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The Journal of Immunology, 2007, 179, 8059-8068
Copyright © 2007 by The American Association of Immunologists, Inc.
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Chronic Antigen Stimulation In Vivo Induces a Distinct Population of Antigen-Specific Foxp3–CD25– Regulatory T Cells1

Wiebke Hansen2,*, Astrid M. Westendorf{dagger}, Simone Reinwald{dagger}, Dunja Bruder{dagger}, Stefanie Deppenmeier{ddagger}, Lothar Groebe{dagger}, Michael Probst-Kepper§, Achim D. Gruber{ddagger}, Robert Geffers{dagger} and Jan Buer*

* Institute of Medical Microbiology, University Hospital Essen, Essen, Germany; {dagger} Department of Mucosal Immunity, Helmholtz Centre for Infection Research, Braunschweig, Germany; {ddagger} Department of Veterinary Pathology, Free University, Berlin, Germany; and § Junior Research Group for Xenotransplantation, Department of Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The concept of immune regulation/suppression has been well-established and, besides thymus-derived CD4+CD25+ regulatory T (TR) cells, it became clear that a variety of additional peripherally induced TR cells play vital roles in protection from many harmful immune responses including intestinal inflammation. In the present study, we have analyzed in vivo-induced Ag-specific CD4+ TR cells with respect to their molecular and functional phenotype. By comparative genomics we could show that these Ag-specific TR cells induced by chronic Ag stimulation in vivo clearly differ in their genetic program from naturally occurring thymus-derived CD4+CD25+ TR cells. This distinct population of induced TR cells express neither CD25 nor the TR-associated transcription factor Foxp3. Strikingly, CD25 is not even up-regulated upon stimulation. Despite the lack in Foxp3 expression, these in vivo-induced CD25– TR cells are able to interfere with an Ag-specific CD8+ T cell-mediated intestinal inflammation without significant increase in CD25 and Foxp3 expression. Thus, our results demonstrate that in vivo-induced Ag-specific TR cells represent a distinct population of Foxp3–CD25– TR cells with regulatory capacity both in vitro and in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The immune system is a highly effective and dynamic cellular network that has been evolved to protect the host from a huge variety of foreign, potentially harmful microorganisms. To avoid self-destructive immune responses, one of the major tasks of the immune system is to discriminate between self and nonself. Peripheral tolerance to self-Ags, in addition to clonal deletion and induction of anergy, involves active suppression mediated by regulatory T (TR)3 cells (1, 2, 3).

A number of different types of TR cells have been described (4, 5, 6), and these may, for convenience, be defined as natural or induced TR cells. The naturally occurring CD4+CD25+ TR cells are the best established subset present among thymocytes and peripheral T cells playing a pivotal role in the prevention of autoimmunity (7, 8, 9). Among the induced TR cells, a broad variety of different subsets have been described including T regulatory cell type 1 cells (10) or Th3 cells (11) differing from naturally occurring CD4+CD25+ TR cells by acting in a cell-contact independent, but cytokine-dependent, manner in vitro. Like the naturally occurring CD4+CD25+ TR cells, the heterogeneous populations of induced TR cells exhibit suppressive capacity both in vitro and in vivo, show no proliferation upon stimulation in vitro, and produce low levels of IL-2. They can be generated from naive T cells by deliberate administration of Ag in the presence or absence of immunomodulating cytokines such as IL-10 or TGF-β (12) or Ag targeting via the DEC205 mAb to immature DCs (13, 14). Even chronic Ag stimulation in vivo by prolonged s.c. infusion of low doses of hemagglutinin (HA) peptide by means of osmotic pumps can transform mature Ag-specific T cells into regulatory CD4+CD25+ T cells (15).

Chronic Ag stimulation in mice transgenic for the MHC class II-restricted TCR specific for HA, with concomitant HA expression on hemopoietic cells as a self-Ag (TCR-HA x Ig-HA mice), does not lead to thymic deletion of HA-specific T cells by negative selection. In contrast, Ag-specific T cells accumulate in the periphery (16). These HA-specific CD4+ T cells (detected by the clonotypic 6.5 mAb) have been described to be "anergic" in terms of in vitro-induced proliferation and secrete significant levels of IL-10 (17). It has been shown that the whole CD4+6.5+ T cell population isolated from TCR-HA x Ig-HA mice suppressed tissue destruction by naive CD4+6.5+ T cells (isolated from TCR-HA mice) (18) and, most recently, we could demonstrate that these CD4+6.5+ TR cells are able to reduce wasting disease induced by HA-specific CD8+ T cells in mice expressing the cognate Ag under control of the gut-specific Villin promoter (19). It has been suggested that the specific expression of the cognate Ag by nonactivated APCs in the hemopoietic system in these double-transgenic (dtg) TCR-HA x Ig-HA mice resulted mostly in CD4+CD25– TR cells, which could be differentiated from mature Ag-specific T cells in the absence of a functioning thymus and without tutoring by other T cells (20).

Over the last few years, several molecules such as CTLA-4, glucocorticoid-induced TNFR (GITR), CD103, neuropilin 1 (Nrp1), and G protein-coupled receptor 83 (GPR83) were shown to be highly expressed by naturally occurring CD4+CD25+ TR cells and therefore might be important for their function (21, 22, 23, 24, 25). Until now, little has been known about the expression pattern of these molecules in the heterogenous population of induced TR cells. The transcription factor Foxp3, assumed to function as a TR cell lineage specification factor, is expressed at relatively high levels in TR cells and was shown to orchestrate their development and function (26, 27, 28). Mutations of the Foxp3 gene in both mice and humans are associated with severe immunopathology (29, 30, 31). Whereas Foxp3 has clearly been shown to be expressed in naturally occurring, thymus-derived CD4+CD25+ TR cells at high levels, it remains to be clarified to which extent in vivo-induced TR cells express Foxp3 and whether the level of Foxp3 expression is necessarily linked to the regulatory phenotype of these cells.

In this study, we demonstrate that chronic Ag stimulation in vivo in TCR-HA x Ig-HA dtg mice induces a distinct subset of Ag-specific CD4+Foxp3–CD25– TR cells with regulatory capacity both in vitro and in vivo. This distinct TR cell population exhibits a quite different genetic program compared with naturally occurring CD4+CD25+ TR cells; most strikingly, they do not express elevated levels of CD25 and the TR-associated transcription regulator Foxp3. Even upon stimulation in vitro or in vivo, no significant increase in CD25 and Foxp3 expression could be observed suggesting that chronic Ag stimulation in vivo leads to the generation of a new member of the TR cell family.


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

TCR-HA-transgenic mice express a TCR{alpha}β specific for the peptide 110–120 from influenza HA presented by I-Ed (32). Ig-HA mice express the HA transgene under the control of the Ig{kappa} promoter and enhancer elements in hemopoietic cells (16). Villin-HA mice express the HA transgene under control of the gut-specific Villin promoter (33). CL4-TCR-transgenic mice express a TCR{alpha}β that recognizes the HA-peptide 512–520 presented by MHC class I (H-2Kd) (34). Mice were bred and maintained in the animal facility at the Helmholtz Centre for Infection Research under specific pathogen-free conditions. BALB/c mice were obtained from Harlan Breeders. All animal experiments were performed in accordance with institutional, state, and federal guidelines.

Abs and flow cytometry

The mAb 6.5 (anti-TCR-HA) was purified from hybridoma supernatant. Anti-CD3 (2C11), anti-CD28, anti-CD4 (GK1.5), anti-CD25 (PC61), anti-PD1 (J43), anti-Klrg1 (2F1), anti-CTLA4 (UC10–4F10-11), anti-CD103 (M290), anti-IL-2 (JES6-5H4), and anti-IFN-{gamma} (XMG1.2) were obtained from BD Biosciences, anti-GITR was purchased from R&D Systems. Abs were used unlabeled or as biotin, FITC, PE, or CyChrome conjugates. For intracellular IL-2, IFN-{gamma}, and CTLA4 staining, cells were stimulated with 0.01 µg/ml PMA and 1 µg/ml ionomycin in the presence of 5 µg/ml brefeldin A. After 4 h, cells were fixed with 2% paraformaldehyde for 15 min and permeabilized with 0.1% Nonidet P40 for 4 min before intracellular staining. Foxp3 staining was performed using the PE-coupled anti-Foxp3 (FJK-16s) staining kit from eBioscience, according to the manufacturer’s recommendations. Flow cytometric analyses were done on a FACSCalibur with CellQuest software (BD Biosciences). For gene expression profiling and proliferation experiments, labeled cells were separated with a MoFlow cell sorter (Cytomation) and purity was >97%.

Cytometric bead array (CBA)

Quantification of cytokines in culture supernatants was done using the CBA kit (BD Bioscience). Data acquisition was performed by flow cytometry using the FACSCalibur. Acquired data were analyzed by BD Biosciences CBA software.

Preparation of lamina propria lymphocytes (LPL)

LPL were isolated as described previously (33). Briefly, the small intestine was cut into small pieces followed by sequential stirring in medium to remove mucus and the epithelial layer. LPL were released by digestion at 37°C with collagenase. Lymphocytes were collected by density centrifugation.

Proliferation assay

For antigenic stimulation, 2.5 x 104 sorted 6.5+CD4+CD25+ and 6.5+CD4+CD25– T cells isolated from TCR-HA mice as well as from TCR-HA x Ig-HA-transgenic mice were cultured alone or cocultured with 2.5 x 104 6.5+CD4+CD25– T cells isolated from TCR-HA mice in the presence of irradiated 2.5 x 105 BALB/c splenocytes with or without 10 µg/ml of the MHC class II HA-peptide HA 110–120 for 72 h. In some coculture experiments, a transwell system with 0.2-µm pore size (Nunc) was used or assays were supplemented with 50 µg/ml anti-IL-10 (JES5-2A5; BioSource International) or 50 µg/ml anti-TGF-β-1,2,3 (1D11; R&D Systems). Proliferation assays were done in triplicate in 200 µl of IMDM medium. Cells were pulsed with 1 µCi/well [3H]thymidine for the final 6 h of the experiment and [3H]thymidine incorporation was measured by scintillation counting.

T cell activation

For Ag-specific T cell stimulation, RBC-depleted splenocytes from TCR-HA mice were stimulated with 10 µg/ml of the MHC class II HA 110–120 peptide for either 16 h (TCR-HA 16h TA) or 3 days (TCR-HA 3d TA), respectively. Subsequently, cells were harvested and CD4+6.5+CD25+ T cells were sorted and used for RNA preparation. For Foxp3 and CD25 expression analysis, sorted 6.5+CD4+CD25– T cells isolated from TCR-HA and TCR-HA x Ig-HA mice were stimulated in vitro with 10 µg/ml of their cognate Ag in the presence of 50 U/ml recombinant human IL-2 and irradiated BALB/c splenocytes as APCs for different time points before analyzing Foxp3 and CD25 expression by FACS on CD4+6.5+ T cells.

DNA microarray analysis

For RNA amplification, the first round was done according to Affymetrix without biotinylated nucleotides using the Promega P1300 RiboMax kit for T7 amplification. For the second round of amplification, the precipitated and cleaned aRNA was converted to cDNA using random hexamers (Pharmacia). Second-strand synthesis and probe amplification was done like in the first round except that there was incubation with RNase H before first strand synthesis to digest the aRNA and the use of the T7T23V oligo for initiation of the synthesis of the second strand. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5 µg of each biotinylated cRNA preparation were fragmented and placed in a hybridization mixture containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MOE430A for 16 h. After hybridization, the GeneChips were washed, stained, with streptavidin-PE and read using an Affymetrix GeneChip fluidic station and scanner. Analysis was done with gene expression software GCOS 1.2 (Affymetrix) and Genesis 1.6 (35).

Real-time RT-PCR

Total RNA was prepared from sorted T cells using the RNeasy kit (Qiagen) following cDNA synthesis by Superscript II Reverse Transcriptase (Invitrogen Life Technologies) and OligodT mixed with random hexamer primers according to the manufacturer’s recommendations. Real-time RT-PCR was done in an ABI PRISM cycler (Applied Biosystems) using a SYBR Green PCR kit from Stratagene and specific primers for CCL5 (5'-GCT CCA ATC TTG CAG TCG TGT TT-3' and 5'-GAC CGG AGT GGG AGT AGG GGA TTA-3'), IL-21 (5'-CCC GTG TAC CGC CCT AAG ATA-3'and 5'-AGC AGC AAA CTC AGC AAC CAA T-3'), GPR83 (5'-ACC CTC CCC AGT TCC TTC CTT CAG-3' and 5'-GGC CAC AAC GGG TTC CAC AGA T-3'), and RPS9 (5'-CTG GAC GAG GGC AAG ATG AAG C-3' and 5'-TGA CGT TGG CGG ATG AGC ACA-3'). A threshold was set in the linear part of the amplification curve, and the number of cycles needed to reach it was calculated for every gene. Relative mRNA level were determined by using included standard curves for each individual gene and further normalization to the housekeeping gene RPS9. Melting curves established the purity of the amplified band.

Membrane labeling of T cells with CFSE

Sorted T cells were resuspended in RPMI 1640 at a concentration of 1 x 107 cells/ml and incubated with 2.5 µM CFSE (Molecular Probes) for 8 min at 37°C. For additional incubation of 5 min, two volumes of FCS were added. After washing in complete medium followed by washing with PBS, CFSE-labeled cells were used for adoptive transfer experiments.

Adoptive transfer

CD8+ T cells were isolated from CL4-TCR splenocytes using the MACS CD8 T cell isolation kit according to the manufacturer’s recommendations (Miltenyi Biotec). For cotransfer experiments, splenocytes isolated from TCR-HA or TCR-HA x Ig-HA mice were stained with anti-6.5, anti-CD4, and anti-CD25 and separated by cell sorting. Purified cells were washed and resuspended in PBS. A total of 2 x 106 CD8+ T cells were injected i.v. either alone or coinjected with 2 x 106 6.5+CD4+CD25– T cells isolated from TCR-HA or TCR-HA x Ig-HA mice in Villin-HA-transgenic mice. Four or 14 days posttransfer, intestine probes were used for histological analysis and lymphocytes were isolated from the lamina propria (LPL); the mesenteric lymph nodes (MLN) and spleen were used for FACS analysis.

Histology

Organs were immersion fixed with buffered formalin, embedded in paraffin, sectioned at 4-µm thickness, and stained with H&E. The sections were evaluated in a blinded fashion by a board-certified pathologist (A. D. Gruber). The histopathological scoring was evaluated as described (19).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Chronic Ag stimulation in vivo induces CD4+CD25– TR cells with regulatory properties in vitro

It has been described that chronic Ag stimulation in vivo observed in transgenic mice expressing both a class II-restricted TCR-{alpha}/TCR-β specific for HA and HA under control of the Ig-{kappa} promoter (TCR-HA x Ig-HA) led to the accumulation of Ag-specific (6.5+) T cells in the periphery (16). Apostolou et al. (20) showed that not only the CD4+6.5+CD25+ T cell subset but also the CD4+6.5+CD25– T cells isolated from TCR-HA x Ig-HA mice exhibit a regulatory phenotype in vitro. To investigate the nature of these in vivo-induced Ag-specific TR cells in more detail, in particular their immune-regulatory and molecular properties, we first analyzed their proliferative and suppressive capacity in vitro. For this purpose, we isolated 6.5+CD4+CD25+ T cells (dtg CD25+ TR) and 6.5+CD4+CD25– T cells (dtg CD25– TR) from TCR-HA x Ig-HA dtg mice and compared their proliferative activity with Ag-specific thymus-derived 6.5+CD4+CD25+ TR cells (single-transgenic (stg) TR) and naive 6.5+CD4+CD25– T cells (stg naive T (TN) isolated from TCR-HA stg mice. To analyze whether an additional stimulus might influence the proliferative as well as suppressive properties of dtg CD25– TR cells isolated from TCR-HA x Ig-HA mice, we include dtg CD25– TR cells prestimulated with their cognate Ag overnight. For the sake of clarity, we use synonyms for the different T cell subsets analyzed in the present work which are summarized in Table I. Sorted T cells were stimulated in vitro with the cognate HA-peptide 110–120. Under these conditions, only stg TN cells proliferated in an Ag-specific manner, whereas stg TR cells, dtg CD25+ TR cells, and dtg CD25– TR cells do not exhibit proliferative capacity upon antigenic stimulation even if they were prestimulated overnight (Fig. 1A). Moreover, after in vitro stimulation, stg TR cells, dtg CD25+ TR cells, dtg CD25– TR cells, and prestimulated dtg CD25– TR cells were able to suppress proliferation of naive stg TN cells as depicted in Fig. 1B.


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  		Table I. Description of analyzed T cells with synonyms used within the present study

 

Figure 1
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  		FIGURE 1. Regulatory phenotype of in vivo-induced dtg CD25– TR cells. A, HA-specific dtg CD25– TR cells do not proliferate upon Ag stimulation in vitro. A total of 2.5 x 104 sorted stg TR and stg TN cells isolated from TCR-HA mice as well as dtg CD25+ TR, dtg CD25– TR isolated from TCR-HA x Ig-HA mice and dtg CD25– TR cells preactivated overnight (dtg CD25– TR stim.) were stimulated in the presence of 2.5 x 105 irradiated BALB/c splenocytes as APCs with 10 µg/ml HA peptide for 72 h. B, Ag-specific dtg CD25– TR cells suppress proliferation of naive stg TN cells. A total of 2.5 x 104 sorted stg TN cells were stimulated with 10 µg/ml HA peptide and 2.5 x 105 irradiated APCs either alone or in the presence of 2.5 x 104 stg TR cells, dtg CD25+ TR, dtg CD25– TR, or prestimulated CD25– TR cells at a ratio of 1:1. C, Suppressive capacity of dtg CD25– TR cells is cell-cell contact dependent. A total of 5 x 104 sorted naive stg TN cells isolated from TCR-HA mice were cocultured with 5 x 104 sorted dtg CD25– TR cells isolated from TCR-HA x Ig-HA mice or, D, with Ag-specific prestimulated dtg CD25– TR cells and were stimulated in the presence of APCs with 10 µg/ml HA peptide. Cocultures were either separated by a transwell system or 50 µg/ml anti-IL-10 mAb or 50 µg/ml anti-TGF-β mAb was added. Proliferation was measured by [3H]thymidine incorporation and data are presented as means for triplicate wells from at least two independent experiments. Proliferation of stg TN cells was set to 100%. E, Induced dtg CD25– TR cells secrete only low amounts of IL-2 and IFN-{gamma}. A total of 2.5 x 104 sorted stg TR and stg TN cells isolated from TCR-HA mice as well as dtg CD25+ TR and dtg CD25– TR isolated from TCR-HA x Ig-HA mice were stimulated in the presence of 2.5 x 105 irradiated BALB/c splenocytes and 10 µg/ml HA 110–120 peptide. Supernatants were collected after 72 h and the amounts of secreted IL-2 and IFN-{gamma} were quantified using the CBA from BD Biosciences. Experiments were performed in triplicate and the mean value is shown.

 
To investigate by which mechanism dtg CD25– TR cells elicit their suppressive activity in vitro, we performed coculture experiments either separating the TR cell populations from responder T cells by a semipermeable membrane or by adding IL-10 or TGF-β-neutralizing Abs. As shown in Fig. 1, C and D, dtg CD25– TR cells as well as prestimulated dtg CD25– TR cells lost their suppressive capacity in the absence of cell-cell contact, whereas the inhibitory function was not affected by neutralizing the cytokines IL-10 and TGF-β.

It is well-established that proliferation of TR cells depends on exogenous IL-2 in vitro, but TR cells fail to produce this cytokine by themselves (36, 37). To further characterize the phenotype of in vivo-induced dtg CD25– TR cells, we analyzed them with respect to their potential to produce proinflammatory cytokines upon Ag recognition in vitro. Therefore, sorted stg TR cells, dtg CD25+ TR cells, dtg CD25– TR cells, and stg TN cells were stimulated in the presence of APCs with the HA-peptide 110–120 and culture supernatants were used for CBA. As shown in Fig. 1E, all TR populations failed to secrete significant amounts of IL-2 and IFN-{gamma}. Thus, dtg CD25– TR cells induced by chronic Ag stimulation in vivo exhibit a regulatory phenotype in vitro despite of the absence in CD25 expression.

Molecular signature of Ag-specific induced dtg CD25– TR cells

To define the molecular signature of Ag-specific in vivo-induced dtg CD25– TR cells in comparison to naturally occurring CD25+ TR cells, we performed comparative gene expression profiling by Affymetrix microarray analysis. Sorted splenic wild-type (WT) TR cells, stg TR cells, dtg CD25+ TR cells, dtg CD25– TR cells, in vitro-stimulated stg 16h TA cells, stg 3d TA cells as well as stg TN cells, were included in the experiment and analyses were performed in triplicates. Normalized signal intensity data were calculated by GCOS 1.2 software. To determine significant expression changes, we applied a statistical multiclass response comparison embedded in the SAM 1.21 software module (38). By this approach, we identified ~3500 genes whose expression levels differ between analyzed groups applying the {delta} parameter of 0.206675294 minimizing the false discovery rate to 0.05%. For further downstream analysis using the GENESIS 1.6 software suite (35), we calculated, for each T cell population from triple values, the mean expression levels of individual genes. Normalization of gene expression profiles was achieved by median centring of the individual signal intensities for each gene. With respect to their expression profile within the various TR cells in comparison to naive TN cells or recently activated TA cells, differentially expressed genes were subdivided into 10 gene clusters depicted in Fig. 2A. Expression levels of individual genes were presented with respect to the median of expression intensities shown as baseline in Fig. 2A.


Figure 2
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  		FIGURE 2. Comparative gene expression profiling of different TR cell subsets. Affymetrix array analysis of cDNA from WT TR, stg TR, dtg CD25+ TR, dtg CD25– TR cells, in vitro-stimulated stg TA as well as stg TN sorted T cells. A, k-mean cluster analysis of genes with at least a 1.5-fold difference in inducible gene expression compared with in vitro-activated stg TA as well as stg TN cells. Approximately 3500 regulated genes were included in the cluster analysis and subdivided into 10 gene clusters according to their expression profile in the different T cell subsets analyzed by Genesis software. On the basis of this gene clustering, a selection of genes up-regulated by, B, CD4+CD25+ TR cells isolated from BALB/c (WT TR), TCR-HA (stg TR), and TCR-HA x Ig-HA (dtg CD25+ TR) mice or, C, selectively expressed by dtg CD25– TR cells is depicted as heat map. Red indicates induction of gene expression, green indicates repression. The brighter the color the stronger the gene regulation factor (+2, bright red; –2, bright green). Black indicates no changes. Results were obtained from T cells isolated from pooled individual mice (n ≥ 3) and Affymetrix gene chip analyses were performed in triplicate.

 
Most of the regulated genes (~2000) were differentially expressed by activated TA cells. However, we have also identified two clusters containing genes either up-regulated by CD25+ TR cell subsets (Fig. 2B) or specifically expressed by in vivo-induced dtg CD25– TR cells (Fig. 2C). In accordance with the literature, we identified the transcription regulator Foxp3, known to be involved in the development and function of thymus-derived CD4+CD25+ TR cells (26, 27), to be differentially expressed in the CD25+ TR cell subsets (Fig. 2B). Molecules coexpressed with Foxp3 and therefore suggested to share the genetic program of monoclonal as well as polyclonal CD25+ TR cells were, for example, CD83, Nrp1, killer cell lectin-like receptor G1 (Klrg1), and GPR83. These results are well in line with the current knowledge about molecules expressed by naturally occurring TR cells.

Strikingly, despite their regulatory function in vitro (Fig. 1), we could not detect these molecules including the TR-specific transcription factor Foxp3 in in vivo-induced dtg CD25– TR cells. A selection of genes predominantly expressed by this distinct population of dtg CD25– TR cells is depicted in Fig. 2C. Among these molecules, we identified the chemokine ligands Scya3 (CCL3, MIP-1{alpha}) and Scya5 (CCL5, RANTES), playing a role in chemotaxis and different immune responses as mediator of virus-induced inflammation (39) and suppressive factors for HIV infection (40). In addition, we found the cytokines IL-10, which has been shown previously to be secreted by CD4+6.5+ T cells isolated from TCR-HA x Ig-HA mice (17), and IL-21, the newest member of the {gamma}c-related cytokine family within this gene cluster.

To confirm the gene expression pattern detected by Affymetrix gene chip analysis, we performed FACS as well as real-time RT-PCR analysis of selected genes. Additionally, we have included expression analysis of TR cell-associated molecules namely CTLA4, GITR, and CD103. These genes are known to also be up-regulated upon stimulation of T cells and thereby could not be detected within our "TR cell" clusters (Fig. 2, B and C). As depicted in Fig. 3, all CD25+ TR cell populations analyzed (WT TR, stg TR, and dtg CD25+ TR cells) express elevated levels of Klrg1, GITR, CD103, and GPR83 compared with their naive counterparts. Whereas in vivo-induced dtg CD25– TR cells do not up-regulate these molecules, they express high amounts of PD1 (45%) and CTLA4 (34%) comparable to dtg CD25+ TR cells. Furthermore, we could detect high mRNA levels of CCL5 and IL-21 specifically in dtg CD25– TR cells. Similar results were obtained by the Affymetrix gene expression analysis.


Figure 3
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  		FIGURE 3. A, Expression analysis of TR-associated molecules by FACS. Freshly isolated splenocytes from BALB/c mice, TCR-HA mice, and TCR-HA x Ig-HA mice were stained for 6.5, CD4, CD25 and PD1, Klrg1, CTLA4 (i.c.), GITR, or CD103. Cells were gated on 6.5, CD4, and CD25 expression or CD4 and CD25 expression for BALB/c splenocytes and analyzed regarding the expression of PD1, Klrg1, CTLA4, GITR, or CD103. Number of positive cells is indicated as percentage. Data shown are of one representative of at least two experiments using cells isolated from at least two mice (n = 2). B, Real-time RT-PCR analysis of CCL5, IL-21, and GPR83 expression. Total RNA was isolated from sorted cell populations as indicated, reverse transcribed, and mRNA expression levels of CCL5, IL-21, and GPR83 were analyzed by real-time RT-PCR. Relative mRNA amounts were normalized with respect to expression level in naive stg TN cells (fold change set to 1). RPS9 mRNA expression served as a housekeeping control. Two individual experiments were done in duplicate and data are from pooled individual mice (n ≥ 3).

 
In summary, these data suggest that chronic Ag stimulation in vivo induces a distinct subset of TR cells exhibiting a quite different genetic program than CD4+CD25+ naturally occurring TR cells.

CD25 and Foxp3 expression pattern in in vivo-induced dtg CD25– TR cells

On the basis of our extensive gene expression profiling, we could not detect Foxp3 expression in Ag-specific dtg CD25– TR cells. To confirm this interesting finding, we analyzed Foxp3 expression on protein level by intracellular FACS analyses in Ag-specific stg TR cells and stg TN cells isolated from TCR-HA mice, dtg CD25+ TR cells, and dtg CD25– TR cells isolated from TCR-HA x Ig-HA mice, as well as naturally occurring WT TR cells and WT TN cells. Consistent with the Affymetrix gene chip data, Ag-specific CD4+CD25+ TR cells isolated from TCR-HA mice (stg TR) and polyclonal WT TR cells expressed elevated levels of Foxp3 protein (74 and 92%, respectively) (Fig. 4A). Interestingly, within the dtg CD25+ TR cells isolated from TCR-HA x Ig-HA mice only 42% express Foxp3. In addition, our Affymetrix gene chip analysis revealed that these dtg CD25+ TR cells exhibit a slightly distinct gene expression profile than naturally occurring CD4+CD25+ TR cells (WT TR and stg TR cells). One might speculate that these dtg CD25+ TR cells represent an intermediate population "in the middle" of naturally occurring CD4+CD25+ TR cells and induced dtg CD25– TR cells. This might be traced back to the chronic Ag stimulation in TCR-HA x Ig-HA mice. In contrast to thymus-derived TR cell subsets, dtg CD25– TR cells generated by chronic Ag stimulation in vivo produced significantly lower amounts of Foxp3 (5%) similar to nonregulatory naive stg TN cells (3%). Real-time RT-PCR analysis revealed a slight increase in Foxp3 mRNA expression by induced dtg CD25– TR cells (data not shown), which might reflect known varieties between mRNA level and protein expression. However, these results indicate that chronic Ag stimulation in vivo induces a distinct subset of TR cells with regulatory capacity in vitro despite the lack of Foxp3 protein expression.


Figure 4
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  		FIGURE 4. In vivo-induced Ag-specific dtg CD25– TR cells do not express Foxp3 and CD25 even upon stimulation. A, Foxp3 expression in freshly isolated lymphocytes. CD4+CD25+ and CD4+CD25– sorted splenocytes isolated from BALB/c mice or 6.5+CD4+CD25+ and 6.5+CD4+CD25– sorted splenocytes isolated from TCR-HA or TCR-HA x Ig-HA were stained for Foxp3. Cells were gated for 6.5, CD4, and CD25 expression and analyzed regarding intracellular Foxp3 expression by FACS. One representative experiment of three independent experiments is shown. B, Foxp3 and CD25 (C) are not up-regulated by dtg CD25– TR cells upon Ag-specific stimulation in vitro. Sorted 6.5+CD4+CD25– splenocytes isolated from TCR-HA x Ig-HA mice (dtg CD25– TR) or isolated from TCR-HA mice (stg TN) were cultured in the presence of 10 µg/ml HA peptide, APCs, and 50 U/ml IL-2. At different time points indicated, cells were harvested, stained, and gated for 6.5 and CD4 expression and analyzed for their Foxp3 (B) or CD25 expression (C). One representative experiment of two independent experiments is shown.

 
It is well-established that some TR subpopulations are adaptively induced from CD25– T cells as a consequence of a certain mode of Ag exposure, especially in a particular cytokine milieu (41). Therefore, we asked whether induced Foxp3–CD25– TR cells might change their phenotype toward Foxp3+CD25+ TR cells upon antigenic stimulation in vitro; under these terms they exhibited their suppressive capacity in vitro (Fig. 1). For this purpose, sorted dtg CD25– TR splenocytes isolated from TCR-HA x Ig-HA mice as well as sorted naive CD25– stg TN cells isolated from TCR-HA mice were stimulated in vitro with 10 µg/ml HA peptide and cultured in the presence of APCs and IL-2 for 24, 48, and 96 h before analyzing CD25 and Foxp3 expression by FACS. As shown in Fig. 4, B and C, dtg CD25– TR cells generated by chronic Ag stimulation in vivo up-regulated neither Foxp3 (Fig. 4B) nor CD25 (Fig. 4C) expression upon stimulation in vitro. Similar results were obtained after polyclonal activation with anti-CD3 and anti-CD28 in the presence of IL-2 (data not sown). As expected, stg TN cells showed increased levels in CD25 expression and only marginal Foxp3 expression upon stimulation in vitro (Fig. 4, B and C). However, upon in vitro stimulation, dtg CD25– TR cells expressed slightly more Foxp3 than TCR-HA TN cells, which is well in line with the Foxp3 expression pattern in the corresponding unstimulated T cell subsets (Fig. 4A).

Ag-specific induced dtg CD25– TR cells are able to control CD8+ T cell immune responses in vivo

As Ag-specific induced Foxp3–CD25– TR cells isolated from TCR-HA x Ig-HA mice have been shown to inhibit proliferation of naive T cells despite the lack in Foxp3 expression, we were interested in their suppressive capacity in vivo. Most recently, we have demonstrated that the whole CD4+6.5+ T cell population isolated from TCR-HA x Ig-HA mice was able to interfere with CD8+ T cell-induced colitis in Villin-HA mice, expressing their cognate Ag under control of the gut-specific Villin promoter (19). However, these experiments were performed with CD4+6.5+ T cells irrespective of CD25 expression. To investigate whether the suppressive capacity observed in these cotransfer experiments was dependent on the presence or expansion of Foxp3+CD25+ TR cells or whether induced Foxp3–CD25– TR cells (dtg CD25– TR cells) were also able to interfere with severe wasting disease, we cotransferred 2 x 106 of these dtg CD25– TR cells with 2 x 106 HA-specific CD8+ T cells isolated from CL4-TCR mice into Villin-HA mice and analyzed the disease severity by histological analysis 4 days posttransfer. As controls, we included Villin-HA mice that received 2 x 106 naive stg TN cells isolated from TCR-HA mice in addition to 2 x 106 HA-specific CD8+ T cells as well as Villin-HA mice that received 2 x 106 HA-specific CD8+ T cells alone. As depicted by histological scoring in Fig. 5A, Ag-specific dtg CD25– TR cells significantly reduced the severity of wasting disease in contrast to naive stg TN cells isolated from TCR-HA stg mice, which further exacerbated the CD8+ T cell-induced colitis. By this we could show that not only the CD25+ TR cell population within the CD4+6.5+ T cell pool is responsible for the described regulatory activity in vivo (18, 19), but also the quite distinct dtg CD25– TR cell population. From this data, one might argue that cotransferred dtg CD25– TR cells simply delay the onset of CD8+ T cell-mediated wasting disease in Villin-HA mice. However, histological analysis of the small intestine at day 14 clearly showed that Villin-HA mice receiving HA-specific CD8+ T cells in the presence of dtg CD25– TR cells exhibited a reduced severity in wasting disease (Fig. 5A).


Figure 5
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  		FIGURE 5. Induced dtg CD25– TR cells interfere with CD8+ T cell-mediated wasting disease in vivo. A total of 2 x 106 purified CD8+ T cells isolated from CL4-TCR mice were injected i.v. in Villin-HA mice or nontransgenic littermates either alone or cotransferred with 2 x 106 sorted CD4+6.5+CD25– T cells isolated from TCR-HA stg mice (stg TN) or TCR-HA x Ig-HA dtg mice (dtg CD25– TR), respectively. Four days posttransfer, mice were scarified. A, H&E staining of small intestine probes paraffin-embedded were analyzed at day 4 or 14 and used for histological evaluations. Mean values from four to six animals analyzed are shown. The Student t test was used to assess the significance of differences (**, p < 0.01; ***, p < 0.001). B, A total of 2 x 106 sorted and CFSE-labeled 6.5+CD4+CD25– T cells isolated from TCR-HA (stg TN) or TCR-HA x Ig-HA (dtg CD25– TR) mice were coinjected with 2 x 106 purified CD8+ T cells isolated from CL4-TCR mice i.v. in Villin-HA mice. At day 4, lymphocytes were reisolated from the lamina propria (LPL), MLN, and spleen and stained for the expression of CD4 and the clonotypic TCR (6.5). Dot blots show the expression of CFSE on gated CD4+6.5+ T cells. One representative experiment of two independent experiments with similar results is shown. C, FACS analysis of IFN-{gamma} and IL-2 expression in CD4+6.5+ T cells adoptively cotransferred with HA-specific CD8+ T cells reisolated from the lamina propria (LPL), MLN, and spleen of Villin-HA mice. One of at least two experiments with similar results is shown. D, FACS analysis of Foxp3 and CD25 expression in MLN cells isolated from Villin-HA mice receiving CD8+ T cells isolated form CL4-TCR mice either cotransferred with 6.5+CD4+CD25– T cells isolated from TCR-HA (stg TN) or TCR-HA x Ig-HA (dtg CD25– TR) mice, respectively. Cells were gated on CD4 and 6.5 expression. One representative experiment of four independent experiments with similar results is shown.

 
As it is well-known that TR cells might change their behavior and phenotype in vivo (42), we next analyzed the proliferative capacity, cytokine profile as well as CD25 and Foxp3 expression of induced dtg CD25– TR cells upon suppression of Ag-specific CD8+ T cell responses to their cognate Ag in vivo.

Therefore, we adoptively cotransferred 2 x 106 CFSE-labeled dtg CD25– TR cells or stg TN cells with 2 x 106 HA-specific CD8+ T cell into Villin-HA mice and analyzed the CFSE dye profile of CD4+6.5+ T cells reisolated from the lamina propria (LPL), draining lymph nodes (MLN), and spleen 4 days posttransfer (Fig. 5B). The majority of dtg CD25– TR cells exhibited proliferative activity in the lamina propria (LPL) and the MLN, whereas the number of nondividing T cells was higher in the peripheral lymphoid organ (spleen) (Fig. 5B).

Next, we analyzed the cytokine profile of adoptively transferred Ag-specific CD4+ T cells. Intracellular staining of reisolated CD4+6.5+ dtg CD25– TR and stg TN cells revealed that 13% of naive stg TN cells isolated from the MLN produced IL-2, in contrast to induced dtg CD25– TR cells (3%) (Fig. 5C). Nearly no IFN-{gamma} production was detected by reisolated stg TN cells or by dtg CD25– TR cells 4 days posttransfer.

As shown in Fig. 4, in vivo-induced dtg CD25– TR cells do not express Foxp3 even upon stimulation in vitro. Therefore, we were interested in the Foxp3 as well as CD25 expression in dtg CD25– TR cells upon interfering with CD8+ T cell-mediated wasting disease in vivo. For this purpose, we reisolated lymphocytes from MLN from Villin-HA mice, which received either dtg CD25– TR cells or stg TN cells together with HA-specific CD8+ T cells and analyzed the expression of CD25 and Foxp3 on CD4+6.5+ T cells by FACS. As shown in Fig. 5D, we observed ~1.5–2 times higher expression of CD25 as well as Foxp3 in reisolated dtg CD25– TR cells in contrast to CD25– naive stg TN cells originally derived from TCR-HA stg mice. However, the ratio of about two times higher Foxp3 expression between both T cell types remained stable when compared with Foxp3 expression in freshly isolated CD4+6.5+CD25– T cells from TCR-HA (stg TN) and TCR-HA x Ig-HA (dtg CD25– TR) mice (Fig. 4A) as well as upon Ag stimulation in vitro (Fig. 4B). These data suggest that even during active immunosuppression in vivo, induced dtg CD25– TR cells do not up-regulate CD25 and Foxp3.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Over the last few years it became clear that, besides the thymus-derived naturally occurring CD4+CD25+ TR cells, a broad variety of induced TR cells exist which could be induced from naive CD4+ T cells by different approaches. In the present study, we could demonstrate that chronic Ag stimulation in vivo leads to the generation of Ag-specific CD4+Foxp3–CD25– TR (dtg CD25– TR) cells. Interestingly, the expression of CD25 and Foxp3 is not induced upon stimulation in vitro or upon active immunosuppression in the course of a CD8+ T cell-mediated intestinal inflammation.

Comparative gene expression profiling of induced dtg CD25– TR cells revealed that these TR cells clearly differ from naturally occurring CD4+CD25+ TR cells. We identified CCL5 (Scya5) and the cytokine IL-21 to be specifically expressed by in vivo dtg CD25– TR cells. CCL5 is a CC chemokine with chemoattractant properties, whereas its function in T cell immunity still remains controversial. Anti-CCL5 Ab treatment was shown to decrease Th1 responses, while increasing Th2-type hypersensitivity in mice (43). In contrast, extended exposure of T cells to CCL5 decreased the ability of T cells to produce IFN-{gamma} and TNF-{alpha} (44). Moreover, upon CCL5 exposure, T cells were less efficient at TCR translocation and lipid raft clustering (44), suggesting that CCL5 may impact T cell effector function by modulating the ability to create a functional immunological synapse.

Recently, Knoechel et al. (45) have described a population of anergic T cells induced in dtg mice expressing T cells recognizing their cognate Ag, expressed in a soluble form under control of the metallothionein promoter I. These T cells failed to proliferate upon stimulation in vitro and up-regulated the expression of genes which we also found to be up-regulated by induced dtg CD25– TR cells isolated from TCR-HA x Ig-HA mice, namely CCL5 (Scya5), IL-21, and Tnfsf6. The anergic T cell population described by Knoechel et al. (45) did not exhibit suppressive capacity; quite to the contrary they caused pathologic effector cell responses in vivo. The authors suggested that these anergic T cells responded to their cognate Ag, but failed to become fully activated, underlining a possible role of CCL5 in creating a functional immunological synapse as suggested by Cridge et al. (44).

In the present study, we identified the cytokines IL-10 and IL-21 to be highly expressed by in vivo-induced Ag-specific CD25– TR cells. However, despite the high expression of the immunosuppressive cytokine IL-10 also published by Buer et al. (17), IL-10 seems not to play a key role in the suppressive capacity of these cells. The addition of a blocking anti-IL-10 mAb does not abrogate the inhibitory effect of induced dtg CD25– TR cells on the proliferative activity of naive T cells in coculture experiments (Fig. 1C).

IL-21 was recently discovered as a member of the common {gamma}-chain family of cytokines, with pleiotropic effects on proliferation, differentiation, and effector function of B, T, NK, and dendritic cells (DCs). Ectopic expression of IL-21 in conventional T cells led to an impaired proliferative capacity of these cells, but did not confer a regulatory phenotype in vitro (data not shown). However, it was supposed that IL-21 is a novel immunomodulatory cytokine, whose regulation of any given immune response is highly dependent on the surrounding environmental context. Brandt et al. (46) reported about an inhibitory function of IL-21 on the maturation of bone marrow DCs very similar to the inhibitory effects seen from IL-10. In vitro differentiation of DCs in the presence of IL-21 resulted in DCs with an immature phenotype (Ref. 46 and our own observation). With regard to the high expression of IL-21 by in vivo-induced CD25– TR cells, one might speculate about a T cell extrinsic effect leading to the induction of immature DCs which are known to induce TR cells and thereby might in turn lead to the induction of Ag-specific TR cells. This would be a possible explanation for the regulatory capacity of 6.5+CD25+ (dtg CD25+ TR) as well as 6.5+CD25– TR (dtg CD25– TR) cells isolated from TCR-HA x Ig-HA mice, whereas Apostolou et al. (20) suggested that the expression of the cognate HA Ag by hemopoietic cells, in particular B cells, was responsible for the induction of 6.5+CD25– TR cells in the TCR-HA x Ig-HA dtg mice. However, further investigations are required to elucidate the precise role of CCL5 and IL-21 in induced TR cells.

We could further demonstrate that PD1 and CTLA4 were highly expressed in common by naturally occurring CD4+CD25+ TR cells as well as by induced dtg CD25– TR cells (Fig. 3A). The PD1 receptor interacts with two new B7 family members, PD-L1 and PD-L2. Engagement of PD1 with its ligand inhibits TCR-mediated proliferation and cytokine production. In addition, the inhibitory effects of CD4+CD25+ TR cells on the proliferation of naive T cells is prevented by a blocking anti-PD-L1 Ab, suggesting that the PD-L-PD1 pathway might have a role in CD4+CD25+ TR cells (47). CTLA4 is a homolog of CD28 and described to be a negative regulator of T cell activation to attenuate T cell response. In vitro blockade of CTLA4 neutralizes the suppressive function of TR cells and, in addition, the in vivo administration of nonactivating anti-CTLA4 mAb abolishes the protective activity of naturally occurring TR cells in inflammatory bowel disease (48). These data suggest that CTLA4 seems to be necessary for TR cell function. However, whether PD1 and CTLA4 are involved in the suppressive function of in vivo-induced Ag-specific dtg CD25– TR cells analyzed in the present study remains to be elucidated.

The most interesting finding from our comparative gene expression analysis was that in vivo-induced dtg CD25– TR cells do not express elevated levels of the transcription factor Foxp3, which has been described to be essential for the development and function of naturally occurring CD4+CD25+ TR cells. Even during immunosuppression of intestinal inflammation, we detected only a slight increase in Foxp3 expression in induced dtg CD25– TR cells comparable to the increase of Foxp3 in stg TN cells. However, we could not exclude that the small Foxp3+ population within the induced dtg CD25– TR cells is responsible for the suppressive capacity in vitro and in vivo. But similar amounts of naive stg TN cells also express and/or up-regulate Foxp3 and these Foxp3+ T cells were not sufficient to confer suppressive activity to naive stg TN cells. In contrast, cotransfer of these T cells with HA-specific CD8+ T cells into Villin-HA mice led to a more severe wasting disease.

Recent reports have described different induced CD4+CD25+ TR cell types with regulatory capacity although these T cells express only marginal amounts or no Foxp3 (12, 49). Despite similarities in the lack in Foxp3 expression, these induced TR cell types differ from the HA-specific dtg CD25– TR cells analyzed in the present study with regard to CD25 expression. Nicolson et al. (50) reported that repeated intranasal peptide administration led to the induction of a specific subset of TR cells, which exhibited a similar phenotype as the herein analyzed HA-specific dtg CD25– TR cells. They did neither express Foxp3 nor CD25, produced no IL-2, but secreted IL-10 in response to Ag stimulation. However, in contrast to dtg CD25– TR cells induced by chronic Ag stimulation in vivo, these TR cells became CD25+ upon stimulation. Taken together, these findings suggest that high Foxp3 expression seems not to be a prerequisite for the suppressive capacity of induced CD4+ TR cells and, moreover, underline the importance of the mode in self-Ag expression for the induction of the different induced TR cells. Pancreatic expression of soluble self-Ag leads to the induction of anergic T cells (45), intranasal application induces Foxp3–CD25– TR cells becoming CD25+ upon stimulation (50), and constitutive expression of self-Ags on hemopoietic cells leads to the induction of Foxp3–CD25– TR cells as demonstrated in the present study.


   Acknowledgments
 
We are very grateful to Silvia Prettin and Tanja Toepfer for excellent technical assistance and the staff of the animal facility at the Helmholtz Centre for Infection Research for mouse colony management.


   Disclosures
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 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 grants from the Deutsche Forschungsgemeinschaft to J.B. and D.B. (SFB621). Back

2 Address correspondence and reprint requests to Dr. Wiebke Hansen, Institute of Medical Microbiology, University Hospital Essen, Hufelandstrasse 55, D-45122 Essen, Germany. E-mail address: wiebke.hansen{at}uk-essen.de Back

3 Abbreviations used in this paper: TR, regulatory T; HA, hemagglutinin; GITR, glucocorticoid-induced TNFR; Nrp1, neuropilin 1; GPR, G protein-coupled receptor; CBA, cytometric bead array; LPL, lamina propria lymphocyte; MLN, mesenteric lymph node; dtg, double transgenic; stg, single transgenic; WT, wild type; DC, dendritic cell; TN, naive T. Back

Received for publication November 29, 2006. Accepted for publication October 3, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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