Anti-CD25 Antibody-Mediated Depletion of Effector T Cell Populations Enhances Susceptibility of Mice to Acute but Not Chronic Infection This information is current as of October 1, 2021. Kevin N. Couper, Paula A. Lanthier, Georgia Perona-Wright, Lawrence W. Kummer, Wangxue Chen, Stephen T. Smiley, Markus Mohrs and Lawrence L. Johnson J Immunol 2009; 182:3985-3994; ;

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Anti-CD25 Antibody-Mediated Depletion of Effector T Cell Populations Enhances Susceptibility of Mice to Acute but Not Chronic Toxoplasma gondii Infection1

Kevin N. Couper,2* Paula A. Lanthier,* Georgia Perona-Wright,* Lawrence W. Kummer,* Wangxue Chen,† Stephen T. Smiley,* Markus Mohrs,* and Lawrence L. Johnson*

Natural regulatory T cells (Tregs) constitutively express the IL-2R ␣-chain (CD25) on their surface. Consequently, administration of anti-CD25 Abs is a commonly used technique to deplete Treg populations in vivo. However, activated effector T cells may also transiently express CD25, and are thus also potential targets for anti-CD25 Abs. In this study using Toxoplasma gondii as a model proinflammatory infection, we have examined the capacity of anti-CD25 Abs to target effector T cell populations during an inflammatory episode, to determine to what extent that this action may modulate the outcome of disease. Anti-CD25 Ab-treated C57BL/6 mice displayed sig- nificantly reduced CD4؉ T cell IFN-␥ production during acute T. gondii infection and exhibited reduced weight loss and liver pathology Downloaded from during early acute infection; aspects of infection previously associated with effector CD4؉ T cell responses. In agreement, anti-CD25 Ab administration impaired parasite control and caused mice to succumb to infection during late acute/early chronic stages of infection with elevated tissue parasite burdens. In contrast, anti-CD25 Ab treatment of mice with established chronic infections did not markedly affect brain parasite burdens, suggesting that protective T cell populations do not express CD25 during chronic stages of T. gondii infection. In summary, we have demonstrated that anti-CD25 Abs may directly abrogate effector T cell responses during an inflammatory episode, highlighting important limitations of the use of anti-CD25 Ab administration to examine Treg function during inflammatory http://www.jimmunol.org/ settings. The Journal of Immunology, 2009, 182: 3985–3994.

atural regulatory T cells (Tregs)3 constitutively express have obtained results consistent with the depletion of regulatory high levels of the IL-2R ␣-chain (CD25) and until re- cell populations, as evidenced by augmented protective immune cently this marker was the most commonly used to iden- responses and enhanced control (for example see Refs. N ϩ tify these cells. Natural Tregs, which comprise 5–10% of CD4 T 5–8), whereas other studies have observed no difference in immu- cells in naive mice, have potent regulatory capacity and have been nity following anti-CD25 administration, possibly due to the dis-

shown to have important roles in a number of autoimmune and ease model used or the strain and immune status of mice in the by guest on October 1, 2021 infectious diseases (reviewed in Refs. 1, 2). Importantly, however, study (for example see Refs. 9–13). In addition, anti-CD25 Abs CD25 is not a specific marker of natural Tregs and it may be are also routinely used in vitro for the positive selection of natural expressed by a number of other cell populations, including acti- Tregs from naive and infected mice for the subsequent use in adop- vated T and B cells, monocytes, and some dendritic cells (re- tive transfer systems or in vitro suppression assays. In general, viewed in Ref. 3). Moreover, the identification of the transcription these experiments have almost entirely focused on the effects of factor Foxp3 as a lineage specific marker for natural Tregs has anti-CD25 Abs on CD4ϩ natural Tregs, even though many cells enabled more accurate differentiation of regulatory and effector other than natural Tregs, including effector T cells, could poten- CD4ϩ T cell populations, and has shown that populations of Ϫ ϩ tially be targeted by anti-CD25 treatment (3). Thus, anti-CD25 CD25 Foxp3 Tregs also exist (4). treatment in vivo may have effects beyond simply depleting CD25- Nevertheless, administration of anti-CD25 Abs remains a com- expressing natural Tregs or affecting their regulatory capability, monly used method to deplete natural Tregs in vivo. Some studies potentially leading to inaccurate conclusions on the role of natural Tregs in any particular disease setting. *Trudeau Institute, Saranac Lake, NY 12983; and †Institute for Biological Sciences, In this study, we use oral Toxoplasma gondii infection as a National Research Council of Canada, Ottawa, Ontario, Canada model of inflammatory disease to directly investigate the effect of Received for publication September 16, 2008. Accepted for publication January anti-CD25 Ab treatment on the effector arm of the immune re- 18, 2009. sponse. The T. gondii infection model has several useful features The costs of publication of this article were defrayed in part by the payment of page for this purpose. During acute infection, which lasts 1–2 wk, strong charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. proinflammatory innate and adaptive responses develop as rapidly 1 This work was supported by a Public Health Service Grant AI061587 from the proliferating single cell tachyzoites develop from dormant en- National Institute of Allergy and Infectious Diseases and by funds provided by the cysted parasites and disseminate from the intestine to liver, lung, Trudeau Institute. brain, and other sites. Cooperation among neutrophils, NK cells, 2 Address correspondence and reprint requests to Dr. Kevin N. Couper at the current address: Immunology Unit, Department of Infectious and Tropical Diseases, London and macrophages is required for the production of IL-12 and School of Hygiene and Tropical , Keppel Street, London WC1E 7HT, U.K. IFN-␥, which then play critical protective roles during both acute E-mail address: [email protected] and chronic infections (14–19). However, in susceptible mouse 3 Abbreviations used in this paper: Treg, regulatory T cell; yFP, yellow fluorescent strains such as C57BL/6 (B6), type I cytokines cause immunopa- protein. thology in small intestine and liver that is not evident in resistant ϩ Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 mouse strains (e.g., BALB/c) (20). Notably, CD4 cells, although www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803053 3986 EFFECT OF ANTI-CD25 ON T. gondii INFECTION not required for control of parasites in the first week or so of acute to note that fluorochrome-labeled 7D4 was used for flow cytometric de- infection, are nonetheless important mediators of the immunopa- tection of CD25 expression in all experiments in which PC61 was admin- thology seen in C57BL/6 mice (20). istered in vivo to deplete CD25-expressing cells; PC61 does not inhibit 7D4 detection of cell surface CD25 (27). Following the development of sufficient adaptive T cell-me- ϩ Ϫ ϩ diated immune responses, the acute stage of infection is con- Isolation of CD4 CD25 and CD4 CD25high T cells trolled, and tachyzoites are cleared in the intestine, liver, and CD4ϩ T cells were purified from the spleens and brains of C57BL/6 mice lungs and parasites revert to a semidormant state within cysts in using anti-CD4 MidiMacs beads according to the manufacturer’s instruc- ϩ the brain. In this chronic infection phase, acquired immune tions (Miltenyi Biotec). Following separation, CD4 cells were stained ϩ ϩ with anti-CD4 (GK1.5) and anti-CD25 (PC61) and sorted into CD4ϩ mechanisms dependent on CD4 and CD8 T cells and B cells, Ϫ ϩ high ␥ ␣ CD25 and CD4 CD25 populations using a FACSVantage cell sorter. along with IFN- and TNF- are required to control tachyzoite The sorted cell populations were routinely Ͼ99% pure. dissemination and to prevent the development of toxoplasmic encephalitis (21–24). Thus, activated effector CD4ϩ T cells me- In vitro restimulation experiments diate damaging immunopathology early during acute infection, CD4ϩCD25high and CD4ϩCD25Ϫ cells, obtained as described, were cul- yet contribute to parasite control during late acute/chronic tured individually or at a ratio of 1:1. Cells were either stimulated with stages of infection and prevent the development of encephalitis naive APCs and anti-CD3 Ab (1 ␮g/ml) or with T. gondii-pulsed APC (without anti-CD3 Ab costimulation). APCs were prepared by gamma during chronic infection. irradiation (3000 rad) of spleen cells obtained from naive B6 mice. Our results support the conclusion that anti-CD25 Ab adminis- Separately, T. gondii primed APC were prepared to measure parasite- ϩ tration directly targets CD25 expressing effector CD4 T cells specific T cell responses. RH strain tachyzoites, maintained through during acute T. gondii infection. We show that anti-CD25 Ab ad- continuous in vitro culture in fibroblast monolayers, were irradiated at ministration, while depleting ϳ40% of Foxp3ϩ natural Tregs, also 15,000 rad and added to APC generated from naive mice as indicated. Downloaded from ϩ Irradiated tachyzoites and APC were cultured for 12 h at 5% CO2 and significantly reduces effector CD4 T cell numbers and diminishes 37°C before being washed and added at required cell concentration to wells ␥ ϩ IFN- production during acute T. gondii infection, ameliorating including purified CD4 cells. The cells were then incubated at 5% CO2 early immune-mediated pathology but significantly impairing dis- and 37°C for 60 h. Supernatants were then harvested to assay cytokine production and proliferation was determined by pulsing cells with 0.2 ␮Cu/ ease control and elevating mortality during late acute/early chronic 3 3 T. gondii well [ H]thymidine (Amersham Biosciences) for an extra 12 h. [ H]Thy- stage infection. Thus, our results strongly imply that midine incorporation and proliferation was measured using a beta counter. anti-CD25 Abs should be used with caution in vivo during highly http://www.jimmunol.org/ proinflammatory disease models, and that the potential effects on ELISA and real-time PCR all cells that can express CD25 should be considered when they are Serum IFN-␥ levels were measured by ELISA as previously described used to examine the function and importance of natural Tregs. (18). Tachyzoites were quantified by a real-time PCR-based method (28) based on the expression of the tachyzoite-limited Sag1 gene (29). RNA was Materials and Methods isolated from tissues, cDNA prepared, amplified, and quantitated using the following primer to probe sets: T. gondii Sag1 (forward) TTTCCGAAGG Mice CAGTGAGACG, (reverse) GGATCCGATGCCATAGCG, probe TTGC ␥ C57BL/6J, Thy1.1 C57BL/6 (B6), and bicistronic IFN-␥ reporter (Yeti) CGCGCCCACACTGATG; IFN- (forward) CATTGAAAGCCTAGAA AGTCTGAATAAC, (reverse) TGGCTCTGCAGGATTTTCATG, probe male mice (25) were used between 6 and 12 wk of age. Mice were bred in by guest on October 1, 2021 the Trudeau Institute Animal Breeding Facility or obtained from The Jack- TCACCATCCTTTTGCCAGTTCCTCCAG; IL-10 (forward) GAAGAC son Laboratory or Taconic Farms. All experiments were reviewed and CCTCAGGATGCGG, (reverse) ACCTGCTCCACTGCCTTGCT, probe approved by the Trudeau Institute Institutional Animal Care and Use Com- TGAGGCGCTGTCATCGATTTCTCCC; Foxp3 (forward) CCCAGGA mittee. The Trudeau Institute is fully accredited by the Association for the AAGACAGCAACCTT, (reverse) TTCTCACAACCAGGCCACTTG, Assessment and Accreditation of Laboratory Animal Care. probe ATCCTACCCACTGCTGGCAAATGGAGTC; and GAPDH (for- ward) CTCGTCCCGTAGACAAAATGG, (reverse) AATCTCCACTTT Parasites and infections GCCACTGCA, probe CGGATTTGGCCGTATTGGGCG. Values were normalized to GAPDH expression. IFN-␥ message levels in brain are

ME49 cysts were obtained from brains of chronically infected B6 mice. shown as fold change (log10) relative to control mAb-treated infected Infections were initiated by peroral administration of 10 cysts in 0.1 ml mice. Tachyzoite numbers were calculated relative to a standard curve of diluted brain suspension as previously described (26). Sham-infected generated by inoculating known numbers of tachyzoites into uninfected mice received similarly diluted brain suspension containing no cysts. liver samples. Weight loss was monitored as a sign of morbidity and is expressed as the percentage of baseline starting weight. Mice were monitored daily Histological exam for survival. On day 8 postinfection, livers and ileums from anti-CD25 Ab-treated Ab treatments mice and control Ab-treated mice were removed and processed for H&E staining. Liver and ileal lesions were semiquantitatively scored based Mice were given i.p. injections of 1 mg of anti-CD25 (clone PC61) or a on our previous scoring systems (28, 30) as follows: liver damage 1, control rat IgG (clone HRPN) obtained from BioXcell (previously Bio- degeneration and necrosis of individual hepatocytes occasionally seen; Express). Abs were administered 5–7 days before infection or during in- 2, clusters or small aggregates of hepatocyte degeneration and necrosis; fection on days stated in the experiment. PC61 administration promoted 3, medium to large aggregates of hepatocyte necrosis; 4, large areas of long-lasting depletion/neutralization of CD25 expression (longer than 2 hepatocyte necrosis with loss of normal liver anatomic architecture; wk) in naive mice, consistent with previously published reports (27). En- intensity of the inflammatory response: 1, small inflammatory infiltrates dotoxin levels of clones PC61 and HRPN were below 1 EU/mg. with a few inflammatory cells; 2, medium-sized inflammatory infiltrates with a small to moderate number of inflammatory cells; 3, large in- Flow cytometry flammatory infiltrates with a moderate to large number of inflammatory Single cell suspensions were prepared from spleens, liver, blood, and mes- cells; 4, extensive infiltration with a large number of inflammatory cells. enteric lymph nodes. Brains were gently mashed using a Teflon pestle, cell The ileal lesions were scored for the degeneration and necrosis of vil- populations separated on a Percoll gradient, and lymphocytes recovered for lous epithelial cells, transmural inflammation, and lamina propria and analysis. Cells were counted using a hemacytometer. The surface Ag phe- crypt inflammation and an integer score between 0 (unremarkable) and notype was determined by flow cytometry using the following fluoro- 4 (severe) was assigned. chrome-conjugated Abs: anti-CD4 (clone GK1.5), anti-CD25 (clone PC61 Statistics or clone 7D4), or anti-CD44 (clone IM7). Cells were preincubated with Fc block (clone 2.4G2) before staining. All Abs were obtained from BD Bio- Unless otherwise noted, group means were compared by Student’s t test. sciences. Foxp3 staining was performed using anti-Foxp3 (FJK-16s) Ab Intergroup survivals were compared by log-rank test. All statistical anal- according to the manufacturer’s instructions (eBioscience). It is important yses were performed using GraphPad Prism software. The Journal of Immunology 3987 Downloaded from http://www.jimmunol.org/

FIGURE 1. Anti-CD25 Ab administration significantly modulates the outcome of acute T. gondii infection. Groups of five B6 males were given 1 mg of control mAb (clone HRPN) or anti-CD25 (clone PC61) 5 or 7 days before oral infection with 10 ME49 cysts. A, Changes in body weight were monitored by guest on October 1, 2021 p Ͻ 0.05 for significant difference. B, Compiled survival ,ء .and are presented as a percentage change relative to starting weight. Data show mean Ϯ SD data from three experiments comparing anti-CD25-treated with control T. gondii-infected B6 male mice. Survivals differed significantly (p Ͻ 0.01) by log-rank test. C, Brain cysts of three anti-CD25-treated B6 male mice, and five controls 28 days after mice were sham-infected or infected with 10 ME49 cysts. Horizontal bar denotes median values. D, On day 8 postinfection, livers and ileums were removed and stained with H&E for histological examination of tissue pathology with arrows marking areas of inflammation or necrosis (also marked by an asterisk). E and F, On day 8 postinfection, livers (E) and ileums (F) were removed for real-time PCR (Taqman) quantification of tissue parasite burdens. Results shown are mean Ϯ SD of the group (n ϭ 5) and .p Ͻ 0.01 ,ءء .are representative of two independent experiments

Results Anti-CD25 Abs modulate development of immune-mediated Anti-CD25 Ab increases susceptibility of mice to late acute pathology and significantly impair parasite control during acute T. gondii infection T. gondii infection The administration of anti-CD25 Ab (clone PC61) significantly in- As anti-CD25 Ab administration elevated mortality, modulated creased the susceptibility of mice to acute oral T. gondii infection (Fig. weight loss, and increased brain cyst numbers, we next examined 1). The majority of C57BL/6 mice treated with PC61 succumbed to whether anti-CD25 Ab administration impeded tachyzoite control infection by day 20 of infection, and this correlated with a biphasic or limited acute stage immunopathology. In agreement with this modulation of weight loss (Fig. 1, A and B). PC61 pretreated mice hypothesis, we observed significantly reduced liver pathology in exhibited reduced weight loss during early acute T. gondii infection, anti-CD25 Ab-treated mice compared with control Ab-treated with significant differences compared with control Ab treated mice on mice on day 8 of infection, with ameliorated inflammatory cell days 6–8 of infection ( p Ͻ 0.01). However, in contrast to control infiltration (predominantly mononuclear cells and neutrophils) Ab-treated mice that survived acute infection and recovered weight, throughout the parenchymal and portal areas and significantly re- PC61-treated mice were unable to resolve the acute infection and duced levels of hepatic coagulation necrosis (Fig. 1D and Table I). exhibited significantly elevated weight loss during mid to late acute In addition, moderately severe perivasculitis, vasculitis, and fibrin- infection on days 15–18 ( p Ͻ 0.05) (Fig. 1A). These data are con- ous thrombosis was observed in medium to large blood vessels of sistent with previous studies in which the depletion of proinflamma- control Ab-treated mice, which was relatively absent in anti-CD25 tory CD4ϩ T cells ameliorated weight loss and pathology during early Ab-treated mice (Fig. 1D). Nevertheless, parasite control in the acute infection but caused enhanced weight loss during late acute liver was relatively unaffected by anti-CD25 Ab treatment, as com- infection (31). Surviving PC61 pretreated mice also harbored signif- parable tachyzoite numbers were observed in control Ab- and icantly increased brain cyst numbers compared with control mice on PC61-treated mice on day 8 postinfection (Fig. 1E), indicating that day 30 postinfection ( p Ͻ 0.033) (Fig. 1C). parasite control in the liver during early acute T. gondii infection 3988 EFFECT OF ANTI-CD25 ON T. gondii INFECTION

Table I. Tissue pathology during acute T. gondii infection following anti-CD25 Ab administration

Liver Lesionsa

Treatment Liver damage Inflammatory responses Ileum Lesionsa

Control mAb 3.0 (3.0–4.0) 3.0 (2.0–3.0) 1.0 (1.0–2.0) Anti-CD25 1.0 (1.0–2.0) 1.0 (1.0–1.0) 3.0 (2.0–3.0)

a Median score (with range) from five individual mice. is CD25-independent. In contrast, significantly elevated pathology was observed in the ileum of anti-CD25 Ab-treated mice compared with control Ab-treated mice on day 8 postinfection; extensive infiltration of a moderate number of mononuclear cells was ob- served in the lamina propria of anti-CD25 Ab-treated mice, which was absent in control Ab-treated mice (Fig. 1D and Table I). El- evated pathology was directly correlated with increased parasite burdens in the ileum of anti-CD25 Ab-treated mice on day 8 postinfection (Fig. 1F), suggesting that the failure of parasite con- Downloaded from trol, rather than partial depletion or inactivation of natural Tregs following anti-CD25 Ab administration, was responsible for the increased pathology evident in the ileum. Combined, these data highlight the pathological role of CD25-expressing effector cells in mediating hepatic pathology during early acute T. gondii infection and demonstrate the importance of CD25-expressing effector cells http://www.jimmunol.org/ for control of T. gondii parasites in the ileum during the acute stage of infection.

CD25 is transiently up-regulated on CD4 T cells during acute T. gondii infection To examine the kinetics of CD25 expression during T. gondii infection, to establish when it is up-regulated and on which cells, we determined the expression level of CD25 on CD4ϩ by guest on October 1, 2021 and CD8ϩ splenic lymphocytes on various days during acute T. gondii infection. Fig. 2A shows the marked increase between days 4 and 12 of infection in both the absolute number of total splenic CD4ϩ T cells and the number of splenic CD4ϩ T cells expressing high levels of CD25. A similar transient up-regula- tion of CD25 on splenic CD8ϩ T cells was also observed be- tween days 4 and 12 of infection, before absolute numbers of splenic CD8ϩ T cells and CD8ϩCD25high T cells returned to baseline preinfection levels (results not shown). The expansion of CD4ϩ T cells expressing CD25 during acute T. gondii infection could potentially have been due to either the proliferation of CD4ϩCD25high Tregs (present in naive mice) or due to the up-regulation of CD25 on activated precursor CD25Ϫ naive CD4ϩ T cells. Thus, to investigate the ability of CD4ϩ CD25Ϫ T cells to up-regulate CD25 during acute T. gondii infec- ϩ ϩ Ϫ tion we purified and adoptively transferred Thy1.1 CD4 CD25 FIGURE 2. Transient up-regulation of CD25 on effector T cells during ϩ cells into congenic Thy1.2 mice before oral T. gondii infection. acute T. gondii infection. A, Total number of splenic CD4ϩ T cell and Importantly, significant up-regulation of CD25 expression was ob- CD4ϩCD25high T cells was calculated on selected days following oral T. served on the CD4ϩCD25Ϫ donor T cell population (Fig. 2B). gondii infection. CD25 detection was performed using the clone PC61 ϩ Moreover, the kinetics (and magnitude) of CD25 up-regulation on anti-CD25 Ab. Error bar shows mean Ϯ SD. Number above bar for CD4 ϩ CD25high cells indicates the percentage of that population within the total the donor CD4 T cells mirrored the up-regulation of CD25 in the ϩ ϩ ϩ Ϫ ϩ CD4 T cell population. B, Cell sorted Thy1.1 CD4 CD25 cells were total CD4 T cell population in wild-type mice (Fig. 2A), indicat- ϩ adoptively transferred into Thy1.2 B6 mice before oral T. gondii infec- ing that CD4ϩCD25Ϫ cells are the primary precursor cells of the ϩ high tion. The frequency of adoptively transferred donor cells expressing CD25 CD4 CD25 cells generated during acute T. gondii infection. in the spleen was determined on selected days postinfection. C and D,In ϩ high Although there were significantly more CD4 CD25 spleen a separate experiment, groups of five B6 males were infected as in A, and cells in infected mice than in sham-infected mice on day 8 postin- spleens harvested on day 8 to analyze the percentage of CD4ϩCD25high fection, there was no significant difference in the number of total cells that coexpressed Foxp3. The number of CD4ϩCD25high cells and CD4ϩFoxp3ϩ cells, or the number of CD4ϩFoxp3ϩ cells express- CD4ϩFoxp3ϩ cells (C) and representative dot plots (D) gating on CD4ϩ T ing CD25 (Fig. 2C), suggesting that the majority of CD4ϩ cells showing CD25 expression vs Foxp3 expression in naive and T. gondii- ϩ .p Ͻ 0.01 ,ء .CD25high T cells were not Tregs, as shown from day 8 of infection infected mice. Mean ϩ SD is indicated for CD4 populations The Journal of Immunology 3989

versely, CD4ϩCD25high cells derived from acutely infected mice failed to suppress CD4ϩCD25Ϫ cell responses, produced signifi- cant quantities of IFN-␥ and proliferated rapidly following mito- genic and T. gondii antigenic challenge (Fig. 3, C–F). Importantly, CD4ϩCD25high T cells derived from acutely infected mice pro- duced more IFN-␥ and proliferated more rapidly than infection- derived CD4ϩCD25Ϫ cells following specific T. gondii stimula- tion (Fig. 3, E and F), suggesting that during acute T. gondii infection, the majority of parasite-specific effector CD4ϩ T cells are found within the CD4ϩCD25high T cell population.

Reduced numbers of effector T cells and suppressed IFN-␥ production in anti-CD25 Ab-treated T. gondii-infected mice Numerous studies have shown that anti-CD25 Ab (clone PC61) treatment leaves mice essentially devoid of cells expressing CD25 for an extended period (27, 32), an observation we confirm in this study during T. gondii infection (Fig. 4); CD25 expressing splenic CD4ϩ T cells were almost completely undetectable following anti- CD25 Ab administration (detection day 9 postinfection with Ab Downloaded from injected i.p. 4 days before infection) (Fig. 4A). These results were largely reproduced when examining mesenteric lymph node cells (data not shown). Importantly, and consistent with previous reports (27, 32), PC61 administration failed to induce complete depletion of Foxp3ϩ Tregs, instead promoting a maximal 40% reduction in Foxp3-expressing cell numbers (Fig. 4, B and C). As we have http://www.jimmunol.org/ demonstrated that during acute stage T. gondii infection the over- ϩ FIGURE 3. CD4 T cells that up-regulate CD25 expression during whelming majority of CD4CD25high cells display effector rather acute T. gondii infection are Th1 effector cells that produce high levels of than regulatory characteristics, these results directly indicate that ␥ ϩ Ϫ ϩ high IFN- . CD4 CD25 and CD4 CD25 splenic T cells were sort purified anti-CD25 Abs primarily deplete effector T cells during T. gondii from naive mice (A and B)orT. gondii-infected mice (B–F) (day 10 of infection. In agreement, anti-CD25 Abs principally targeted acti- infection). CD25 detection was performed using the PC61 clone anti-CD25 ϩ Ϫ ϩ high vated T cell populations, as shown by the significant decrease in Ab. CD4 CD25 and CD4 CD25 cells were cultured with naive APC ϩ and anti-CD3 Ab (A–D)orT. gondii-pulsed APC (E and F). A, C, and E, the number of CD4 T cells expressing high levels of the activa- ␥ tion marker CD44 following PC61 administration (28.9 Ϯ 11.6 ϫ IFN- production was assayed in supernatant by ELISA. B, D, and by guest on October 1, 2021 F, Proliferation was determined by [3H]thymidine incorporation. Results 106 in control mAb-treated vs 13.3 Ϯ 4.5 ϫ 106 in anti-CD25- shown are the mean Ϯ SD of the group (n ϭ 3) and are representative of treated) (Fig. 4A). two independent experiments. #, p Ͻ 0.05 for significant difference Consequently, to examine whether the depressed effector T cell ϩ Ϫ ϩ p Ͻ 0.05 for sig- T. gondii ,ء .between CD4 CD25 and CD4 CD25high cultures ϩ Ϫ ϩ Ϫ ϩ responses observed during infection following anti-CD25 nificant difference between CD4 CD25 and CD4 CD25 and CD4 Ab administration correlated with a functional defect in proinflam- high CD25 cocultures. matory immune responses, we next determined the levels of the prototypic type I cytokine IFN-␥ in T. gondii-infected mice treated with anti-CD25 Abs. Our results show that PC61 administration ϩ ϩ (Fig. 2D). Moreover, a significant number of CD4 Foxp3 cells reduced systemic plasma IFN-␥ levels (detected on day 8 postin- ϩ were found within the CD25low subset of CD4 cells, revealing fection) compared with control Ab-treated infected mice (Fig. 4D). ϩ Ϫ the presence of Foxp3 CD25 Tregs during T. gondii infection Although the reduction in IFN-␥ production failed to reach sta- and in naive uninfected mice (Fig. 2C). tistical significance in any individual experiments, a reproduc- ␥ ϩ high ible reduction in IFN- production was observed in four sepa- CD4 CD25 T cells are primarily effector T cells during rate experiments, with the percentage of reductions relative to acute T. gondii infection controls of 11, 20, 27, and 52%. Furthermore, the reduced se- It is well established that CD4ϩCD25high T cells derived from rum IFN-␥ levels were paralleled by significantly reduced lev- naive mice express high levels of Foxp3 and exhibit potent regu- els of IFN-␥ mRNA in the liver of anti-CD25 Ab-treated mice latory capabilities. In contrast, extremely strong systemic proin- on day 8 postinfection (Fig. 4E). flammatory immune responses develop during T. gondii infection CD4ϩ T cells, specifically Th1 cells that express T-bet and cop- and the majority of CD4ϩCD25high cells do not express Foxp3 roduce IFN-␥, have also been shown to be the primary source of (Fig. 2), indicating that these cells may exert effector rather than host-protective IL-10 during T. gondii infection (33). Importantly, regulatory functions. To address this likelihood, we purified CD4ϩ however, the expression level of IL-10 mRNA in the liver was CD25Ϫ and CD4ϩCD25high cells from uninfected mice and mice comparable in anti-CD25 Ab- and control-treated mice (Fig. 4F), acutely infected with T. gondii and assessed the ability of these indicating that the regulatory IL-10 response was largely intact in cells to respond to mitogenic (anti-CD3) and T. gondii Ag-specific anti-CD25 Ab-treated mice. Combined, these data definitively stimulation. As expected, CD4ϩCD25high cells derived from un- demonstrate that in vivo administration of anti-CD25 Abs can neg- infected mice closely matched the functional characteristics of nat- atively effect proinflammatory immune responses during an in- ural Tregs; they failed to produce IFN-␥ or proliferate following flammatory disorder, and that CD25 expression is up-regulated on mitogenic stimulation and suppressed proliferation and IFN-␥ pro- T. gondii-specific activated effector T cells, which are required for duction by CD4ϩCD25Ϫ cells ( p Ͻ 0.05) (Fig. 3, A and B). Con- effective proinflammatory type 1 responses. 3990 EFFECT OF ANTI-CD25 ON T. gondii INFECTION Downloaded from http://www.jimmunol.org/ FIGURE 4. Anti-CD25 administration depletes activated CD25ϩ T cells and significantly reduces IFN-␥ production. Groups of five B6 males were administered 1 mg of anti-CD25 (clone PC61) or control mAb (clone HRPN) i.p. 5 days before oral infection with 10 ME49 cysts. A, The number of splenic-activated CD4ϩ T cells were calculated on day 9 postinfection by measuring CD25 and CD44 expression. CD25 detection was performed using the 7D4 clone anti-CD25 Ab. B, Representative dot plots showing Foxp3 expression on splenic CD4ϩ T cells on day 8 postinfection. C, The number of splenic CD4ϩFoxp3ϩ cells on day 8 postinfection in control Ab- and anti-CD25 Ab-treated mice. D–F, The level of IFN-␥ and IL-10 in PC61- and HRPN-treated mice was determined on day 9 postinfection in plasma (D) by ELISA and in liver (E and F) by real-time PCR (Taqman). Results shown .p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .are mean Ϯ SD of the group (n ϭ 5) and are representative of two independent experiments

Anti-CD25 Ab administration suppresses IFN-␥ production by mice did not coexpress yFP, suggesting that these cells are either by guest on October 1, 2021 CD4ϩ T cells Tregs or activated T cells that have failed to produce IFN-␥ (Fig. To examine the T cell populations affected by anti-CD25 Ab admin- 5A). Irrespective of this finding, our results in Fig. 3 clearly show ϩ high istration leading to impaired IFN-␥ expression, we used IFN-␥ bicis- that splenic CD4 CD25 T cells predominantly exert effector tronic reporter mice (YETI) (25), enabling the detection and quanti- rather than regulatory functionality during acute stage T. gondii fication of cell-specific IFN-␥ production directly ex vivo, negating infection. the requirement for subsequent restimulation in vitro. Importantly, In support of our hypothesis that anti-CD25 Ab administration yellow fluorescent protein (yFP) expression in YETI mice not only directly suppresses proinflammatory protective immune responses ␥ during acute T. gondii infection, we observed significantly reduced marks cells actively producing IFN- , but also marks cells that have ϩ expressed IFN-␥, providing an accurate representation of both current frequency of yFP-positive CD4 T cells in the liver, mesenteric and historical cellular function (25). As expected, the majority of lymph node, blood, and spleen of T. gondii-infected mice treated CD4ϩ T cells in naive mice were found to be yFP-negative (25), with anti-CD25 Abs as compared with control Ab-treated infected indicating that few CD4ϩ T cells were polarized type 1 effector/mem- mice (Fig. 5B). Similarly, significantly reduced frequency of yFP- ϩ ory cells (Fig. 5A). The CD4ϩ yFP-positive cells found in liver, positive CD8 T cells were observed in the liver of T. gondii- spleen, blood, and mesenteric lymph nodes in naive mice displayed infected mice treated with anti-CD25 Abs as compared with control heterogeneity in CD25 expression, suggesting the existence of a num- Ab-treated infected mice (results not shown). In contrast anti- ber of different populations of IFN-␥-producing CD4ϩ T cells at var- CD25 Ab administration did not significantly reduce yFP expres- ϩ ious stages of cellular activation. All yFP-positive cells irrespective of sion by CD8 T cells in the spleen, mesenteric lymph node, or ϩ CD25 expression or tissue/organ location were, however, uniformly blood, suggesting that effector CD8 T cells are less affected by ϩ CD44high (results not shown). anti-CD25 Ab than CD4 T cells (results not shown). ϩ In agreement with Fig. 2, up-regulation of CD25 on CD4ϩ T Heterogeneous expression of yFP by CD4 T cells occurs dur- cells was observed on day 8 postinfection in all tissues examined ing T. gondii infection, as shown in infected, isotype control group and this response correlated with significant up-regulation of yFP (Fig. 5) and as published in Mayer et al. (34). Importantly, the expression; importantly in all the tissues examined the majority of expression level of yFP is directly correlated to the capacity to yFP-expressing CD4ϩ T cells coexpressed CD25 (Fig. 5A). Thus, produce IFN-␥ following in vitro restimulation, with cells express- virtually all CD4ϩCD25high cells derived from liver and blood ing highest levels of yFP capable of producing most IFN-␥ protein were yFP-positive, marking CD4CD25high cells as predominantly (34). Interestingly, the highest level of yFP expression was rou- effector rather than Tregs in these tissues during acute T. gondii tinely observed in CD4ϩ T cells expressing highest levels of CD25 infection. In contrast, a number of CD4ϩCD25high cells derived (Fig. 5A), marking these potent IFN-␥-producing cells as principal from spleen and mesenteric lymph nodes of T. gondii-infected targets of anti-CD25 Abs. Thus, anti-CD25 Ab administration The Journal of Immunology 3991 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Anti CD25 Ab administration suppresses IFN-␥ production by CD4ϩ T cells. Groups of three bicistronic IFN-␥-enhanced yFP (IFN-␥-yFP) reporter male mice (Yeti) were given 1 mg of anti-CD25 (clone PC61) or control mAb (clone HRPN) i.p. 5 days before oral infection with brain homogenate containing 10 ME49 cysts or sham uninfected brain homogenate. A, Representative dot plots showing yFP expression relative to CD25 expression (using 7D4 to detect) on CD4ϩ T cells from spleen, mesenteric lymph, liver, and blood on day 8 postinfection. B, Relative frequency of CD4ϩ T cells expressing yFP in anti-CD25 Ab-treated and control Ab-treated T. gondii-infected and sham-infected mice on day 8 postinfection. Results shown are mean Ϯ SD of .p Ͻ 0.05 ,ء .the group (n ϭ 3) and are representative of two independent experiments significantly reduced the yFPhigh CD4ϩ T cell subsets in the liver or brain express CD25 during chronic infection, and whether these and blood. A reduction in the frequency of yFPhigh CD4ϩ T cells cells may also be affected by anti-CD25 Ab administration. Addi- was also observed in the mesenteric lymph node and spleen, but tionally, it has previously been shown that natural Tregs that co- the reduction failed to reach statistical significance (results not express Foxp3 and CD25 can suppress protective immunity during shown). Combined, these results demonstrate that anti-CD25 Abs a number of infections, including infection, en- can directly target effector IFN-␥-producing CD4ϩ T cells during abling parasite persistence and the establishment of chronic infec- acute T. gondii infection leading to systemic reductions in IFN-␥ tions (35, 36). Thus, CD25-expressing CD4ϩ T cells may also play production and impaired protective T cell immunity. an important regulatory role during chronic T. gondii infection, lowering proinflammatory responses, moderating immune-medi- Anti-CD25 Ab administration does not impair resistance to ated pathology but impairing parasite control. As a result, anti- chronic stage T. gondii infection CD25 Ab administration during chronic T. gondii infection could It is well established that anti-parasitic CD4ϩ T cells and CD8ϩ T potentially augment anti-parasitic immune responses and enhance cells are required for the control of parasite reactivation in the parasite control. In support of this hypothesis, Foxp3 mRNA levels brain and prevention of Toxoplasma encephalitis during chronic T. in the brain were increased during chronic infection compared with gondii infection (22). However, the activation status of these uninfected control levels (results not shown). Moreover, we have CD4ϩ and CD8ϩ T cells is relatively unknown. Consequently it is shown that CD25 up-regulation on effector T cells is transient dur- unclear whether protective CD4ϩ and CD8ϩ T cells in the spleen ing T. gondii infection and occurs primarily during the acute phase 3992 EFFECT OF ANTI-CD25 ON T. gondii INFECTION

shown) and brain (Fig. 6C) 5 and 10 days postadministration (days 5 and 10 postadministration were examined in separate experi- ments with results from day 5 postadministration shown). How- ever, Foxp3 levels were not significantly reduced in anti-CD25 Ab-treated mice (Fig. 6C). Importantly, it has previously been shown that Foxp3ϩ cells that survive anti-CD25 Ab treatment are functionally inactive (32), indicating that anti-CD25 Ab adminis- tration should still ablate Treg functionality during chronic T. gon- dii infection even if all Foxp3ϩ T cells are not eliminated. None- theless, Ab administration did not significantly alter the level of weight loss (relative to preantibody administration baseline levels) or mortality over a 30-day period posttreatment (Fig. 6, D and E) and this correlated with unaltered parasite control (measuring in- vasive tachyzoite forms of parasite) and IFN-␥ production in the brain of anti-CD25 Ab-treated mice compared with control treated groups (Fig. 6F). Combined these results demonstrate that CD25- expressing natural Tregs do not suppress anti-parasitic immune responses during chronic T. gondii infection and are not required to limit excessive inflammation. In support of this conclusion, the frequency of CD4ϩFoxp3ϩ cells in the brain (of total CD4ϩ T Downloaded from cells) was not elevated in chronically infected mice compared with naive mice. Thus, the increase in brain Foxp3 mRNA expression during chronic infection is due to the increase in total CD4ϩ T FIGURE 6. Anti-CD25 Ab administration does not affect control of cells that migrate to and reside in brains of chronically infected ϩ Ϫ ϩ high chronic T. gondii infection. A and B, CD4 CD25 and CD4 CD25 T mice, rather than specific localization and migration of Tregs to the cells were sort purified from spleen (A) or brain (B) of mice chronically site of infection, as is observed during L. major infection (35). http://www.jimmunol.org/ infected with T. gondii (day 50 postinfection). CD4ϩCD25Ϫ and ϩ high CD4 CD25 cells were cultured with T. gondii-pulsed APC for 60 h and Discussion IFN-␥ production was assayed in supernatant by ELISA. Results shown are Ϯ ϭ In this study, we demonstrate that during acute T. gondii infection the mean SD of the group (n 3) and are representative of two inde- ϩ pendent experiments. #, p Ͻ 0.05 for significant difference between transient up-regulation of CD25 expression occurs on CD4 T -p Ͻ 0.05 for significant dif- cells during the expansion phase of the T cell response. Impor ,ء .CD4ϩCD25Ϫ and CD4ϩCD25high cultures ϩ ference between CD4ϩCD25Ϫ and CD4ϩCD25Ϫ and CD4ϩCD25high co- tantly, the vast majority of CD4 CD25high cells generated during cultures. C–F, The 1 mg of anti-CD25 Ab (clone PC61) or control Ab was acute T. gondii infection do not coexpress Foxp3, clearly defining administered to mice chronically infected with T. gondii (day 56 postin- the cells as effector cells rather than Tregs. Anti-CD25 Abs di- by guest on October 1, 2021 fection). C, At 5 days postantibody administration, the number of total rectly target these CD25-expressing effector CD4ϩ T cell popula- brain CD4ϩ T cells, CD4CD25high (using 7D4 to detect), and ϩ ϩ tions leading to impaired proinflammatory immune responses, CD4 Foxp3 cells were enumerated to determine the effectiveness of anti- exemplified by significantly reduced T cell-derived IFN-␥ produc- -p Ͻ 0.05 for significant difference be ,ء .CD25 Ab-mediated depletion tion. Thus anti-CD25 Ab administration initially reduced the se- tween control Ab- and anti-CD25 Ab-treated groups. Weight loss (D) and survival (E) were followed following Ab administration. Weight values are verity of T cell-dependent immune-mediated pathology during expressed as the percentage weight relative to preantibody administration early acute T. gondii infection, but led to the failure of parasite -p Ͻ 0.05 for significant difference between control Ab- control and ultimately death in a large number of anti-CD25 Ab ,ء .(day 56 weights) and anti-CD25 Ab-treated groups. F, The effect of anti-CD25 Ab admin- treated mice. In contrast, anti-CD25 Ab administration did not af- istration on brain parasite control and immune responses were determined fect parasite control during chronic T. gondii infection, suggesting 30 days postantibody administration by quantifying parasite (tachyzoite) that CD25-expressing T cells do not significantly regulate anti- and IFN-␥ levels by real-time PCR. Results are shown as fold change parasitic immunity during the later chronic stage of T. gondii relative to control Ab-treated mice. None of the differences were statisti- infection. cally significant in this or a replicate experiment. The results observed in this study are entirely consistent with a direct effect of anti-CD25 Ab administration on proinflammatory effector T cell populations. Thus, although (as expected) anti- of infection, suggesting that CD4ϩCD25high cells are functionally CD25 Ab administration reduced the number of Foxp3ϩ natural disparate during acute and chronic stages of T. gondii infection Tregs, it also reduced circulating IFN-␥ levels (in all four exper- (Fig. 2). In agreement with a regulatory rather than effector role of iments examined) and resulted in significantly reduced IFN-␥ CD25-expressing cells during chronic infection, CD4ϩCD25high mRNA levels in the liver. Using IFN-␥ yFP reporter (YETI) mice, cells isolated from both brain and spleen during chronic infection we subsequently showed that anti-CD25 Ab administration leads failed to produce significant quantities of IFN-␥ following T. gon- to reduced IFN-␥ production by CD4ϩ T cells in the liver, spleen, dii antigenic restimulation (as compared with CD4ϩCD25Ϫ cells) blood, and mesenteric lymph node. Consequently our results differ and splenic CD4ϩCD25high cells moderately suppressed IFN-␥ from those obtained by Lund et al. (37), where specific natural production by CD4ϩCD25Ϫ cells (Fig. 6, A and B). Treg depletion reduced localized proinflammatory immune re- To directly determine the relative importance of CD25-express- sponses at the mucosal site of HSV infection, but enhanced proin- ing natural Tregs during chronic T. gondii infection, we adminis- flammatory immune responses in tissue draining lymph nodes. tered anti-CD25 Abs to chronically infected mice (day 56 postin- Therefore, whereas in their model, the specific depletion of natural fection) and determined whether Ab administration significantly Tregs (performed in Foxp3 diphtheria toxoid receptor transgenic modulated disease control. Anti-CD25 Ab administration effec- mice) inhibited effector T cell migration to nonlympoid tissue sites tively neutralized CD25 expression in the spleen (results not of infection, we show that anti-CD25 Ab administration promoted The Journal of Immunology 3993 systemic suppression of effector T cell responses. Combined, these chronic infection (data not shown). In addition, CD4ϩCD25high data demonstrate the lack of specificity of anti-CD25 Abs to target cells isolated from the spleen and brain of chronically infected natural Tregs compared with the more recently generated Foxp3- mice displayed regulatory rather than effector phenotypes follow- dtr system (38). ing in vitro Ag-specific stimulation. Thus, CD25-expressing CD4ϩ It has previously been demonstrated that effector CD4ϩ T cells T cells are primarily regulatory rather than effector cells during are responsible for the onset of immune-mediated pathology and chronic T. gondii infection and effector CD4ϩ and CD8ϩ T cells severe weight loss in B6 mice following oral T. gondii infection. do not require CD25 expression to mediate parasite control during (20, 31, 39, 40). Consistent with this demonstration, anti-CD25 Ab the later stages of infection. Importantly, these results also dem- administration modulated body weight loss during acute T. gondii onstrate that natural Tregs do not control anti-parasitic immune infection and significantly reduced the severity of liver pathology responses during chronic T. gondii infection, as administration of on day 8 of infection, indicating a direct effect on effector T cell anti-CD25 Abs did not augment parasite control. Consequently, it populations. Nevertheless, it has also previously been reported that is unlikely that natural Tregs are the source of IL-10 that is re- Th1 CD4ϩ T cells that coproduce IFN-␥ are the primary source of quired for the limitation of immune-mediated pathology during host-protective IL-10 during T. gondii infection (33). Conse- chronic T. gondii infection (44). quently, given the effect of anti-CD25 Ab administration on CD4ϩ Most studies that use anti-CD25 Ab administration to investi- T cell-derived IFN-␥ production, it was foreseeable that Ab ad- gate Tregs assume that the Ab affects only that subset and, for the ministration could also have impaired IL-10 production, and that most part, these studies have disregarded effects on cells other than this secondary Ab effect may have contributed to the enhanced Tregs that can up-regulate CD25 in response to infection. This susceptibility of anti-CD25 Ab-treated mice during acute T. gondii decision is surprising because Ag-stimulated effector T cells (and infection. Our results show that IL-10 mRNA levels were not sig- others) up-regulate CD25 and IL-2 drives T cell proliferation in Downloaded from nificantly reduced in the liver following anti-CD25 Ab adminis- response to Ag stimulation. Notably, anti-CD25 Abs have been tration, suggesting that CD25 expression is not critical for the pro- used clinically to dampen autoimmunity and reduce rejection of duction of host-protective IL-10 during acute T. gondii infection. transplants (45), uses that run counter to the notion that anti-CD25 These results appear to further underline the effects of anti-CD25 augments immunity. The reason for the pronounced effect of anti- Ab administration on effector proinflammatory responses, rather CD25 Ab on effector T cell populations during acute T. gondii than regulatory based responses during acute T. gondii infection. infection, but not in other models is unclear, but is likely correlated http://www.jimmunol.org/ As we did not specifically examine the cellular source or protein with the extreme polarized systemic proinflammatory immune re- levels of IL-10 additional experiments are, however, required to sponse that develops during T. gondii infection. As CD25 is up- determine the extent that anti-CD25 Ab treatment alters IL-10 pro- regulated on a significant percentage of the CD4ϩ and CD8ϩ T duction by Th1 CD4ϩ T cells during infection. Interestingly, al- cell population during acute T. gondii infection, it is foreseeable though we highlight the importance of CD25 expression on effec- that any effect of anti-CD25 Ab treatment on effector T cell pop- tor T cells during the acute stage of primary T. gondii infection, ulations is more pronounced during acute T. gondii infection, and administration of anti-CD25 Abs did not affect vaccine-induced might not be as apparent in other models. protection during secondary challenge infection. Thus, anti-CD25 In conclusion, we have shown that during acute T. gondii in- by guest on October 1, 2021 Ab administration to mice previously infected with the noncyst fection, anti-CD25 Abs directly target effector CD4ϩ T cell pop- forming attenuated TS-4 strain of parasites did not inhibit protec- ulations, significantly modulating the proinflammatory immune tive immune responses and anti-CD25 Ab- and control-treated response to T. gondii infection. Although initially this limits im- vaccinated mice displayed equivalent ability to prevent brain cyst mune-mediated pathology, it also leads to the failure to control development (results not shown). These results indicate that parasite dissemination and encystment and finally death. Thus, al- CD25-expressing effector T cell populations are not critically re- though highlighting the role of CD25 expressing T cells for the quired for vaccine-induced protection to T. gondii infection. control of acute T. gondii infection, we importantly also demon- Despite the pathological role of CD4ϩ T cells during early acute strate the significant limitations of using anti-CD25 Abs in vivo T. gondii infection, it is well established that effector CD4ϩ T cells during inflammatory diseases to investigate the importance of and CD8ϩ T cells are required for protection and parasite control Tregs. On the basis of our findings, we propose that studies that during late acute and chronic stages of infection (41–43). Conse- use anti-CD25 Abs during inflammatory settings should take note quently, as anti-CD25 Ab treatment reduced the number of acti- of the effects on the entire T cell population, not solely those ex- vated (CD44high) CD4ϩ spleen cells by more than 50% on day 9 pressing markers associated with Tregs. of infection, it is not surprising that anti-CD25 Ab treated mice displayed elevated parasite levels in the ileum on days 8 and 15 Acknowledgments postinfection, exhibited more brain cysts on day 30 post infection We thank Katja Mohrs for technical assistance. We also thank Dr. Julius and succumbed more rapidly to late/acute and chronic stage infec- Hafalla and Dr. Brian de Souza for critical reading of the manuscript. tion. Nonetheless, we have also demonstrated that anti-CD25 Ab administration during established chronic stage of T. gondii infec- Disclosures tion did not affect brain-localized parasite control or anti-T. gondii The authors have no financial conflict of interest. immunity, suggesting that anti-CD25 Abs do not negatively affect protective T cell responses at this stage. In agreement, transient References expression of CD25 on effector T cells was observed only during 1. Belkaid, Y. 2007. Regulatory T cells and infection: a dangerous necessity. Nat. ϩ Rev. Immunol. 7: 875–888. acute T. gondii infection and CD25 expression on splenic CD4 2. Sakaguchi, S., M. Ono, R. Setoguchi, H. Yagi, S. Hori, Z. Fehervari, J. Shimizu, ϩ ϩ ϩ ϩ and CD8 T cells during chronic stages of infection was compa- T. Takahashi, and T. Nomura. 2006. Foxp3 CD25 CD4 natural regulatory T rable to that observed in naive mice. Moreover, brain infiltrating cells in dominant self-tolerance and autoimmune disease. Immunol. 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