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The Journal of Immunology, 2006, 177: 209-215.
Copyright © 2006 by The American Association of Immunologists

G Protein-Coupled Receptor 83 Overexpression in Naive CD4+CD25– T Cells Leads to the Induction of Foxp3+ Regulatory T Cells In Vivo1

Wiebke Hansen*, Karin Loser{dagger}, Astrid M. Westendorf*, Dunja Bruder*, Susanne Pfoertner*, Christiane Siewert{ddagger}, Jochen Huehn{ddagger}, Stefan Beissert{dagger} and Jan Buer2,*,§

* Department of Mucosal Immunity, German Research Centre for Biotechnology, Braunschweig, Germany; {dagger} Department of Dermatology, University of Münster, Münster, Germany; {ddagger} Experimental Rheumatology, Charité University Medicine Berlin, Berlin, Germany; and § Institute of Medical Microbiology, Hannover Medical School, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Foxp3 functions as a lineage specification factor for the development of naturally occurring thymus-derived CD4+CD25+ regulatory T (Treg) cells. Recent evidence suggests that naive Foxp3–CD4+CD25– T cells can be converted in the periphery into Foxp3+ Treg cells. In this study, we have identified the G protein-coupled receptor (GPR)83 to be selectively up-regulated by CD4+CD25+ Treg cells of both murine and human origin in contrast to naive CD4+CD25– or recently activated T cells. Furthermore, GPR83 was induced upon overexpression of Foxp3 in naive CD4+CD25– T cells. Transduction of naive CD4+CD25– T cells with GPR83-encoding retroviruses did not confer in vitro suppressive activity. Nevertheless, GPR83-transduced T cells were able to inhibit the effector phase of a severe contact hypersensitivity reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Most strikingly, this in vivo acquisition of suppressive activity was associated with the induction of Foxp3 expression in GPR83-transduced CD4+ T cells under inflammatory conditions. Our results suggest that GPR83 might be critically involved in the peripheral generation of Foxp3+ Treg cells in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Naturally occurring CD4+CD25+ regulatory T (Treg)3 cells play a crucial role in establishing and maintaining immunological tolerance with their dysfunction, leading to severe or even fatal immunopathology in a broad spectrum of patients (1, 2). Therefore, these cells represent good targets for the rational design of strategies to induce or abrogate immunological tolerance to self and nonself Ags.

Accumulating evidence suggests that naturally occurring CD4+CD25+ Treg cells represent a dedicated T cell lineage that matures within the normal thymus and that Foxp3 functions as the Treg cell lineage specification factor (3). Despite a lot of efforts, the molecular bases for Treg cell functions have not been unraveled yet. Several modes of suppression by Treg cells have been suggested, ranging from immunosuppressive cytokines IL-10 and TGF-beta to cell-cell contact via the inhibitory molecule CTLA-4 (4). However, although these modes of action appear instrumental for Treg function in some models (5, 6), they seem dispensable in others (7, 8). Elucidation of Treg mechanisms and manipulation of this subset would be greatly facilitated by the identification of Treg-selective cell surface receptors that modulate their function. Besides CD25, a number of other cell surface molecules, including CTLA-4 (9), glucocorticoid-induced TNF receptor (GITR) (10, 11), LAG-3 (12), or integrin {alpha}Ebeta7 (CD103) (13, 14), have been reported to identify T cells harboring suppressive activity. However, all of these markers are also expressed on subsets of conventional T cells or upon their activation and, therefore, are not suitable for the isolation and characterization of Treg cells, especially from sites of ongoing immune responses. Neuropilin-1 (Nrp1), a molecule more usually associated with axon guidance, was very recently identified by our group to be constitutively expressed by natural CD4+CD25+ Treg cells and, interestingly, is down-regulated on conventional T cells upon activation (15).

Whereas thymic origin of naturally occurring Foxp3+CD4+CD25+ Tregs is well established, naive Foxp3–CD4+CD25– T cells can also be converted in the periphery into Foxp3+ Treg cells (16, 17, 18). In sublethally irradiated wild-type hosts, the homeostatic expansion of CD4+CD25– T cells leads to the partial conversion into CD4+CD25+ Foxp3-expressing cells with potent suppressor activity (19). Furthermore, naive CD4+CD25– T cells can be instructed in vivo to become Foxp3-expressing CD25+ suppressor cells by chronic Ag exposure (16). However, both regulatory mechanisms and signals underlying the transcriptional control of Foxp3 expression as well as the consecutive conversion of naive CD4+CD25– T cells into de facto Foxp3+ Treg cells remain to be elucidated in more detail.

It is well established that Treg cells of different origins were capable of interfering with a broad variety of immune responses upon adoptive transfer (20, 21, 22, 23). Most recently, Loser et al. (24) have shown that by using Foxp3 transfection, Treg cells can be in vitro generated from naive T cells in numbers sufficient enough for the in vivo treatment of severe T cell-mediated allergic contact hypersensitivity (CHS) responses of the skin.

In the present study, we have identified G protein-coupled receptor (GPR)83 as a cell surface molecule that is selectively up-regulated by both murine and human Treg cells. GPR83 (also termed GIR, human GPR72) is an orphan GPR most homologous to the tachykinin and neuropeptide Y (NPY) receptors (25, 26). It was originally identified among genes induced by glucocorticoids and cAMP in the T cell line WEHI-7TG (27), and later it was shown to be induced by dexamethasone in murine thymocytes (28). It encodes a putative seven-transmembrane-domain protein (25) that is highly expressed in mouse brain and thymus (27, 29). Interestingly, GPR83 turned out to be functionally involved in mediating suppressive activity in vivo. Transfer of GPR83-transduced, naive CD4+CD25– T cells was able to inhibit the effector phase of a severe CHS reaction of the skin, indicating that GPR83 itself or GPR83-mediated signals conferred suppressive activity to conventional CD4+ T cells in vivo. Reanalysis of these GPR83-transduced T cells from protected mice revealed the conversation of Foxp3– into Foxp3+ CD4+ T cells in vivo. Interestingly, the in vivo environment alone seems not to be sufficient for the induction of Foxp3+ because GPR83-transduced CD4+ T cells transferred into healthy control mice or Ag-specific GPR83-infected T cells transferred into recipients, followed by immunization with the cognate Ag remained Foxp3–. These results suggest that GPR83 is critically involved in the peripheral conversion to Foxp3+ Treg cells in an inflamed environment in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

TCR-hemagglutinin (HA) transgenic mice (30), DO11.10 TCR transgenic mice (31), BALB/c mice (Harlan), C57BL/6 mice (Harlan), and IL-10tm1Cgn mice (IL-10 knockout (KO), deficient in IL-10; The Jackson Laboratory) were housed and bred under specific pathogen-free conditions. B6.PL mice (C57BL/6 Thy-1.1+) were kindly provided by the Bundesinstitut für Risikoforschung. All animal experiments were performed in accordance with institutional, state, and federal guidelines.

Antibodies

The mAb 6.5 (anti-TCR-HA) was purified from hybridoma supernatant and used in FITC-labeled form. Anti-CD3 (2C11), anti-CD28 (37.51), anti-CD4 (L3T4), anti-CD25 (PC61), anti-CD8 (53-6.7), anti-CD44 (IM7), and anti-Thy-1.2 were obtained from BD Biosciences; anti-D011.10 TCR (KJ1.26) from Caltag Laboratories; and anti-Foxp3 (FJK-16s) from eBioscience, and were used unlabeled or as FITC, allophycocyanin, CyChrome, or PE conjugates.

Cell separation and flow cytometry

Murine CD4+CD25– were enriched from the whole spleen by negative selection using an AutoMACS (Miltenyi Biotec). Human CD4+CD25+ and CD4+CD25– T cells were separated from PMBCs using the regulatory human T cell isolation kit and an AutoMACS separation unit (Miltenyi Biotec) following the manufacturer’s instructions. Purity of the enriched cell fractions was >90%, as determined by flow cytometry. For gene expression analysis, proliferation and adoptive transfer experiment labeled cells were separated using a MoFlow cell sorter (DakoCytomation), and purity was >97%. Foxp3 staining was performed using the PE anti-Foxp3 staining kit from eBioscience, according to the manufacturer‘s recommendations. Flow cytometry analyses were done on a FACSCalibur flow cytometer with CellQuest software (BD Biosciences).

T cell activation

Splenic CD4+CD25– T cells or CD4+CD25+ T cells from BALB/c were FACS sorted and cultured in the presence of 0.75 µg/ml anti-CD3 (plate bound), 1 µg/ml anti-CD28 (soluble), and 50 U/ml IL-2. Different time points after stimulation, cells were recovered for RNA preparation. Alternatively, CD4+CD25– splenocytes from BALB/c, C57/BL6, IL-10KO, or DO11.10 mice were stimulated with 0.75 µg/ml anti-CD3 (plate bound) and 1 µg/ml anti-CD28 (soluble) for 48 h prior retroviral infection. For Ag-specific T cell stimulation, RBC-depleted splenocytes from TCR-HA mice were stimulated with 10 µg/ml HA110–120 for either 16 h or 3 days, respectively. Subsequently, cells were harvested; labeled with anti-CD4, anti-CD25, and 6.5 (anti-TCR-HA); sorted; and used for RNA preparation.

Retroviral infection

cDNA encoding murine GPR83 or Foxp3 was amplified by RT-PCR from mouse CD4+CD25+ sorted splenocytes or whole spleen, respectively, using specific primers (GPR83, 5'-GGA GCT CAG CCC TTG TGC-3' and 5'-TTG TGC CTG TTC TTT TCT GAG C-3'; and Foxp3, 5'-GGA CAA GGA CCC GAT GCC CAA CC-3' and 5'-CCC TGC CCC CAC CAC CTC TGG-3'), cloned into pCR2.1 TOPO (Invitrogen Life Technologies), sequenced, and inserted into a murine stem cell virus-based retroviral vector encoding enhanced GFP (eGFP) under control of an internal ribosomal entry site. These constructs or the empty control vector were used to stably transfect the ecotropic GPE-86+ packaging cell line. Concentrated and filtrated (0.45 µm) retrovirus-containing culture supernatants supplemented with 20 mM HEPES and 8 µg/ml Polybrene were used to infect stimulated CD4+CD25– T cells by centrifugation at 500 x g for 2 h. Thereafter, cells were transferred to six-well plates and incubated at 37°C and 5% CO2. After 24 h, half of the culture medium was exchanged and 50 U/ml IL-2 was added.

Proliferation assay

A total of 5 x 104 sorted CD4+CD25+ and CD4+CD25– splenocytes isolated from BALB/c mice and 5 x 104 GPR83-transduced or control vector-infected T cells sorted 1 wk postinfection was cultured either alone or with 5 x 104 CD4+CD25– T cells isolated from BALB/c mice as responder in the presence of 2.5 x 105 irradiated BALB/c splenocytes as APCs with 1 µg/ml anti-CD3 for 72 h. Proliferation assays were performed in triplicates in 200 µl of IMDM medium containing 10% FCS. Cells were pulsed with 1 µCi/well [3H]thymidine for the final 8 or 18 h of the experiment, and [3H]thymidine incorporation was measured by scintillation counting.

Real-time RT-PCR

Total RNA was prepared from sorted cell populations using the RNeasy kit (Qiagen) following DNase digestion (Qiagen) and cDNA synthesis by Superscript II reverse transcriptase and oligo(dT) mixed with random hexamer primers (Invitrogen Life Technologies), according to the manufacturer’s recommendations. Real-time RT-PCR was performed in an ABI PRISM cycler (Applied Biosystems) using a SYBR Green PCR kit from Stratagene and specific primers for GPR83 (5'-ACC CTC CCC AGT TCC TTC CTT CAG-3' and 5'-GGC CAC AAC GGG TTC CAC AGA T-3'), Foxp3, IL-10, TGF-beta (32), Nrp1, and RPS9, as described previously (15).

Adoptive transfer of T cells

CHS experiments with BALB/c, C57BL/6, or B6.PL mice were performed, as described elsewhere (24). Briefly, mice were sensitized to 2,4-dinitro-1-fluorobenzene (DNFB) on day 0. On day 4, 1 x 106 sorted GPR83-, Foxp3-, control virus-transduced, or nontransduced CD4+CD25– T cells were injected i.v. into each recipient mouse 24 h before elicitation of CHS responses. For immunization, 100 µg of OVA-peptide/mouse emulsified in CFA was i.p. injected in wild-type mice 24 h after transfer of 2.5 x 106 sorted OVA-specific control virus or GPR83-infected naive T cells. Two days later, KJ1.26+CD4+ T cells were analyzed for Foxp3 expression.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
GPR83 is highly expressed by Treg cells of different origin

We initially sought to define a general Treg signature, a set of genes specifically expressed by naturally occurring polyclonal and Ag-specific Treg cells. For this purpose, we performed extensive gene expression profiling of naturally occurring polyclonal Foxp3+CD4+CD25+ Treg cells isolated from BALB/c mice, monoclonal Foxp3+CD4+CD25+ Treg cells of known Ag specificity isolated from TCR-HA mice, as well as CD4+ T cells recently activated with their specific Ag to their naive Foxp3–CD4+CD25– T cell counterpart using whole genome Affymetrix MOE430 microarrays. By this approach, we identified genes that are coregulated with Foxp3, i.e., that are highly expressed on monoclonal and polyclonal Treg cells without being up-regulated upon T cell activation (15). Among these genes associated with Foxp3-dependent transcriptional control in naturally occurring Treg cells, we found the GPR83 to be coexpressed with Foxp3. These findings are well in line with recently published microarray data of Treg cells identified by a fluorescent protein reporter knocked in the Foxp3 locus (3). To investigate the coregulation of GPR83 and Foxp3 in more detail, we quantified GPR83 mRNA amounts in Foxp3+ polyclonal and Ag-specific CD4+CD25+ Treg cells in comparison with their naive or recently activated CD4+CD25– counterparts by real-time RT-PCR. As shown in Fig. 1A, GPR83 was found to be highly up-regulated in naturally occurring Foxp3+ Treg cells (11-fold) and Ag-specific CD4+CD25+Foxp3+ Treg cells (5-fold) in contrast to recently activated T cells, which even show a 2- to 10-fold down-regulation of GPR83 mRNA.


Figure 1
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  		FIGURE 1. GPR83 is up-regulated in Treg cells of different origin. Total RNA was prepared from sorted freshly isolated, retrovirally infected, or in vitro-activated T cell populations, as indicated, and reverse transcribed, and mRNA expression levels of GPR83 were analyzed by real-time RT-PCR. Relative mRNA amounts were normalized with respect to expression levels in naive, unstimulated, or control (RV-eGFP) counterparts (fold change set to 1). RPS9 mRNA expression served as a housekeeping control. Results are from pooled individual mice (n ≥ 3). A, Sorted CD4+CD25+, CD4+CD25–, and Ag-stimulated HA-specific T cells were isolated from TCR-HA mice and analyzed for GPR83 expression by real-time RT-PCR in comparison with CD4+CD25+, CD4+CD25– T cells isolated from BALB/c mice. B, Normalized GPR83 expression in Foxp3-encoding retrovirus or control virus-infected naive T cells as well as sorted CD4+CD25+ and CD4+CD25– T cells isolated from BALB/c mice. C, CD4+CD25+ and CD4+CD25– T cells were isolated from BALB/c mice and stimulated in vitro with anti-CD3, anti-CD28, and IL-2 for 24, 48, and 96 h, respectively, before GPR83 expression analysis. D, GPR83 expression in sorted double-negative (CD4–CD8–) (fold change = 1), double-positive (CD4+CD8+), and single-positive (CD4+CD8–; CD4–CD8+) thymocytes isolated from BALB/c mice.

 
We next analyzed whether ectopic expression of Foxp3 in naive CD4+CD25– T cells induces GPR83 expression in these cells. To this end, naive CD4+CD25– T cells were infected with a Foxp3-encoding retrovirus conferring regulatory function to the infected cells (data not shown). Real-time RT-PCR analysis indicated elevated GPR83 levels in Foxp3-transduced T cells that were similar to those observed in the naturally occurring Treg cells (Fig. 1B). Extending our analysis to activated polyclonal CD4+CD25– T cells revealed that GPR83 mRNA expression is further down-regulated upon stimulation in vitro. In contrast, naturally occurring CD4+CD25+ Treg cells exhibited a 3-fold increase in GPR83 mRNA content 24 h upon T cell stimulation (Fig. 1C).

Most recently, it was shown that GITR, CTLA-4, and Foxp3 expression is initiated at the double-positive stage of thymic development; thus, Treg cells seem to be positively selected at the CD4+CD8+ differentiation stage (33, 34). In line with these reports, we could detect increasing GPR83 expression levels along thymic development and elevated GPR83 expression in the double-positive compartment (6-fold up-regulation in comparison with the double-negative stage) and CD4+ single-positive stage (17-fold up-regulation) in contrast to double-negative and CD8+ single-positive thymocytes (Fig. 1D).

In summary, we could clearly demonstrate that GPR83 is predominantly expressed by naturally occurring polyclonal, Ag-specific, and Foxp3-transduced Treg cells in contrast to naive and recently activated ones, thereby exhibiting a similar expression pattern as Foxp3 also during development of Treg cells in the thymus.

Considering the GPR83 expression profile in murine Treg cells, the question arises whether GPR83 is regulated in a similar fashion in human CD4+CD25+ Treg cells. For this purpose, we isolated CD4+CD25+ and CD4+CD25– T cells from peripheral blood of seven healthy donors by MACS sorting and analyzed GPR83 and Foxp3 expression by real-time RT-PCR. As shown in Fig. 2, GPR83 is 2- to 7-fold up-regulated in all individual human CD4+CD25+ Treg cell populations analyzed and also coregulated with Foxp3 much like it was shown above for murine Treg cells (Fig. 1).


Figure 2
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  		FIGURE 2. GPR83 expression in human CD4+CD25+ Treg cells. MACS-sorted CD4+CD25+ and CD4+CD25– T cells were isolated from seven healthy donors and analyzed with a pool of eight healthy donors for GPR83 and Foxp3 expression by real-time RT-PCR. Relative expression levels in CD4+CD25+ T cells were normalized to CD4+CD25– T cells (fold change = 1). RPS9 served as housekeeping control.

 
In vitro characterization of GPR83-transduced naive T cells

To better define the biological function of GPR83 expression by Treg cells, we constructed murine stem cell virus-based retroviral vectors encoding GPR83 and eGFP under control of an internal ribosomal entry side (RV-GPR83). In addition, an empty control vector was generated that contained only eGFP (RV-eGFP) (Fig. 3A). Retroviral vectors were stably transfected into GPE-86+ packaging cells, and virus-containing supernatants were used to infect naive CD4+CD25– T cells.


Figure 3
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  		FIGURE 3. In vitro analysis of GPR83-transduced CD4+CD25– T cells. A, Schematic drawing of MCSV-based retroviral vector constructs encoding GPR83 and eGFP under control of an internal ribosomal entry side (RV-GPR83) and the empty control vector (RV-eGFP). B, Sorted CD4+CD25+ and CD4+CD25– T cells isolated from BALB/c mice or sorted eGFP+ T cells 1 wk postinfection with retroviral vectors encoding GPR83 and eGFP (RV-GPR83) or the control vector (RV-eGFP) were cultured alone (left) or cocultured with CD4+CD25– T cells (right) in the presence of irradiated APCs with ({blacksquare}) or without ({cjs2108}) 1 µg/ml anti-CD3 for 72 h. Proliferation was measured by [3H]thymidine incorporation; the data represent one of three independent experiments as mean from triplicate wells. C, Real-time RT-PCR analysis for GPR83, Foxp3, Nrp1, IL-10, and TGF-beta was performed using reversely transcripted RNA isolated from sorted eGFP+ GPR83-transduced (RV-GPR83), Foxp3-transduced (RV-Foxp3) naive T cells 1 wk postinfection and freshly isolated CD4+CD25+ T cells. Expression levels in GPR83-transduced and Foxp3-transduced T cells were normalized for each gene analyzed with respect to expression levels in control virus-infected cells (fold change = 1), whereas expression in CD4+CD25+ T cells was normalized to CD4+CD25– T cells. RPS9 mRNA expression served as housekeeping gene control. Mean values from at least two independent experiments are shown.

 
One week postinfection, eGFP+ T cells (~20%) were FACS sorted, resulting in 99% purity, as determined by FACS reanalysis (data not shown). To address whether GPR83-transduced T cells have acquired characteristics of naturally occurring CD4+CD25+ Treg cells, we performed in vitro proliferation assays and investigated the suppressive capacity in coculture experiments. Naive T cells infected with RV-eGFP served as controls. As shown in Fig. 3B (left panel), GPR83-transduced T cells exhibited proliferative capacity comparable to freshly isolated CD4+CD25– naive T cells, whereas CD4+CD25+ Treg cells showed an anergic phenotype. Furthermore, GPR83-transduced T cells in contrast to naturally occurring CD4+CD25+ Treg cells were not able to inhibit proliferation of naive CD4+CD25– T cells in coculture experiments (Fig. 3B, right panel). Similar results were obtained upon allogenic stimulation in an MLR type assay system (data not shown). Thus, GPR83 transduction did not confer suppressive capacity in vitro. Moreover, when we analyzed the expression of several genes associated with Treg cell function by quantitative real-time RT-PCR from the retroviral infected cells, we observed that overexpression of GPR83 did not result in an increase of Foxp3, Nrp1, and TGF-beta mRNA expression, but induced a 10-fold up-regulation in IL-10 mRNA (Fig. 3C). Reanalysis of GPR83-infected T cells upon allogenic stimulation cocultured with or without congenic naive T cells in the course of an MLR revealed also no induction in Foxp3 expression (data not shown).

GPR83-transduced naive T cells acquire suppressive activity in vivo

It might be possible that GPR83 itself or GPR83-mediated signals confer suppressive activity to conventional CD4+ T cells only under conditions encountered in vivo, as mechanisms used by Treg cells to interfere with ongoing immune responses are much more complex (35). We therefore examined the capacity of GPR83-transduced T cells to inhibit the effector phase of a CHS reaction leading to severe skin inflammation that is T cell mediated and dependent on dendritic cells (DCs) (24, 36).

Groups of naive BALB/c mice were epicutaneously sensitized to DNFB; i.v. injected with GPR83-transduced (RV-GPR83), Foxp3-transduced (RV-Foxp3), and control virus-infected (RV-eGFP) or noninfected (mock) T cells; and subsequently ear challenged with DNFB. Ear swelling was assessed as a measure of CHS response. As shown in Fig. 4A, mice treated with mock-infected naive CD4+CD25– T cells or control virus-infected T cells showed a normal CHS response upon challenge. Interestingly, mice that were adoptively transferred with GPR83-transduced T cells developed a significantly reduced CHS response, which was comparable to the group receiving Foxp3-transduced T cells (RV-Foxp3).


Figure 4
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  		FIGURE 4. GPR83-infected CD4+CD25– T cells inhibit the effector phase of severe CHS. Animals were sensitized with DNFB; i.v. injected with 1 x 106 noninfected (mock), Foxp3-transduced (RV-Foxp3), GPR83-transduced (RV-GPR83), or control virus-transduced (RV-eGFP) CD4+CD25– T cells isolated from BALB/c (A) or IL-10KO (B) mice; and ear challenged. As negative control, mice were not sensitized but challenged, and as positive control, mice were sensitized and challenged without injecting any cells. Ear swelling was evaluated 36 h after challenge and is expressed as difference between the challenged right ear and the unchallenged left ear. Data are shown as mean ± SD of eight mice in two independent experiments. Student’s t test was used to assess the significance of differences.

 
The ability of GPR83-transduced cells to inhibit T cell responses in the CHS reaction could be related to their capacity to make IL-10 (Fig. 3C). We therefore studied the influence of this immunosuppressive cytokine on the reduced CHS response observed in the skin after transfer of GPR83-transduced T cells (Fig. 4A). To this end, MACS-sorted CD4+CD25– T cells isolated from IL-10-deficient (IL-10KO) mice were activated in vitro and infected with GPR83-, Foxp3-encoding, or control retroviruses. Six days postinfection, these transduced T cells were analyzed for their capacity to inhibit the CHS response. As shown in Fig. 4B, IL-10-deficient, GPR83-transduced CD4+ T cells were able to significantly reduce the CHS response comparable to GPR83-transduced T cells from wild-type mice.

Active suppression in vivo was accompanied by the conversion of GPR83-transduced Foxp3– into Foxp3+ T cells

To elucidate the molecular mechanism by which GPR83-infected naive CD4+ T cells acquired their suppressive capacity in vivo, we analyzed Foxp3 expression in GPR83-transduced and control virus-infected CD4+ T cells reisolated from mice undergoing the CHS response as well as healthy recipients.

For this purpose, C57BL/6 Thy-1.1+ (B6.PL) mice were sensitized with DNFB and i.v. injected with 7 x 106 GPR83-transduced (RV-GPR83) or control virus-infected (RV-eGFP) CD4+Thy-1.2+ congenic T cells. We could not detect any Foxp3 expression by both infected T cell populations before adoptive transfer as determined by FACS analysis shown in Fig. 5 (upper panel, left). Two days after ear challenge with DNFB, Foxp3 expression was again quantified by FACS analysis on CD4+Thy-1.2+ T cells reisolated from the draining lymph nodes as well as the spleen. As depicted in Fig. 5A, ~20% of the GPR83-transduced T cells become Foxp3+ in the draining lymph node, in contrast to control virus-infected T cells. Interestingly, we could also observe an induction of Foxp3 expression to the same extent in GPR83-transduced T cells reisolated from the spleen and unaffected mesenteric lymph nodes (MLN) (Fig. 5A). Therefore, we wondered whether the in vivo environment alone is sufficient to induce Foxp3 expression in GPR83-transduced T cells rather than inflammatory conditions. However, transfer of 7 x 106 GPR83- or control virus-infected CD4+Thy-1.2+ T cells in congenic Thy-1.1+ wild-type mice and reisolation at day 3 did not confer any Foxp3 protein expression in GPR83-transduced T cells, as shown in Fig. 5B. To investigate the in vivo induction of Foxp3+ Treg cells by GPR83 overexpression in more detail, we transferred 2.5 x 106 OVA-specific control virus or GPR83-infected Foxp3–KJ1.26+CD4+ T cells in wild-type mice before immunization with the cognate OVA peptide in CFA (Fig. 5C, upper panel, right). Reanalysis of the Ag-specific, retroviral infected T cell subsets isolated from cervical lymph nodes (CVLN) and MLN as well as the spleen of immunized mice exhibited no significant up-regulation of Foxp3 in Ag-specific GPR83-transduced T cells (Fig. 5C).


Figure 5
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  		FIGURE 5. In vivo suppression during inflammatory immune responses involves Foxp3 conversion in GPR83-transduced T cells. Intracellular Foxp3 stainings of sorted eGFP+ control virus (RV-eGFP) or GPR83-transduced (RV-GPR83) CD4+ T cells isolated from C57BL/6 Thy-1.2+ mice (upper panel, left) or KJ1.26+ T cells isolated from DO11.10 mice (upper panel, right) 1 wk upon infection. A, C57BL/6 Thy-1.1+ mice were sensitized with DNFB, i.v. injected with 7 x 106 GPR83-transduced (RV-GPR83) or control virus-infected (RV-eGFP) C57BL/6 Thy-1.2+CD4+ T cells, and ear challenged. Forty-eight hours postchallenge with DNFB, CVLN, splenocytes (spleen), and MLN cells were isolated and analyzed for Thy-1.2 and Foxp3 expression by flow cytometry. B, 7 x 106 GPR83-transduced (RV-GPR83) or control virus-infected (RV-eGFP) C57BL/6 Thy-1.2+CD4+ T cells were i.v. injected in healthy C57BL/6 Thy-1.1+ mice. At day 3, Foxp3 and Thy-1.2 expression was analyzed by intracellular FACS staining. C, Wild-type BALB/c mice were immunized with OVA-peptide/CFA 1 day after transfer of 2.5 x 106 eGFP+KJ1.26+ control virus (RV-eGFP) or GPR83-transduced (RV-GPR83) T cells. After 48 h, Foxp3 expression was assessed on KJ1.26+ T cells reisolated from CVLN, spleen, and MLN by flow cytometry.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the present study, we could identify GPR83 as a cell surface molecule selectively up-regulated by Treg cells of both murine and human origin, which seems to be directly involved in mediating Treg cell function. Moreover, we could demonstrate the induction of GPR83 upon ectopic expression of Foxp3 within peripheral, naive CD4+ T cells, indicating that GPR83 is linked to the genetic program orchestrated by Foxp3 in Treg cells. These data are well in line with more indirect studies between Foxp3 and GPR83 expression in genetically tagged, transgenic mice, expressing a chimeric GFP-Foxp3 fusion protein. Foxp3gfp+ T cells isolated from these mice also exhibited elevated levels of GPR83 (3). Although CD4+CD25+ Treg cells represent a well-characterized population of T cells playing a pivotal role in the control of a broad spectrum of immune responses, the basic molecular mechanisms underlying thymic development remain mostly elusive. However, more recently it was shown that GITR, CTLA-4, and Foxp3 expression is initiated at the double-positive stage of thymic development; thus, thymic Treg cells seem to be positively selected at the CD4+CD8+ differentiation stage (33, 34). According to these reports, we could not detect GPR83 expression within bone marrow or in the early thymic double-negative compartment, whereas double-positive as well as CD4+ single-positive thymocytes exhibit elevated levels of GPR83 expression.

Several actual studies revealed the peripheral conversion of naive CD4+CD25– T cells into CD4+CD25+ Treg cells, suggesting that thymic selection seems not to represent the only mode by which Treg cells can be generated. In this study, we have illustrated an involvement of GPR83 in the peripheral induction of Treg cells in vivo. Ectopic expression of GPR83 in peripheral, naive T cells induces Foxp3 expression and confers suppressive capacity for a T cell-dependent immune response to a similar extent as Foxp3-transduced T cells.

In vitro studies have reported acquisition of suppressor activity and Foxp3 expression in the bulk populations of cultured murine and human CD4+CD25– T cells upon activation in the presence of nanomolar amounts of rTGF-beta1 (5, 37). Although these studies opened up an important avenue for pharmacologic in vitro manipulation of T cell function for potential therapeutic purposes, there is no evidence to date that such high concentrations of TGF-beta1 are ever attainable in vivo and that such a conversion of CD4+CD25– into CD4+CD25+ T cells expressing Foxp3 can occur under physiologic conditions or in the course of autoimmune and inflammatory disease. In another approach, extrathymic de novo generation of Treg cells was achieved by prolonged s.c. infusion of low doses of peptide by means of osmotic pumps. By this, mature T cells converted into CD4+CD25+ Treg cells in vivo that confer specific immunologic tolerance upon challenge with Ag (16). Interestingly, we could also detect high levels of both Foxp3 and GPR83 within these cells (data not shown). Furthermore, Mahnke et al. (17) have reported that Abs directed against DEC-205 can target Ags to immature DCs. Presentation of Ags by these immature DCs leads to the generation of Treg cells capable of suppressing allergy (17) as well as autoimmune diabetes (32). Thus, a prerequisite for achieving the conversion of naive T cells into Treg cells in vivo is Ag presentation under subimmunogenic conditions and knowledge of the ligand or the Ag involved, unfortunately, not yet determined for most diseases encountered in the clinic. However, despite the existence of a number of protocols, which describe the peripheral generation of Treg cells from naive T cells in vivo, the molecular mechanisms governing this conversion, especially the induction of Foxp3 expression, remain still unknown.

Results of the present study suggest that GPR83 is involved in such a conversion of naive CD4+CD25– T cells into CD4+Foxp3+ Treg cells during inflammatory immune responses in vivo. Interestingly, despite exhibiting suppressive capacity under inflammatory conditions in vivo, overexpression of GPR83 within naive T cells does not confer regulatory function in vitro and does not induce Foxp3 expression per se or upon allogenic stimulation. Furthermore, neither the in vivo situation itself nor a single immunization with the cognate Ag is sufficient to initiate the conversion of Foxp3– to Foxp3+ Treg cells in vivo. This discrepancy suggests that GPR83 might represent a Treg cell-specific surface receptor, whose interaction with a yet unknown ligand, which is not present under in vitro or noninflammatory conditions in vivo, turns on Foxp3 expression and eventually leads to the observed suppressive capacity in vivo. In fact, our results suggest that the induction of Foxp3+ Treg cells by GPR83 overexpression depends on a more complex in vivo situation as it is true for CHS immune responses, known to be DC dependent, CD8+ as well as CD4+ T cell derived, and mediated by a broad variety of different cytokines (36). As shown in Fig. 5, we could detect conversion of GPR83-transduced CD4+Foxp3– T cells into CD4+Foxp3+ T cells not only in the draining lymph nodes, but also in the spleen and unaffected lymph nodes during the CHS response. One might speculate that the induction of Treg cells occurs directly at the side of immune response, within the inflammatory environment, followed by emigration to the periphery. This assumption was supported by the fact that neither GPR83-transduced T cells adoptively transferred to healthy congenic recipients nor Ag-specific GPR83-overexpressing T cells transferred to wild-type mice followed by a single immunization with the cognate Ag undergo conversion with regard to Foxp3 expression. Preliminary data by Fontenot et al. (38) from GPR83-deficient mice do not provide any hint toward the role of GPR83 in autoimmune-related phenotypes. Keeping our results in mind, it would be interesting to analyze the effect of GPR83 deficiency in the context of inflammation. However, identifying molecules that interact with and/or trigger GPR83 is clearly a priority.

GPR83 belongs to the family of GPR with high similarities to the tachykinin receptors (25) and closely related to the Y receptor family (26). One might speculate about NPY-like molecules as putative ligands for GPR83 due to its predicted function as a NPY receptor. NPY is distributed throughout the whole body and participates in the regulation of multiple physiological and psychological processes, and has also been described to play a role in neuroimmune interactions (39, 40). Kawamura et al. (41) have shown that NPY inhibits the IFN-{gamma} production by Th1 clones and of freshly isolated T cells. Furthermore, it was demonstrated that exogenous NPY significantly suppresses the clinical course of experimental autoimmune encephalomyelitis (39). T cells isolated from these protected mice exhibit low IFN-{gamma} production as well as reduced proliferative capacity. One might figure out a mechanism by which NPY interacts with GPR83-positive T cells, resulting in an up-regulation of Foxp3 and thereby generating Treg cells, enabling the suppressive effect demonstrated in the experimental autoimmune encephalomyelitis model.

In summary, we could demonstrate that GPR83 is directly linked to Foxp3 expression. All different Foxp3+ T cell subsets analyzed in this study exhibited elevated levels of GPR83. Retroviral expression of Foxp3 induces GPR83 transcription in peripheral naive T cells. Strikingly, transduction of GPR83 confers suppressive activity in vivo and results in Foxp3 induction upon an assumed triggering with a to date unknown ligand present under inflammatory conditions. The identification of this ligand or the generation of a stimulatory anti-GPR83 Ab might facilitate the development of improved strategies for the treatment of a broad spectrum of inflammatory and autoimmune diseases; blocking this receptor might also be an attractive target to interfere with the induction of Treg cells through tumors.


   Acknowledgments
 
We are very grateful to Michael Probst-Kepper for critical discussion of the manuscript; Robert Geffers and Tanja Toepfer for the microarray analysis; Lothar Groebe for cell sortings; Silvia Prettin for excellent technical assistance; Bastian Pasche for providing the IL-10-deficient mice; and staff of the animal facility at the Gesellschaft für Biotechnologische Forschung for mouse colony management.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Deutsche Forschungsgemeinschaft Grants SFB621 (to J.B. and D.B.), SFB650 (to J.H.), and SFB293 (to S.B.). Back

2 Address correspondence and reprint requests to Dr. Jan Buer, Department of Mucosal Immunity, German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany. E-mail address: jab{at}gbf.de Back

3 Abbreviations used in this paper: Treg, regulatory T; CHS, contact hypersensitivity; CVLN, cervical lymph node; DC, dendritic cell; DNFB, 2,4-dinitro-1-fluorobenzene; eGFP, enhanced GFP; GITR, glucocorticoid-induced TNF receptor; GPR, G protein-coupled receptor; HA, hemagglutinin; KO, knockout; MLN, mesenteric lymph node; NPY, neuropeptide Y; Nrp1, neuropilin-1. Back

Received for publication December 19, 2005. Accepted for publication April 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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