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Experience-Driven Development: Effector/Memory-Like _E⁺Foxp3⁺ Regulatory T Cells Originate from Both Naive T Cells and Naturally Occurring Naive-Like Regulatory T Cells¹

Christiane Siewert^*, Uta Lauer^*, Sascha Cording^*, Tobias Bopp, Edgar Schmitt, Alf Hamann^* and Jochen Huehn^2,*

^* Experimentelle Rheumatologie, Charité Universitaetsmedizin Berlin, Campus Mitte, Berlin; and Institut fuer Immunologie, Johannes-Gutenberg-Universitaet, Mainz, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Naturally occurring Foxp3⁺CD25⁺CD4⁺ regulatory T cells (Treg) have initially been described as anergic cells; however, more recent in vivo studies suggest that Tregs vigorously proliferate under both homeostatic as well as inflammatory conditions. We have previously identified a subset of murine CD4⁺ Tregs, which is characterized by expression of the integrin _Eβ₇ and which displays an effector/memory-like phenotype indicative of Ag-specific expansion and differentiation. In the present study, the _E⁺ Treg subset was found to contain a large fraction of cycling cells under homeostatic conditions in healthy mice. Using an adoptive transfer system of Ag-specific T cells, we could demonstrate that the vast majority of transferred natural, naive-like CD25⁺CD4⁺ Tregs acquired expression of the integrin _Eβ₇ upon tolerogenic application of Ag via the oral route. In addition, using the same system, Foxp3⁺ Tregs could be de novo induced from conventional naive CD25^–CD4⁺ T cells, and this conversion was associated with concomitant expression of _E. These findings suggest that Tregs expressing the integrin _E are effector/memory Tregs with a high turnover rate that can develop in the periphery upon Ag contact under tolerogenic conditions, both from thymic-derived CD25⁺CD4⁺ Tregs with a naive-like phenotype as well as from conventional naive T cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The CD4⁺ regulatory T cells (Treg)³ are powerful regulators of immune responses in organ-specific autoimmunity, allergic responses, allograft rejection, and host responses against pathogens (1). In addition, they control systemic homeostasis and total lymphocyte numbers (2). The transcription factor Foxp3 appears to be the most specific marker identifying naturally occurring CD25⁺ and CD25^– CD4⁺ Tregs generated as a distinct lineage in the thymus (3). We have recently shown that within the murine CD4⁺ Treg compartment, the markers CD25 and _E may serve to identify phenotypically and functionally distinct Treg subsets. Using these markers we were able to subdivide the Treg compartment into naive-like _E^–CD25⁺ and effector/memory-like _E⁺CD25⁺ or _E⁺CD25^– cells (4). The differential phenotypes of _E^– and _E⁺ Treg subsets resulted in distinct homing patterns, which were directly coupled to the functional activity of the respective subsets in vivo, indicating that appropriate localization was a prerequisite for suppressive capacity (5).

Initially, Tregs had been characterized as anergic cells, as they do not respond to TCR-mediated stimulation in vitro, and only high amounts of exogenous IL-2 can overcome their hyporesponsiveness (6). Later, it has been demonstrated that Tregs show proliferation in vivo, i.e., they respond to self-peptide presented by unique dendritic cells in tissue-draining lymph nodes (LN) (7, 8, 9, 10, 11, 12). These cells display an activated phenotype and proliferate in normal unmanipulated mice (7, 13). However, a clear correlation between peripheral proliferation of Tregs and acquisition of _E expression has not been established in much detail yet (13).

Apart from the peripheral expansion of thymic-derived Tregs, there is also ample indication that Tregs can be induced de novo from the conventional naive CD4⁺ T cell pool, both in vitro and in vivo. Immunosuppressive cytokines like TGF-β (14, 15, 16) and IL-10 (17), as well as immature dendritic cells (18, 19), have been used to induce Tregs with distinct properties. Various protocols of tolerance induction, i.e., oral, nasal, and i.v. Ag-administration in transfer models of Ag-specific T cells, were also shown to result in the generation of Tregs (16, 20, 21, 22, 23, 24). However, the reported phenotypes of de novo induced Tregs are heterogeneous, particularly with respect to the expression of the Treg marker Foxp3.

The memory phenotype of _E-expressing Tregs suggests Ag-driven induction or expansion in the periphery. However, it was not clear so far whether _E⁺ Tregs originate from naive-like _E^–CD25⁺ Tregs differentiating into an effector/memory phenotype upon Ag-driven activation and proliferation, or whether _E⁺ Tregs can also directly develop from conventional naive T cells under appropriate tolerogenic conditions.

In the present study, we analyzed proliferation of the various Treg subsets and of naive T cells under homeostatic conditions and upon oral feeding of Ag. Adoptive transfer of Ag-specific TCR-transgenic T cell subsets provided clear evidence that _E⁺ effector/memory Tregs can develop both from conventional naive T cells under tolerogenic conditions of Ag delivery as well as by activation and differentiation of naive-like, natural Tregs.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals

BALB/c, C57BL/6, and OVA-specific TCR-transgenic DO11.10 mice were obtained from the breeding facility BfR (Bundesinstitut für Risikobewertung). Thymectomized C57BL/6 mice were provided by L. Klein (Research Institute of Molecular Pathology, Vienna, Austria). DO11.10 x RAG-1^–/– mice were from the breeding facility of the University of Mainz. Mice were 6–10 wk of age unless otherwise stated. All animal experiments were performed under specific pathogen-free conditions and in accordance with institutional, state, and federal guidelines.

Abs, staining, and sorting reagents

The following Abs were produced in our laboratory: anti-FcR II/III (2.4G2), FITC- and Alexa 405-labeled anti-CD4 (GK1.5), biotinylated anti-OVA-TCR (KJ1.26), biotinylated and Alexa 647-labeled anti-_E (CD103; M290), FITC-labeled anti-CD44 (IM7), and FITC-labeled and biotinylated anti-CD62L (Mel-14). The following Abs and secondary staining reagents were purchased from BD Pharmingen: FITC-labeled anti-CD4 (RM4–5), allophycocyanin- and PerCP-Cy5.5-labeled anti-CD25 (PC6.1), biotinylated anti-CD25 (7D4), PE-Cy7-labeled streptavidin, PE-labeled anti-CD44 (IM7), and appropriate isotype controls. The following Abs were purchased from eBioscience: allophycocyanin-Alexa750-labeled anti-CD4 (RM4–5) and Pacific Blue-labeled anti-CD62L (Mel-14). For intracellular detection of BrdU incorporation, the BrdU Flow kit (BD Biosciences) was used. Intracellular staining for Foxp3 was performed by using the Foxp3 staining set with PE- or PE-Cy5-labeled anti-mouse Foxp3 (FJK-16s) from eBioscience. All MicroBeads were purchased from Miltenyi Biotec.

Cell preparation and flow cytometry

For ex vivo analysis of Treg subsets, single-cell suspensions were prepared from indicated, secondary lymphoid organs. Erythrocyte-depleted cells were stained using fluorochrome-labeled reagents, and multicolor analysis was performed using a LSRII (BD Biosciences) and the CellQuest software. Propidium iodide or 4',6-diamidino-2-phenylindole (Sigma-Aldrich) were used for dead-cell exclusion. For intracellular detection of BrdU and Foxp3, cells were fixed and permeabilized according to the manufacturer’s instructions.

Evaluation of homeostatic proliferation by BrdU incorporation

C57BL/6 mice thymectomized at the age of 3 wk were used 4 wk later for the experiment. BrdU (Sigma-Aldrich) was added to the drinking water at 1 mg/ml and changed every 2–3 days. On day 10 after the start of BrdU feeding, mice were killed and cell suspensions were prepared from different lymphoid organs. Cell surface staining was performed, and subsequently cells were fixed, permeabilized, and stained with FITC-labeled anti-BrdU Ab according to the manufacturer’s instructions. Cells were analyzed on a LSRII.

Ag-specific activation in vivo

For Ag-specific activation, lymphocyte subsets from OVA-specific, TCR-transgenic DO11.10 mice were isolated by MACS and transferred into BALB/c recipient mice. Briefly, single cell suspensions from lymphoid organs of DO11.10 mice were depleted of _E⁺ cells, subsequently CD25⁺ cells were isolated with biotinylated anti-CD25 and anti-Biotin MicroBeads. From the MACS-negative fraction naive cells were isolated using anti-CD62L MicroBeads. Sorted cells were labeled with CFSE and 4–6 x 10⁶ _E^–CD25^–CD62L^high or 6–9 x 10⁵ _E^–CD25⁺ cells were injected i.v. into BALB/c mice. One day after adoptive transfer, OVA protein (Sigma-Aldrich) was administered via different routes. The tolerogenic protocol involved continuous feeding of OVA protein at indicated doses in the drinking water for 6 days and preparation of lymphoid organs on day 7. For the immunogenic-Ag administration, recipient mice were injected once with 500 µg OVA protein and 70 µg LPS (Sigma-Aldrich) i.p.; mice were sacrificed on day 5 after immunization. Cell surface staining was performed on single-cell suspensions from different lymphoid organs, followed by fixation, permeabilization, and intracellular staining for Foxp3 according to the manufacturer’s instructions. Cells were analyzed by FACS.

In vitro suppression assay

_E⁺Foxp3⁺CD4⁺ T cells were de novo induced as described above and isolated from the celiac LN draining liver (livLN), pancreas, and stomach. Thereto, single-cell suspensions from 12 to 17 livLNs were pooled, stained for CD4 and KJ1.26, and sorted for CFSE^low KJ1.26⁺CD4⁺ T cells using a FACS Aria (BD Biosciences). CD25⁺ control Tregs, CD62L^highCD25^–CD4⁺ naive responder T cells, and CD90^– APCs were isolated from lymphoid organs of BALB/c mice. Briefly, CD25⁺ cells were isolated with biotinylated anti-CD25 and anti-Biotin MicroBeads. From the MACS-negative fraction, CD4⁺ T cells were enriched using anti-CD4-FITC and anti-FITC MultisortBeads. After release of the beads according to the manufacturer’s instructions, naive CD62L^highCD25^–CD4⁺ T cells were sorted using anti-CD62L MicroBeads. APCs were prepared from the CD4^– fraction by depletion of CD90⁺ cells using anti-CD90 MicroBeads and were irradiated (30 Gy) before culture.

A total of 1 x 10⁴ CD62L^highCD25^–CD4⁺ naive responder T cells were cultured with 2 x 10⁴ APCs in round-bottom microtiter plates with addition of anti-CD3 (1 µg/ml), and unstimulated cells were taken as controls. Cell culture was done with RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FCS (Sigma-Aldrich). Cells were incubated for 48 h, followed by addition of 1µCi of methyl-[³H]thymidine (Amersham Pharmacia Biotech) per well for 20 h to measure proliferation. In coculture assays, 2–3 x 10³ CFSE^low KJ1.26⁺CD4⁺ T cells or CD25⁺ control Tregs were added to naive responder cells and APCs and treated as described above.

Statistical analysis

The results were either represented as single values representing individual mice or as mean ± SD. For statistical analyses, nonparametric tests were used to determine significant differences. For paired observations, the Wilcoxon signed rank test and for unpaired observations, the Mann-Whitney U test were used, respectively. Differences were considered statistically significant when p < 0.05 and highly significant when p < 0.01.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
_E⁺Foxp3⁺ Tregs preferentially express an effector/memory-like phenotype

Our group has previously described a dichotomy within the naturally occurring CD4⁺ Treg population based on thorough phenotyping of _E⁺ Tregs. Expression of _E was found to be highly associated with an effector/memory-like status of Tregs (4). In this study, we confirmed and extended these previous findings regarding the phenotype of _E⁺ and _E^– Treg subsets by analyzing the subset composition of CD4⁺ T cells and Foxp3⁺CD4⁺ Tregs from the spleen of C57BL/6 mice (Fig. 1a). Gating on CD4⁺ T cells revealed that _E^–CD25⁺ cells contained 78.4 ± 7.7%, _E⁺CD25⁺ cells 88.2 ± 6.3%, and _E⁺CD25^– cells 58 ± 9.6% Foxp3⁺ cells (mean ± SD, n = 17), respectively. Conversely, among gated Foxp3⁺CD4⁺ T cells, the frequency of _E^–CD25⁺ cells was 47.3 ± 9.3%, _E⁺CD25⁺ cells 16.6 ± 4.4%, and _E⁺CD25^– cells represented 7.7 ± 2.8% (mean ± SD, n = 17), respectively.

View larger version (34K): [in this window] [in a new window]   FIGURE 1. E+ Tregs preferentially express an effector/memory-like phenotype. Spleen cells from C57BL/6 mice were surface-stained for CD4, CD25, E, and CD62L or CD44, followed by intracellular staining for Foxp3. Cells were analyzed by flow cytometry. a, Representative dot plots showing expression of CD25 and E among CD4+ T cells (left) and CD4+Foxp3+ T cells (right). b, CD4+ T cells were gated according to the expression of CD25 and E as displayed in a. Dot plots show expression of Foxp3 and CD62L or CD44 on indicated subsets. Isotype control staining for Foxp3 is shown (left dot plots). Data are representative of at least two independent experiments with 2–3 individual mice each. Numbers indicate frequency of positive cells among indicated CD4+ T cell subsets.

_E-expressing peripheral Treg subsets show high proliferation under homeostatic conditions

Based on our previous observation that _E⁺ Tregs harbor reduced TCR excision circle numbers, indicative of increased proliferative history (4), we wanted to address the question whether this is the consequence of peripheral proliferation. To exclude any impact of thymic proliferation, we used thymectomized mice for the evaluation of peripheral proliferation by BrdU incorporation. C57BL/6 mice were thymectomized at the age of 3 wk, when the immune system is thought to have fully evolved. Four weeks later, we started to feed the mice with BrdU in their drinking water for a period of 10 days. Mice were sacrificed on day 10 of BrdU feeding, and spleen and LNs were evaluated for BrdU⁺ cells. Surprisingly, we found high frequencies of BrdU⁺ cells in all Treg subsets, indicating that a substantial number of cells had proliferated within the 10 days of BrdU administration. In contrast, the fraction of _E^–CD25^–CD4⁺ T cells, which largely consists of conventional naive cells, showed only low frequencies of BrdU⁺ cells as expected (Fig. 2). Within the Treg subsets, the _E⁺CD25^– cells showed the highest frequency of BrdU-labeled cells in all of the tested lymphoid organs (Fig. 2). _E⁺CD25⁺ cells showed only slightly reduced frequencies of BrdU⁺ cells compared with _E⁺CD25^– cells, and the lowest frequency was detected among _E^–CD25⁺ Tregs. These data correspond to the phenotype of the respective Treg subsets (Fig. 1b) and to our previous observation that _E⁺ Treg subsets have reduced TCR excision circle numbers (4), suggesting that indeed these cells display a high degree of peripheral proliferation under steady-state conditions.

View larger version (19K): [in this window] [in a new window]   FIGURE 2. E-expressing peripheral Treg subsets show high proliferation under homeostatic conditions. C57BL/6 mice were thymectomized 4 wk before start of BrdU feeding for 10 days and subsequent flow cytometric evaluation of proliferation in different lymphoid organs. CD4+ T cells were gated according to the expression of E and CD25 and analyzed for BrdU incorporation. a, Representative histograms of BrdU staining from peripheral lymph node CD4+ T cells of gated E/CD25 subsets from BrdU-fed mice (right). Control staining for BrdU from peripheral lymph node CD4+ T cells of a mouse that did not receive BrdU in the drinking water (left, unfed control). Numbers indicate frequency of positive cells among indicated CD4+ T cell subsets. b, Graphs summarize frequencies of BrdU+ cells in gated E/CD25 subsets from CD4+ T cells isolated from indicated lymphoid organs of BrdU-fed mice. Symbols represent individual mice analyzed in two independent experiments. Lines represent the median. *, p < 0.05; **, p < 0.01.

_E⁺ Treg numbers are stable in the absence of thymic output

When we analyzed the relative distribution of _E/CD25 Treg subsets, we observed highly increased frequencies of _E-expressing Tregs in thymectomized mice compared with non-thymectomized control mice (Fig. 3a). At the same time, the size of the total CD4⁺ compartment was reduced in all organs analyzed (Fig. 3b), presumably due to the lack of thymic output of newly generated T cells. Analysis of the respective cell number of _E/CD25 Tregs (Fig. 3b) revealed that the total number of _E-expressing Tregs in thymectomized mice was similar to control mice and that the high frequency was rather the result of a selective decrease of all other T cells, leading to a skewed ratio between conventional CD4⁺ T cells and _E⁺ Tregs. Interestingly, this shift only affected the effector/memory-like _E⁺ Tregs but not the naive-like _E^–CD25⁺ Tregs, suggesting that the latter subset relies much more on newly generated, thymic-derived Tregs to maintain stable cell numbers in the periphery. Even though _E^–CD25⁺ Tregs showed a considerable degree of peripheral proliferation, this was apparently not sufficient to maintain stable cell numbers in the absence of thymic output, whereas the _E⁺ Tregs kept a stable population size.

View larger version (24K): [in this window] [in a new window]   FIGURE 3. E+ Treg numbers are stable in the absence of thymic output. Thymectomized mice, generated as described in Fig. 2, were analyzed for the absolute cell number and relative distribution of Treg subsets in comparison to sex- and age-matched, non-thymectomized control mice. a, Proportion of E/CD25 subsets among CD4+ T cells from indicated organs of control (open symbols) and thymectomized mice (filled symbols). b, Total cell numbers of total CD4+ T cells, E–CD25– T cells (left), and of E/CD25 Treg subsets (right). Each symbol represents one individual mouse from two independent experiments with indicated median. *, p < 0.05, **, p < 0.01.

In vivo conversion of naive-like CD25⁺ Tregs into _E⁺ Tregs

The previous experiments confirmed that the activated phenotype of _E-expressing Tregs indeed correlates with a high degree of peripheral/homeostatic proliferation in normal, healthy mice. Given that naturally occurring Tregs are thought to originate from the thymus, where they are positively selected to recognition of self-Ag (1), we speculated that acquisition of the effector/memory phenotype could be a consequence of activation by self-Ag in the periphery, i.e., in secondary lymphoid tissue draining the site of respective self-Ag expression (8). In this case, naive-like _E^– Tregs should differentiate into effector/memory-like _E⁺ Tregs upon Ag-specific stimulation in vivo. To address this question, we switched from the analysis of the polyclonal T cell pool to an Ag-specific system, where we were able to follow the immune response to known cognate Ag. We chose the OVA-specific system and first compared the distribution of Treg subsets in TCR transgenic DO11.10 mice with that of age-matched BALB/c mice. Besides a weak reduction in the frequency of _E^–CD25⁺Foxp3⁺ Tregs, no gross differences were observed (Fig. 4a). Next, we isolated naive-like _E^–CD25⁺ Tregs from DO11.10 mice (Fig. 4b). After adoptive transfer of the Ag-specific Tregs into naive BALB/c recipients, we administered the cognate Ag via the oral route, which is known to result in tolerance induction (25), and followed proliferation and phenotype of transferred cells by multicolor FACS analysis (Fig. 4c). When we continuously fed the recipient mice with different doses of OVA protein (4–20 mg/ml) in the drinking water for a period of 6 days, we observed moderate proliferation of the Ag-specific KJ1.26⁺CD25⁺ Tregs in gut-associated lymphoid organs such as the celiac LN draining liver, pancreas, and stomach (liv LN) (26) and mesenteric LNs (mLNs). Hardly any OVA-specific CD25⁺ Tregs were detected in Peyer’s Patches (PP) and spleen (data not shown). We analyzed the transferred CD25⁺ Tregs for the expression of _E and could detect a high degree of coexpression of _E on previously _E^–CD25⁺ cells after Ag-driven proliferation (Fig. 4c). The frequency of _E expression on OVA-specific CD25⁺ Tregs increased with each cell division, as judged by loss of CFSE, and reached similar levels both in livLN and mLN (Fig. 4d). Moreover, converted Tregs displayed down-regulated CD62L levels and up-regulated CD44 expression when compared with nonconverted _E^–CD25⁺ Tregs (Fig. 4e). These results support the concept that upon Ag-specific activation in the periphery, naive-like _E^–CD25⁺ Tregs differentiate into effector/memory Tregs, characterized by expression of _E.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. E is induced on naive-like CD25+CD4+ Tregs upon Ag-specific activation. a, Pooled spleen and LN cells from TCR-transgenic DO11.10 and age-matched BALB/c mice were surface-stained for CD4, CD25, and E, followed by intracellular staining for Foxp3. Cells were analyzed by flow cytometry. Numbers indicate frequency of positive cells among indicated CD4+ T cell subsets. Among gated CD4+Foxp3+ T cells from BALB/c mice, the frequency of E–CD25+ cells was 42.7 ± 1.0%, E+CD25+ cells 17.0 ± 1.2%, and E+CD25– cells represented 10.8 ± 0.2% (mean ± SD, n = 5), respectively. Among gated KJ1.26+CD4+Foxp3+ T cells from DO11.10 mice, the frequency of E–CD25+ cells was 32.6 ± 5.2%, E+CD25+ cells 18.7 ± 2.5%, and E+CD25– cells represented 13.0 ± 2.7% (mean ± SD, n = 5), respectively. b, Pooled spleen and LN cells from DO11.10 mice were depleted of E+ cells and enriched for naive-like CD25+ T cells by MACS. Purified E–CD25+ cells were labeled with CFSE, adoptively transferred into BALB/c recipients, and subsequently stimulated in vivo by oral application of their cognate Ag. Dot plots show staining of CD25 vs E and KJ1.26 vs Foxp3 of CD25+CD4+ DO11.10 T cells before transfer. Numbers indicate frequency of positive cells among indicated CD4+ T cell subsets. c, After 6 days of continuous feeding with indicated concentrations of OVA, mice were sacrificed and lymphocytes were analyzed by flow cytometry. Transferred cells were identified according to CD4+KJ1.26+ staining and CFSE. Dot plots show representative data of E expression and proliferation of re-isolated Tregs in livLN and mLN, after feeding of different doses of OVA. Numbers indicate frequency of positive cells among indicated CD4+ T cell subsets. d, Frequencies of E+ Tregs in indicated lymphoid organs after different doses of OVA in drinking water. Mean ± SD corresponds to three individual mice per group from one of two independent experiments with similar results. e, Histogram plots show representative data of CD62L and CD44 expression of re-isolated E+ (black) vs E– (gray) KJ1.26+CD4+Foxp3+ Tregs in livLN after feeding of 10 mg/ml OVA.

Induction of _E⁺Foxp3⁺ cells from conventional naive T cells

To answer the question whether, in addition to the aforementioned differentiation of naive-like _E^–CD25⁺ Tregs, de novo induction of Foxp3⁺ Tregs from conventional naive T cells might contribute to the pool of _E-expressing effector/memory-like Tregs, we performed corresponding experiments with adoptive transfer of naive _E^–CD25^–CD62L⁺ T cells from DO11.10 mice and OVA administration via the oral route (Fig. 5a). Proliferating transgenic KJ1.26⁺CD4⁺ T cells were detected in livLN, mLN, PPs, and spleen at various doses of Ag (Fig. 5b). Although we observed slightly reduced proliferation in livLN compared with the other organs (Fig. 5b and data not shown), intriguingly, we identified a high proportion of Foxp3⁺ cells among transgenic cells re-isolated from the livLN, predominantly, but not exclusively, on proliferating cells. The induction was dose-dependent and largely confined to this particular LN (Fig. 5, b and c), as the frequency of Foxp3⁺ cells was much lower in mLN and PP cells (Fig. 5c). When we isolated CFSE^lowKJ1.26⁺CD4⁺ T cells from the livLN, which largely were composed of Foxp3⁺ cells (data not shown), and tested their suppressive capacity, we observed that these de novo induced Foxp3⁺ cells were equally efficient in suppressing naive T cell proliferation as ex vivo isolated CD25⁺CD4⁺ Tregs, identifying them as bona fide Tregs (Fig. 5d).

View larger version (28K): [in this window] [in a new window]   FIGURE 5. De novo induction of Foxp3+ cells by Ag-specific activation in vivo. Naive T cells were isolated from DO11.10 mice by depleting of E+ and CD25+ cells and subsequent isolation of CD62L+ cells. Highly pure E–CD25–CD62L+ T cells were labeled with CFSE, adoptively transferred into BALB/c recipients, and subsequently stimulated in vivo by oral application of their cognate Ag. a, Dot plots show staining of CD62L vs E and Foxp3 vs KJ1.26, respectively, of CD4+ DO11.10 T cells before transfer. b, After 6 days of continuous feeding with indicated concentrations of OVA, mice were sacrificed and lymphocytes were analyzed by flow cytometry. Transferred cells were identified according to CD4+KJ1.26+ staining and CFSE. Dot plots show representative data of Foxp3 expression and proliferation of re-isolated DO11.10 T cells in indicated lymphoid organs after feeding of different doses of OVA. c, Frequencies of Foxp3+ cells among transferred CD4+KJ1.26+ in indicated lymphoid organs after different doses of OVA in drinking water. Mean ± SD represents 3–4 individual mice per group. d, In vitro suppressive capacity of de novo induced Tregs was determined by measurement of incorporated [3H]thymidine after coculture of CFSElow KJ1.26+CD4+ T cells isolated from livLNs with naive responder cells for 68 h at a 1:5 ratio. Culture of responder cells alone, without (unstimulated) or with addition of soluble anti-CD3 (stimulated), and coculture of naive responder cells with ex vivo isolated CD25+ Tregs served as controls (n = 3–5; mean ± SD; one representative of three independent experiments).

View larger version (39K): [in this window] [in a new window]   FIGURE 6. E as a marker of peripherally generated Foxp3+ Tregs. Naive E–CD25–CD62L+ DO11.10 cells were isolated, CFSE-labeled, and adoptively transferred as described in Fig. 5. a, Recipient mice were continuously fed for 6 days with 10 mg/ml OVA in the drinking water, and induction of Foxp3 and E on transferred DO11.10 cells was analyzed in livLN on day 7. b, Alternatively, recipient mice were immunized by i.p. injection of 500 µg OVA with LPS 1 day after adoptive transfer of naive E–CD25–CD62L+ DO11.10 cells and analyzed for expression of Foxp3 and E on transferred cells in livLN on day 5 after immunization. Representative dot plots showing staining of CFSE vs Foxp3 or E on transferred CD4+KJ1.26+ T cells in livLN after oral application of OVA (a) or i.p. immunization with OVA/LPS (b). Regions indicate gating for individual generations according to CFSE-dilution. Graphs show frequency of E+Foxp3+, E+Foxp3–, and E–Foxp3+ in individual generations of transferred transgenic T cells. Mean ± SD of six individual mice from two independent experiments. *, p < 0.05. c, Histogram plots show representative data of CD62L and CD44 expression of de novo induced E+Foxp3+ Tregs (black) compared with undivided CFSEhigh (gray) transferred CD4+KJ1.26+ T cells in livLN after feeding of 10 mg/ml OVA. d, Dot plots show de novo induction of Foxp3 expression in adoptively transferred CD4+ T cells derived from DO11.10 x RAG-1–/– mice after oral feeding with 10 mg/ml OVA within the livLN. Cells were gated on CFSE+KJ1.26+ cells to identify transferred cells. Representative data from one of five individually analyzed mice are depicted.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

The observed proliferation indicates that a major part of Tregs is constantly cycling under steady-state conditions, presumably in response to their cognate Ags. Our findings are in accordance with results from Fisson and colleagues (7), who demonstrated rapid turnover of transferred polyclonal CD25⁺CD4⁺ Tregs in nonlymphopenic recipients associated with acquisition of an activated phenotype. A proliferative response of CD25⁺ Tregs toward the presence of their cognate Ag in vivo could also be observed in studies using TCR-transgenic models (8, 9), supporting the notion that Treg proliferation requires Ag-specific activation.

In the polyclonal repertoire, the nature of the Ags recognized by Tregs remains elusive; however, it was shown that Tregs display a diverse TCR repertoire (29), which is distinct from conventional T cells and skewed toward high affinity recognition of self-Ag (30, 31). Furthermore, recent findings demonstrated that Tregs and potentially pathogenic self-reactive T cells have intersecting TCR repertoires (32). However, Tregs were also reported to recognize Ags from infectious agents such as Leishmania major (33), and unpublished data from our own group suggest a contribution of stimuli derived from the intestinal microflora to the proliferation of Tregs in specific pathogen-free mice. Taken together, these findings indicate that under steady-state conditions, sufficient Ag sources exist stimulating homeostatic proliferation of Tregs in healthy mice. Clearly, the highest proliferative activity accumulates in the Treg subsets, which displays an effector/memory-like phenotype and is characterized by the expression of _E.

In thymectomized mice, we observed a relative increase in the frequency of _E-expressing Tregs, which was due to the reduced cell count of conventional CD4⁺ T cells resulting in a mild lymphopenia in the T cell compartment. Absolute numbers of _E⁺ Tregs were comparable to non-thymectomized control mice, indicating that thymic output was not necessary to maintain this Treg subset. In contrast, the _E^–CD25⁺ Tregs displayed a comparable reduction in cell numbers following thymic ablation, as the conventional naive T cell pool. Constant numbers of _E⁺ Tregs upon thymectomy appear to contradict the higher sensitivity toward apoptosis of _E⁺CD25⁺ Tregs as compared with _E^–CD25⁺ Tregs, which has recently been described by Stephens and colleagues (13) and which we could confirm using Treg subsets from Foxp3-GFP-reporter mice (data not shown). The observation that numbers of _E⁺ Tregs are maintained in the absence of thymic export despite a high propensity for apoptosis, therefore, suggests that the fate of _E⁺ Tregs is well balanced by a constant peripheral expansion of this population of Tregs.

Our findings support a concept where _E identifies peripherally expanded effector/memory-like Tregs with an activated phenotype, which possibly have differentiated from _E^–CD25⁺ Tregs. Our data are consistent with a previous report by Dujardin et al. (34), who demonstrated a pronounced predominance of _E⁺CD25⁺ Tregs in adult mice that had been thymectomized at day 3 (d3Tx). In the periphery of newborn mice, these authors observed significant numbers of _E^–CD25⁺ Tregs emigrated from the thymus before thymectomy, but not of _E⁺CD25⁺ Tregs, while a 5-fold higher frequency of _E⁺CD25⁺ Tregs was found in adult d3Tx mice. Our study using mice thymectomized at the age of 3 wk, a time point when the T cell compartment has fully evolved, further demonstrates that independence of the _E⁺ Treg subset from thymic output is not restricted to the aforementioned situation in d3Tx mice, where postnatal development of the whole T cell compartment is severely disturbed. Furthermore, when we compared in vivo proliferation in non-thymectomized control mice to thymectomized mice, no differences were observed regarding the frequency of BrdU^high cells, which are indicative of proliferation in the periphery among the various Treg subsets (data not shown). In summary, the above findings clearly indicate that post-thymic proliferation contributes to the development of _E⁺ Treg subsets. The data suggest that _E⁺ Tregs keep proliferating, yet we cannot formally exclude the alternative hypothesis that only _E^–CD25⁺ Tregs proliferate and cease to cycle once _E-expression has been acquired.

To clarify the developmental relationship between the different naturally occurring Treg subsets, we aimed to identify potential precursors of the _E-expressing Treg subset. When we transferred OVA-specific naive-like _E^–CD25⁺ Tregs followed by oral Ag administration, the vast majority of CD25⁺ Tregs previously negative for _E up-regulated expression of this marker, suggesting that tolerogenic, Ag-specific activation of naive-like Tregs leads to proliferation, differentiation, and acquisition of an effector/memory-like phenotype. Under these conditions, transferred CD25⁺ Tregs maintained high levels of Foxp3 and CD25 expression (data not shown). The degree of proliferation of formerly _E^–CD25⁺ Tregs under these tolerogenic conditions was modest as compared with an immunogenic activation in the presence of LPS (data not shown). Nevertheless, it remains elusive how already converted _E⁺CD25⁺ Tregs would respond to Ag-specific activation in this setting. Indirect evidence for a profound proliferative response of _E⁺ Tregs comes from recent data obtained in a polyclonal transfer model, demonstrating that total CD4⁺CD25⁺ cells, but not _E-depleted CD4⁺CD25⁺ cells, displayed loss of CFSE within 7 days after transfer into congenic recipient mice (13).

When we transferred conventional naive _E^–CD25^–Foxp3^–CD62L⁺ DO11.10 T cells and subjected them to the same regimen of oral Ag administration, we observed induction of Foxp3⁺ cells in a dose-dependent fashion. Induction of Foxp3⁺ cells was most prominent in the livLN, where basically all cells acquired Foxp3 expression after two rounds of division, while undivided cells only partially up-regulated Foxp3. Several studies have demonstrated the conversion of nonregulatory, conventional T cells into Tregs under distinct conditions in vivo (19, 20, 24, 35, 36), both by using TCR-transgenic and polyclonal naive precursor cells. In line with our findings, recent data investigating the homeostatic proliferation of transferred CD4⁺Foxp3^eGFP–in RAG-1^–/– recipient mice revealed that those cells that had converted in the GALT into Foxp3^eGFP+ cells were positive for _E-expression (16).

Hultkrantz and colleagues (23) first described an important role for the livLN in the generation of CD25⁺ Tregs, characterized by high levels of _E coexpression and suppressive capacity. However, these authors did not detect Foxp3 mRNA in this subset. This is in contrast to our finding, where we consistently observed expression of Foxp3 on the single cell level as well as suppressive activity of CFSE^lowKJ1.26⁺CD4⁺ cells re-isolated from the livLN. The periportal livLN drains the liver, stomach, and pancreas (26), and several studies have shown an important role for the liver in generating tolerogenic immune responses to food Ag and even in transplantation settings (37, 38). Ag from the intestinal lumen reaches the liver directly via the portal vein, and occlusion of the portal vein prevented the development of oral tolerance (39). De novo induction of Foxp3⁺ Tregs in the livLN, upon oral Ag administration, found in this study underlines the pivotal role of the livLN in the induction for peripheral tolerance. At the same time, immunization with OVA protein i.p. in the presence of LPS did not result in the induction of Foxp3⁺ Tregs, revealing that the specific microenvironment of the livLN is tolerogenic only in the absence of inflammatory stimuli.

The data presented in this study indicated that Foxp3⁺ Tregs could be generated by conversion of previously Foxp3^– non-Tregs. That this was not due to selective outgrowth or survival of small numbers of contaminating Foxp3⁺ T cells was concluded from the following findings: 1) Up-regulation of Foxp3 occurred even on a part of the nondivided CFSE^high fraction of transferred cells, but only in the presence of cognate Ag. This argues against selective outgrowth of the pre-existing 0.12% of Foxp3⁺ cells. 2) The absolute cell numbers recovered after transfer of either 6.2 x 10³ CD25^–Foxp3⁺ cells or 3.5 x 10⁵ CD25⁺Foxp3⁺ cells clearly contradicted the possibility that appearance of Foxp3⁺ Tregs in recipients of CD25^– cells was due to expansion from the pre-existing contaminating CD25^–Foxp3⁺ subset. Otherwise, a dramatically increased expansion and/or survival rate (30- to 70-fold) would have to be assumed, compared with the situation where CD25⁺Foxp3⁺ had been transferred. Although this scenario cannot be formally excluded, it appears to be highly unlikely and was also disproved in a study by Liang and colleagues (36). In addition, our data obtained upon adoptive transfer of CD4⁺ T cells from DO11.10xRAG^–/– mice followed by oral Ag administration confirm de novo induction of Foxp3⁺ cells. Therefore, based on these cumulative findings, we concluded that Foxp3⁺ Tregs were induced from the nonregulatory CD62L^high T cell pool in response to the particular conditions of Ag-specific activation, predominantly in the livLN, following oral administration of cognate Ag.

It is tempting to speculate that TGF-β might be a major factor driving generation of Foxp3⁺ cells in our setting. Several in vitro data have shown induction of Foxp3 upon stimulation in the presence of TGF-β (14, 15). Furthermore, TGF-β is important for maintenance of Foxp3 expression and suppressive function of Tregs in vivo (40, 41). Next to these recent findings, it has been known for some time that TGF-β is a major factor inducing expression of _E on CD4⁺ T cells (42). Therefore _E expression may reflect activation in a microenvironment where local concentrations of active TGF-β were sufficient to both maintain Foxp3 expression in proliferating naive-like CD25⁺ and to generate new Foxp3⁺ cells from conventional naive T cells. Whether retinoic acid is critically involved in the de novo generation of _E⁺ Tregs, as recently suggested for the generation of Foxp3⁺ Tregs (43), remains to be shown.

Expression of _E has also been associated in a number of studies with peripherally induced/expanded Tregs in infection models. Suffia et al. (44) demonstrated a functional role for _E expression in the retention of Tregs at epithelial sites, where _E Tregs suppressed the local response to L. major and were critically involved in the chronicity of the infection. The Tregs were parasite-specific and derived from the naturally occurring Foxp3⁺ Treg pool. Their proliferation and survival depended on the presence of the parasite (33). In other infection models, such as Schistosoma mansoni (45) and Heligmosomoides polygyrus (S. Hartmann, personal communication), Foxp3⁺ Tregs displayed increasing frequencies of _E⁺ Tregs during the course of disease progression.

In summary, our data support the hypothesis that _E expression is not only preferentially associated with an effector/memory-like phenotype of Foxp3⁺ Tregs but, in addition, identifies Tregs that are constantly cycling under steady-state conditions in healthy mice. Furthermore, our results strongly suggest that the _E⁺Foxp3⁺ Treg pool is of diverse origin, consisting of both thymic-derived, expanded/differentiated natural Tregs as well as of de novo induced Foxp3⁺ Tregs, generated upon tolerogenic, Ag-specific activation in distinct anatomical sites of the body. Our findings further enlarge our understanding of the fundamental aspects of Treg biology and support the concept that Foxp3⁺ Tregs with a phenotype indistinguishable from the naturally occurring Foxp3⁺ Treg pool can be generated de novo, under appropriate conditions of Ag-delivery in the periphery, while at the same time the very same conditions promote proliferation/expansion of pre-existing natural Tregs.

	Acknowledgment

 
We thank Dr. Ludger Klein (Ludwig Maximilians Universität Munich, Germany) for providing thymectomized C57BL/6 mice.

	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 Grants SFB 633 and SFB 650 from the Deutsche Forschungsgemeinschaft.

² Address correspondence and reprint requests to Dr. Jochen Huehn, Experimentelle Rheumatologie, Charité Universitaetsmedizin Berlin, Deutsches Rheuma-Forschungszentrum Berlin, Charitéplatz 1, 10117 Berlin, Germany. E-mail address: Huehn{at}drfz.de

³ Abbreviations used in this paper: Tregs, regulatory T cells; LN, lymph node; d3Tx, day 3 thymectomized; livLN, liver-draining lymph node; mLN, mesenteric lymph node; PP, Peyer’s Patch.

Received for publication January 3, 2007. Accepted for publication October 25, 2007.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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