE-labeled anti- mouse CD25 Abs and analyzed using FACS. Ranges in parentheses shown and prevent progression of the disease (50). This suppressive effect in A and B correspond to values for four or five individual mice. C, Rep- of IL-10 is mediated either through enhancement of FasL expres- resentative photomicrographs of H&E-stained thyroid sections for different sion on thyrocytes and induction of activation-induced cell death groups (described in Table I) are shown. (original, ϫ40). Numbers shown of thyroid-infiltrating T lymphocytes (51) or through potent up- in parentheses depict corresponding thyroiditis cellular infiltration index. regulation of antiapoptotic molecules, such as cellular FLIP and 7012 IL-10-PRODUCING CD4ϩCD25ϩ CELLS IN PREVENTING EAT

primarily due to the direct effects of IL-10 on mTg-specific effector T cells. Studies using SCID and TCR transgenic mice are under- way, and they should help elucidate the direct effects of GM-CSF on DCs and/or T cells. In summary, it is likely that GM-CSF induced the expansion of semimatured DCs, and Tg peptide presentation by these DCs led to the expansion of CD4ϩCD25ϩ Treg. IL-10 produced by these Treg inhibited the autoimmune effector functions of mTg-specific T cells with consequent suppression of EAT. These results show the therapeutic potential of GM-CSF in EAT and other autoim- mune diseases with pathogenesis similar to that of EAT. Disclosures The authors have no financial conflict of interest. References 1. Pujol-Borrell, R., I. Todd, M. Londei, A. Foulis, M. Feldmann, and G. F. Bottazzo. 1986. Inappropriate major histocompatibility complex class II expression by thyroid follicular cells in thyroid autoimmune disease and by pan-

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dritic cells induced by vitamin D receptor ligands enhance regulatory T cells Pharmacol. Sin. 24: 885–890. by guest on September 29, 2021