International Immunology Advance Access originally published online on January 30, 2007
International Immunology 2007 19(3):337-343; doi:10.1093/intimm/dxl151
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Treg suppressive activity involves estrogen-dependent expression of programmed death-1 (PD-1)
1 Neuroimmunology Research, Veterans Affairs Medical Center, R&D-31, 3710 SW US Veterans Hospital Road, Portland, OR 97239, USA
2 Department of Neurology, Oregon Health and Science University, Portland, OR 97239, USA
3 Department of Food Hygiene, Faculty of Veterinary Medicine, Warsaw Agricultural University, Warsaw, Poland
4 Department of Molecular Microbiology and Immunology
5 Department of Anesthesiology and Perioperative Medicine, Oregon Health and Science University, Portland, OR 97239, USA
Correspondence to: H. Offner; E-mail: offnerva{at}ohsu.edu
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Abstract |
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Estrogen [17-ß-estradiol (E2)] is a potent driver of the FoxP3+ regulatory T cell (Treg) compartment. Recently, Tregs were further characterized by intracellular expression of the negative co-stimulatory molecule, programmed death-1 (PD-1). To clarify the role of PD-1 versus FoxP3 in E2-enhanced Treg suppression, we evaluated both markers and functional suppression in wild-type, estrogen receptor knockout (ERKO) mice and PD-1 KO mice. We demonstrate that intracellular PD-1 expression is also E2 sensitive, since E2 treatment increased intracellular PD-1 levels in CD4+FoxP3+ cells, and PD-1 expression and Treg suppression were reduced in ERKO mice. Surprisingly, PD-1 KO mice retained normal levels of FoxP3 expression, but Tregs from these mice lacked functional suppression. However, E2 pre-treatment of PD-1 KO mice partially restored functional Treg suppression without enhancing FoxP3 expression. Thus, functional Treg suppression in immunized mice without E2 pre-treatment was more closely linked to PD-1 expression than to FoxP3 expression. However, although enhanced PD-1 expression was E2 dependent, functional suppression was still enhanced by E2 pre-treatment in the absence of PD-1. These data clearly demonstrate that E2 can affect multiple regulatory elements that influence Treg suppression, including both PD-1-dependent and PD-1-independent pathways.
Keywords: autoimmunity, co-stimulation, EAE/MS, T cells, tolerance/suppression
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Introduction |
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The evidence gathered in recent years significantly helped to understand the impact of sex steroids on immunity (1–9). Our studies collectively (10–13) demonstrated that sustained sub-pregnancy levels of 17-ß-estradiol (E2) could prevent clinical and histological signs of experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease, multiple sclerosis (MS). In our recent studies, we were able to link the beneficial effect of hormone therapy to expansion of regulatory T cells (Tregs) (12, 13). It is now well known that alterations in Tregs are involved in pathophysiology of autoimmune disorders, possibly including MS (14–21). Therefore, the understanding of how steroid hormones may change suppressive activity of Treg represents one of the most important issues. So far, our data indicate an E2-dependent increase in FoxP3 expression and expansion of the CD4+CD25+ T cell compartment (12). In addition, enhanced suppressive activity of Tregs was documented in both E2-treated and pregnant mice (13).
Although many groups showed the ability of Treg to suppress the growth of T cells with which they interact, the precise mechanism underlying this process is largely unknown. Thus, in the present study, our goal was to test if the E2 hormone-enhanced suppressive activity of Tregs was related to negative co-stimulatory pathways. Interestingly, a recent finding showed that a programmed death-1 (PD-1) polymorphism was associated with MS disease progression (22). Thus, we evaluated the PD-1-negative co-stimulatory pathway as one possible mechanism that could account for E2-enhanced Treg suppressive activity. At least two independent studies have shown increased expression of PD-1 within the Treg compartment (23, 24). Although it is not clear how increased PD-1 expression may contribute to the suppressive potential, PD-1 was proposed to serve as an additional marker of the Treg sub-population. In a previous report, we further demonstrated that E2 could both potentiate Treg suppression and enhance expression of PD-1 (25).
In the present study, we have investigated intra and extracellular expression of PD-1 in FoxP3+ cells isolated from spleens of mice pre-treated in vivo with pregnancy levels of E2. In addition, we tested the impact of endogenously produced estrogen on PD-1 expression by utilizing estrogen receptor knockout (ERKO) mice. Our current study demonstrated striking differences in the PD-1 expression level in FoxP3+ cells (Tregs) obtained from wild-type (WT) C57BL/6 female and ERKO mice. As shown previously, the majority of PD-1 expression was within the intracellular compartment of Tregs (23). Unlike WT, ERKO mice showed a significant reduction in the expression level of PD-1 among Treg, and this deficiency correlated with decreased suppressive activity of CD4+CD25+ Tregs. These differences were likely influenced most by impaired Esr1 signaling due to alterations in ability to respond to the endogenously produced hormones and the distribution and function of Esr1 among ERKO mice. The final confirmation that PD-1 regulates Treg inhibitory activity was the loss of the ability to suppress the in vitro growth of syngeneic responder cells when the Tregs originated from PD-1 KO donors. However, exogenous E2 could partially restore Treg suppression in these mice, implicating both PD-1-dependent and PD-1-independent pathways.
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Methods |
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Mice
Female (7–12 weeks) C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). ER-
KO (Esr1–/–, ERKO) and estrogen receptor-ß knockout [Esr2–/–, (BERKO)] mice were a gift from Patricia Hurn (Department of Anesthesiology and Perioperative Medicine, Oregon Health and Science University, Portland, OR, USA). PD-1 KO mice on the C57BL/6 background were obtained from Prof. Honjo (Japan). All mice were rested for 7 days prior to being used for any experiments. All presented data represent one of two independent experiments. Animals were housed and cared for according to the institutional guidelines in the Animal Resource Facility at the Veterans Affairs Medical Center (Portland, OR, USA).
Hormone treatment and immunization
For E2 therapy, a 3-mm pellet containing 2.5 mg of E2 (Innovative Research of America, Sarasota, FL, USA) was implanted subcutaneously (dorsally) 7 days before using the mice for experimentation or immunization with encephalitogenic peptide. These pellets are designed to release their contents at a constant rate over 60 days. Serum levels of E2 were monitored by radioimmunoassay as described previously (11). Immunization with myelin oligodendroglia glycoprotein (MOG)-35-55 peptide was done according to standard methods as described previously (11).
Evaluation of FoxP3 and PD-1
The following FITC-, PE- or allophycocyanin-conjugated mAbs were used for cytofluorometric analysis: anti-mouse CD4, anti-mouse CD25 (from BD PharMingen, San Diego, CA, USA) and anti-mouse FoxP3 (clone FJK-16S) mAb from eBioscience. Cells were prepared, stained and data collected using a BD Biosciences FACSCalibur flow cytometer. Data were analyzed on FlowJo software (TreeStar, Inc., Ashland, OR, USA) as previously described. Intracellular staining for FoxP3 of murine cells was performed following the protocol recommended by eBioscience. Briefly, cells were surface stained following standard procedures. After washing, the cells were fixed overnight and washed twice with 2 ml of eBioscience permeabilization buffer. The cells were co-stained for 15 min with FcBlock and IgG CyChrome (BD PharMingen) followed 30 min later with fluorescent-labeled antibodies to PD-1, FoxP3 or isotype control. The cells were then washed twice with 2 ml of permeabilization buffer, re-suspended in 1x PBS, 0.5% BSA and run on a BD FACSCalibur. Data represent 10 000 events, unless otherwise noted.
T cell purification and Treg suppression assay
Single-cell suspensions were prepared from spleens and RBCs were lysed. Unimmunized mice receiving E2 pellets were sacrificed 7 days after implantation. Purified CD4+ cells were obtained by MACS according to the manufacturer's protocols (Miltenyi Biotec, Bergisch Gladbach, Germany). For flow cytometry, cells were stained with FITC–anti-CD4 and APC–anti-CD25 (BD PharMingen). CD4+CD25+ cells were sorted from CD4+CD25– using a FACSVantage (BD Immunocytometry Systems, San Jose, CA, USA). Splenocytes from naive female WT C57BL/6 mice were depleted of T cells by magnetic sorting using CD90 Microbeads (Miltenyi Biotec, Auburn, CA, USA) and used as antigen-presenting cells (APCs). Briefly, cells were incubated on ice for 15 min with microbeads. After wash, cells were sorted using the deplete program on an autoMACS magnetic cell sorter. The negative fraction was washed, re-suspended at 1 x 106 ml–1 in RPMI containing 10% fetal bovine serum (FBS) and irradiated with 1800 rad in a cesium irradiator. The overall purity of T cell-depleted APCs was 
80%. Suppression assays were performed in 96-well flat-bottomed plates (Becton Dickinson) in a final volume of 200 µl per well of RPMI containing 10% FBS. Both APCs and indicator cells (CD4+CD25–) were plated at 0.5 x 104 cells per well in triplicate and CD4+CD25+ suppressor cells were added at the following ratios of indicator:Tregs—1:0, 1:1, 1:0.5, 1:0.25, 1:0.1 and 0:1. Anti-CD3 antibody was added at a final concentration of 0.5 µg ml–1. After 48 h, the plates were pulsed for 18 h with [3H]-thymidine, and the cells harvested on glass fiber filters and assessed for uptake of the labeled thymidine by liquid scintillation. The percent suppression was plotted versus indicator/suppressor cell ratios and a regression line was calculated. I50 was determined as the ratio of indicator/suppressor cells that produced 50% suppression. Suppressive index (SI) = 100 – I50.
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Results |
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E2 treatment augments intracellular PD-1 expression in FoxP3+ Tregs
PD-1 may represent a key pathway in maintaining peripheral tolerance, and has recently been localized in the perinuclear compartment of FoxP3+ Tregs (23). In previous reports, we demonstrated that E2 treatment increased the number of Tregs and their level of expression of FoxP3, resulting in enhanced suppressive activity (12, 13). To determine if the mechanism by which E2 enhances Treg suppression involves modulations in PD-1 expression, we tested co-expression of PD-1 in FoxP3+ cells isolated from WT and estrogen-treated mice. As shown previously, E2 treatment in vivo resulted in an increase in FoxP3 expression (number and fluorescence intensity) in viable CD4+ splenocytes (Fig. 1, two left-hand and upper right hand panels). Furthermore, E2 treatment resulted in a significant up-regulation in intracellular expression of PD-1 in gated FoxP3+ cells compared with naive controls (Fig. 1, lower right-hand panels). These results indicate that intracellular PD-1 expression is estrogen sensitive, and suggest that native expression of PD-1 by Tregs might be altered in Esr1-deficient mice. This was indeed the case, with a striking reduction in intracellular PD-1 expression in FoxP3+ Tregs from ERKO (Esr1 KO) mice, but not in Tregs from BERKO (Esr2 KO) mice compared with WT controls (Fig. 2). In contrast, there were only minor differences in intracellular FoxP3 expression or in extracellular expression of PD-1 among ERKO, BERKO and WT mice, with
2% FoxP3+CD4+ T cells and 3 to 4% of cells expressing extracellular PD-1 in the gated FoxP3+ population from each strain (data not shown). Further evaluation demonstrated reduced suppressive activity of CD4+CD25bright Tregs isolated from ERKO mice compared with BERKO and WT mice (Fig. 3 and Table 1), indicating that functional Treg suppression correlated better with intracellular PD-1 expression levels than with FoxP3 or extracellular PD-1 levels. These results demonstrate that E2 exerts endogenous regulation of intracellular PD-1 expression in vivo through Esr1, a finding that is highly congruous with Esr1-dependent regulation of FoxP3 expression, Treg suppressor function and protection against induction of EAE (26).
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PD-1 but not FoxP3 expression is sufficient to mediate Treg suppression
The E2-mediated increase in PD-1 expression in FoxP3+ Tregs was further investigated in WT and PD-1-deficient (KO) mice immunized with MOG-35–55 peptides in CFA/Ptx. Immunization itself (which induced typical EAE in the WT mice) induced strong expression of PD-1 in FoxP3+ T cells (40%, Fig. 4A, upper left-hand panel) that was comparable to unimmunized but E2-treated WT mice (46%, Fig. 1). Pre-treatment of immunized mice with 2.5-mg pellets of E2 (which protected WT mice from EAE) further enhanced intracellular expression of PD-1 in FoxP3+ T cells (58%, Fig. 4A, upper right-hand panel). To clarify the role of PD-1 versus FoxP3 in Treg suppression, we evaluated both markers and functional Treg suppression in PD-1 KO mice. As is shown in Fig. 4(B), the percentage of FoxP3+CD4+ T cells in immunized PD-1 KO mice (3.8%) was comparable or slightly higher than in immunized WT mice (2.9%), and E2 pre-treatment of PD-1 KO mice did not induce any increase in FoxP3 expression (3.3%). As expected, however, there was essentially no intracellular PD-1 expression in CD4+FoxP3+ T cells from immunized PD-1 KO mice (which developed EAE) or from immunized PD-1 KO mice pre-treated with E2 (which also developed EAE) (Fig. 4A, bottom panels).
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These data from the PD-1 KO mice provided an opportunity to evaluate functional Treg suppression when there was discordant expression of PD-1 versus FoxP3. As is shown in Fig. 4(C), CD4+CD25bright Tregs from immunized WT mice with expected levels of FoxP3 expression (2.8%, Fig. 4B) had normal levels of functional suppression (SI = 60), and pre-treatment with E2 enhanced suppression as expected (SI = 70). However, CD4+CD25bright Tregs from immunized PD-1 KO mice had essentially no detectable suppression (SI = 0), in spite of normal levels of FoxP3 expression (3.8%, Fig. 4B). Interestingly, CD4+CD25bright Tregs from PD-1 KO mice that were pre-treated with E2 had a low but clearly detectable level of suppression (SI = 34), without a further increase in FoxP3 expression (3.3%, Fig. 4B). Thus, functional Treg suppression in immunized mice without E2 pre-treatment was more closely linked to PD-1 expression than to FoxP3 expression. However, although enhanced PD-1 expression was E2 dependent in ERKO mice, functional suppression but not protection against EAE was partially restored by pre-treatment with exogenous E2 in PD-1 KO mice.
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Discussion |
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In this study, we evaluated interactions between E2, FoxP3 and PD-1 that influence functional Treg suppression. Our data clearly demonstrate striking differences in the PD-1 expression level in FoxP3+ cells (Tregs) obtained from WT C57BL/6 female and ERKO mice, and confirmed that the majority of PD-1 expression was within the intracellular compartment of Tregs. Unlike WT, ERKO mice showed a significant reduction in the expression level of PD-1 among Treg, and this deficiency correlated with decreased suppressive activity of CD4+CD25bright Tregs. These differences were likely influenced most by impaired Esr1 signaling due to alterations in ability to respond to the endogenously produced hormones, and the distribution and function of Esr1 in ERKO mice. The final confirmation that PD-1 regulates Treg inhibitory activity was the loss of the ability to suppress the in vitro growth of syngeneic responder cells when the Tregs originated from PD-1 KO donors. However, exogenous E2 could partially restore Treg suppression in these mice without enhancing FoxP3 expression or restoring PD-1 expression, implicating both PD-1-dependent and PD-1-independent pathways.
The importance of intracellular versus extracellular expression of PD-1 as a possible discriminating marker within the CD4+CD25+ fraction for Tregs rather than activated effector cells has been presented and discussed by Raimondi et al. (23). From that work and our present study, it would seem that PD-1 present in Tregs remains in an intracellular compartment when the Tregs are in a resting state, but may increase in extracellular expression when the Tregs are activated (e.g. by anti-CD3 in the functional Treg suppression assay). It remains unclear, however, if the PD-1 transmits inhibitory signals to target cells through a cell–cell contact-dependent mechanism, or if ligation of PD-1 on the Treg surface by PD-L1 and PD-L2 helps to maintain anergy or perhaps induces other surface regulatory molecules in the Treg itself. The latter possibility seems to be more plausible since we observed increased proliferating activity of Treg isolated from PD-1 KO mice (data not shown). In addition, it has been reported that the PD-1 deficiency results in a significant alteration of the final T cell repertoire, either directly or indirectly. C57BL/6 mice deficient for PD-1 develop autoimmune diseases, including lupus-like glomerulonephritis and destructive arthritis, as they age. In addition to the dysregulation of peripheral tolerance (27, 28), the altered T cell repertoire formation in the absence of PD-1 may also increase, due not only to the emergence of mature autoreactive T cells but also to the loss of suppressive activity by Tregs. Together, the data presented here indicate that the level of PD-1 expression in FoxP3+ Tregs correlates with their suppressive activity, and that endogenously produced E2 plays a key role in regulating this process. However, pre-treatment of PD-1 KO mice with exogenous E2 partially restored Treg functional activity without affecting FoxP3 expression levels or restoring PD-1 expression, thus implicating a PD-1-independent pathway. This result is entirely consistent with our previous studies that show an E2 enhancement effect both in Treg and APC function (25, 30).
The functional role of PD-1 in mediating Treg suppression is of potential importance in MS and possibly other autoimmune diseases. In MS patients, the PD-1.3 polymorphism was associated with disease progression, and CD4+ T cells from PD-1.3+ patients had reduced inhibitory activity when PD-1 was ligated with anti-PD-1 antibody compared with patients without PD-1.3 (22). Such differences may explain reduced Treg suppressive activity reported in MS (18, 19, 21, 29), especially in the presence of normal or only slightly reduced levels of FoxP3 expression (18, 21).
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Acknowledgements |
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The authors wish to thank Tasuku Honjo for providing PD-1 KO mice and Eva Niehaus for assistance in preparing and submitting the manuscript. This work was supported by grants NS23444, NS45445 and NS49210 from the National Institutes of Health, National Multiple Sclerosis Society grants RG3405A2, RG3400A4 and RG3794A4, The Nancy Davis MS Center Without Walls and the Biomedical Laboratory R&D Service, Department of Veterans Affairs.
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Abbreviations |
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| APC, antigen-presenting cell |
| BERKO, estrogen receptor-ß knockout |
| EAE, experimental autoimmune encephalomyelitis |
| ER, estrogen receptor |
| ERKO, estrogen receptor- knockout |
| E2, 17ß-estradiol |
| FBS, fetal bovine serum |
| KO, knockout |
| MOG, myelin oligodendroglial glycoprotein |
| MS, multiple sclerosis |
| PD-1, programmed death-1 |
| SI, suppressive index |
| Treg, regulatory T cell |
| WT, wild type |
| l50, the ratio of indicator/suppressor cells that produce 50% of suppression |
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Notes |
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Transmitting editor: L. Steinman
Received 3 November 2006, accepted 27 December 2006.
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