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Th3 Cells in Peripheral Tolerance. II. TGF--Transgenic Th3 Cells Rescue IL-2-Deficient Mice from Autoimmunity¹

Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We developed a transgenic (Tg) mouse that expresses TGF- under control of the IL-2 promoter to investigate Th3 cell differentiation both in vitro and in vivo. We previously found that repetitive in vitro Ag stimulation results in constant expression of Foxp3 in TGF--Tg Th3 cells that acquire regulatory function independent of surface expression of CD25. To examine the differentiation and function of Th3 cells in vivo and to compare them with thymic-derived CD4⁺CD25⁺ regulatory T cells (Treg), we introduced the TGF- transgene into T cells of IL-2-deficient (IL-2^–/–) mice. We found that the induction, differentiation, and function of TGF--derived Foxp3⁺ Th3 cells were independent of IL-2, which differs from thymic Tregs. In an environment that lacks functional CD25⁺ thymic-derived Tregs, expression of the TGF- transgene in IL-2^–/– mice led to the induction of distinct CD25^– regulatory cells in the periphery. These cells expressed Foxp3 and efficiently controlled hyperproliferation of T cells and rescued the IL-2^–/– mouse from lethal autoimmunity. Unlike IL-2^–/– animals, TGF-/IL-2^–/– mice had normal numbers of T cells, B cells, macrophages, and dendritic cells and did not have splenomegaly, lymphadenopathy, or inflammation in multiple organs. Accumulation of Foxp3⁺ cells over time, however, was dependent on IL-2. Our results suggest that TGF--derived Foxp3⁺CD25^+/– Th3 regulatory cells represent a different cell lineage from thymic-derived CD25⁺ Tregs in the periphery but may play an important role in maintaining thymic Tregs in the peripheral immune compartment by secretion of TGF-.

	Introduction

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Thymic-derived CD4⁺CD25⁺ regulatory T cells (Tregs)³ (1, 2) are critical mediators of dominant tolerance and play an important role in the maintenance of self-tolerance. The forkhead family transcription factor Foxp3 acts as the Treg lineage specification factor (3) and mutations of the Foxp3 gene in both mice and humans are associated with pronounced immunopathology (4, 5, 6). Forced expression of the Foxp3 gene in CD4⁺CD25^– non-Tregs induces a Treg-associated phenotype and regulatory function (7, 8). However, when overexpression of Foxp3 is limited in the thymus, the suppressive phenotype is not maintained in the periphery (9), indicating that other factors are responsible for inducing or maintaining CD25⁺ Tregs outside the thymus.

Although functional CD25⁺ Tregs develop in TGF--deficient mice (10), we and others have shown that TGF- induces de novo expression of Foxp3 upon activation in naive CD4⁺CD25^– T cells that leads to generation of CD25⁺ Tregs in vitro (11). Furthermore, using a T cell-specific, inducible TGF--transgenic (Tg) model, we demonstrated that transient overexpression of TGF- upon TCR ligation differentiates Ag-specific Th0 cells into regulatory Foxp3⁺CD25^+/– Th3 cells in vitro (12). Repetitive Ag stimulation results in sustained expression of Foxp3 in Th3 cells that acquire regulatory function independent of surface expression of CD25. Together, these in vitro findings point to an important role for TGF- in the induction of CD25⁺ Tregs in the periphery and the possibility that such converted/induced Tregs (iTreg) might represent a difference lineage of Tregs other than natural, thymic-derived Tregs.

It has been reported that in combination with IL-2, TGF- induces the expansion of CD25⁺ Tregs in vitro and in vivo (13, 14, 15). To separate TGF--induced expansion of CD25⁺ Tregs from TGF--induced differentiation of Foxp3⁺ Tregs in vivo, we used the IL-2-deficient (IL-2^–/–) mouse model. It is now apparent that IL-2 has an indispensable role in maintaining self-tolerance by supporting the in vivo growth, survival, and function of naturally occurring CD25⁺ Tregs (16, 17). Numbers of Tregs are low (18) and dysfunctional (19) in IL-2-signaling deficient mice (IL-2-, IL-2R-, and IL-2R-deficient mice), which succumb to severe systemic inflammation with autoimmune components (20, 21, 22). By crossing the TGF--Tg mouse we generated with the IL-2^–/– mouse on the C57BL/6 background (TGF-/IL-2^–/–), and we examined the differentiation of CD25^– Th3 cells in vivo and their ability to control autoreactivity of T cells in an IL-2-independent fashion. We demonstrate that Th3 cells represent a unique regulatory lineage in the periphery that induces Foxp3⁺ regulatory cells independent of thymic-derived CD25⁺ Tregs.

	Materials and Methods

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Mice

All mice were housed in a specific pathogen/viral-free animal facility at the Harvard Institutes of Medicine. All breeding and experiments were performed in accordance with the guidelines of the Committee on Animals of Harvard Medical School.

The generation, maintenance, and genotyping of TGF-1-Tg mice (on a C57BL/6 background) was described in the previous report. To introduce the TGF- transgene into IL-2^–/– mice (on a C57BL/6 background), TGF-1-Tg females were bred with IL-2^–/– male mice (from The Jackson Laboratory). TGF--Tg^–/IL-2^+/– and TGF--Tg⁺/IL-2^+/– heterozygotes were then interbred to obtain TGF--Tg^–/IL-2^–/– (IL-2^–/–) and TGF--Tg⁺/IL-2^–/– (TGF-/IL-2^–/–) homozygotes. TGF--Tg^–/IL-2^+/+ (IL-2^+/+) littermates were used as controls. IL-2-deficient genes were identified by tail DNA PCR according to instructions from The Jackson Laboratory.

Abs and reagents

The following reagents were obtained from BD Biosciences: purified anti-mouse CD3 (NA/LETM) Ab, purified anti-mouse CD16/CD32 Ab, and FITC-conjugated anti-mouse CD4, CD19, CD44, and CD62L, PE-conjugated anti-mouse CD11c, CD25, and CD69, PerCP-conjugated anti-mouse CD3, allophycocyanin-conjugated anti-mouse CD4 and CD11b, and the respective isotypic control mAbs. 7-amino-actinomycin D was purchased from Calbiochem.

Cell cultures

For proliferation assays, cells were grown in DMEM, 10% FCS supplemented with 5 x 10^–5 M 2-ME (Sigma-Aldrich), 2 µM L-glutamine, 100 U/µg penicillin/streptomycin (BioWhittaker) at 37°C and 5% CO₂ for 60 h. Cells were pulsed with 1 µCi [³H]thymidine for the last 16 h of incubation and thymidine incorporation was measured using a microbeta liquid scintillation and luminescence counter (PerkinElmer). Data are presented as mean ± SD of triplicate wells.

For suppression assay, individual CD25⁺, CD25^–CD4⁺ subsets (0.5 x 10⁶ cells/ml) that were sorted from IL-2^+/+ or TGF-/IL-2^–/– splenocytes were either cultured alone or with IL-2^+/+ CD4⁺CD25^– (0.5 x 10⁶ cells/ml) responder cells in the presence of 0.5 µg/ml anti-CD3. RBC lysed whole spleen cells (irradiated, 4000 rad) of C57BL/6 mice were used as APCs.

Flow cytometry analysis

Cells were resuspended in PBS containing 1% BSA and 0.1% sodium azide (Sigma-Aldrich). For the staining of surface Ags, cells were incubated with FITC-, PE-, PerCP-, or allophycocyanin-conjugated mAbs or their isotype control mAbs as indicated for 20 min on ice. After washing, stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences) and quantified with FlowJo (Tree Star) software. Cells were depicted for the fluorescence intensity of the indicated surface marker.

Quantitative RT-PCR

Naive CD4⁺CD25^– and CD4⁺CD25⁺ cells purified from IL-2^+/+, IL-2^–/–, or TGF-/IL-2^–/– mice were subjected to quantitative RT-PCR using TaqMan kits (Applied Biosystems). GAPDH mRNA levels are used as internal controls. Relative Foxp3 expression (2^{–CT (Foxp3 – GAPDH)} x 1000) was presented as the mean ± SD of triplicate samples, where CT is the cycle threshold.

Histopathology and hematology analysis

Tissue specimens were fixed in Bouin’s fixative (VWR Scientific) and embedded in paraffin. Five-micrometer sections were stained according to standard protocols with H&E. To assess anemia, mice at the indicated age were bleed from the tail vein using heparinized microhematocrit capillary tubes (Fisher Scientific). Each blood sample was analyzed within 1 h using a HEMAVET 950 Hematology Analyzer (Drew Scientific) to obtain a full hematology profile. The hematocrit value and hemoglobin concentration are shown as mean ± SD of each group.

Statistical analysis

One-way ANOVA was used to compare the three groups of mice (wild-type (WT), IL-2^–/–, and TGF-/2^–/–) followed by a Newman-Keuls multiple comparison as a post hoc test when p < 0.05 by ANOVA.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
TGF--producing Tg cells control hyperactivation of lymphocytes in IL-2-deficient mice

After introducing the TGF- transgene into IL-2^–/– mice, we first analyzed the immunological properties of TGF-/IL-2^–/– mice. The cell surface phenotype and in vitro proliferative capacity were evaluated in IL-2^+/+, IL-2^–/–, and TGF-/IL-2^–/– littermates. FACS analysis of lymph node (LN) cells from these three groups is shown in Fig. 1a. Unlike IL-2^–/– mice in which most B cells (CD19⁺) are depleted due to early hyperactivation (22), equal percentages of B cells were observed in IL-2^+/+ littermates and TGF-/IL-2^–/– mice. In IL-2^–/– mice, T cell subsets have an activated phenotype as measured by early and late activation markers CD69 and CD44 and a low level of expression of CD62L. This phenotype is less prominent in TGF-/IL-2^–/– mice (Fig. 1a). We also examined CD8⁺ cells in the LNs and both CD4⁺ and CD8⁺ cells in the spleen and found a similar reversal of the activated phenotype in TGF-/IL-2^–/– mice (data not shown). The reduced expression of activation markers is consistent with a decreased proliferative response to anti-CD3 stimulation by TGF-/IL-2^–/– LN cells compared with both IL-2^–/– and IL-2^+/+ cells ex vivo (Fig. 1b). In addition, whereas an increased number of macrophages (CD11b⁺) and dendritic cells (DCs) (CD11c⁺) were detected in the spleens (Fig. 1c) and LNs of IL-2^–/– mice, these populations were relatively unchanged in TGF-/IL-2^–/– mice when compared with IL-2^+/+ littermates (Table I). Together, these data indicated that TGF-/IL-2^–/– mice do not have hyperreactivate CD4 T cells which in turn induce the hyperactivation of B cells, macrophages, and DCs in IL-2^–/– animals.

View larger version (39K): [in this window] [in a new window]   FIGURE 1. Absence of hyperreactivity of T and B cells from TGF-/IL-2–/– mice. a, Percentage of CD19+ B cells in total population of LN cells, and CD62Llow, CD69+, or CD44+ in gated CD3+CD4+ T populations is examined in IL-2+/+, IL-2–/–, and TGF-/IL-2–/– littermates of 8 wk of age. b, Proliferation of LN cells from the three groups of mice in response to the stimulation of indicated concentrations of soluble anti-CD3 in a 60-h assay. Experiments were repeated with mice of 8, 13, 20 wk of age and similar results were obtained. Data shown are representative of mice 13 wk of age. c, Absolute numbers of T cell (CD3+), B cell (CD19+), macrophage (CD11b+), and DC (CD11c+) subsets in the spleens of the IL-2+/+, IL-2–/–, and TGF-/IL-2–/– mice. Depicted are average ± SD of each group (n = 3) of mice. *, p > 0.05 (IL-2+/+ vs TGF-/IL-2–/–); **, p < 0.01 (IL-2–/– vs TGF-/IL-2–/–).

View this table: [in this window] [in a new window]   Table I. Absolute numbers of each cell population in the lymphoid organs of TGF-/IL-2–/– micea

Control of hyperresponsive T cells in TGF-/IL-2^–/– mice is Foxp3⁺ cell dependent

As described previously, expression of TGF- by T cells converted a fraction of CD4⁺CD25^– T cells into CD4⁺CD25⁺ Foxp3⁺ cells with regulatory function. Thus, it is possible that control of T cell activation seen in the TGF-/IL-2^–/– mice is due to the induction of this population by the TGF- transgene. To investigate whether the restoration of the peripheral CD25⁺ Treg cells was responsible for the correction of lymphoproliferation in IL-2^–/– mice, we examined the expression of the CD25 on CD4⁺ T cells from TGF-/IL-2^–/– mice. At 10 wk of age, there were no detectable CD25⁺ cells in IL-2^–/– or TGF-/IL-2^–/– mice (data not shown). As shown in Fig. 2a, at 20 wk of age, even though we could detect small numbers of CD25⁺ cells in IL-2^–/– and TGF-/IL-2^–/– mice, these populations contained no CD62L⁺ CD25^high cells as in IL-2^+/+ littermates, indicating these were activated cells rather than Tregs which are associated with a naive phenotype. These data are consistent with previous reports that IL-2 is essential for the generation of CD25⁺ Tregs in the thymus, as well as their expansion (23) and function in the periphery (24, 25). Therefore, the correction of the defects associated with IL-2 gene disruption in TGF-/IL-2^–/– mice is due to the induction of Foxp3⁺ Tregs, irrespective of CD25 expression in the peripheral repertoire in TGF-/IL-2^–/– mice.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Immunoregulatory properties of CD4+CD25– cells from TGF-/IL-2–/– mice. a, Splenocytes from IL-2+/+, IL-2–/–, and TGF-/IL-2–/– mice at 20 wk of age were costained with PE-anti-CD25, PerCP-anti-CD3, and allophycocyanin-anti-CD4. CD3+CD4+ cells were depicted for the florescence intensity of CD25. Percentages of CD25 medium- and high-expressing subsets are indicated. b, Four populations of CD3+CD4+ cells from IL-2+/+ (CD25–, CD25+) and TGF-/IL-2–/– (CD25– and CD25+) mice were isolated by FACS sorting. Each population was stimulated with 0.5 µg/ml anti-CD3 and APCs either alone or with equal numbers of WT CD25– T cells as responders. The data are shown as mean ± SD of triplicate wells of [3H] incorporation. Data represent one of two independent experiments with similar results. *, p = 0.39 when responders cocultured with either IL2+/+ CD25+, TGF-/IL-2–/– CD25– or CD25+ subsets are compared. **, p < 0.001 when responder cultures (identified by **) are compared with any of the three cocultures (identified by *).

Because CD4⁺CD25^– cells from TGF-/IL-2^–/– mice possess regulatory function, we asked whether the acquisition of regulatory properties during in vivo differentiation is also correlated with the induction of Foxp3 expression by TGF- as we observed during in vitro differentiation (12). To this end, we monitored the Foxp3 mRNA level at different ages by quantitative RT-PCR. As shown in Fig. 3a, CD25^– cells from TGF-/IL-2^–/– mice expressed higher levels of Foxp3 mRNA compared with those in CD25^– cells from both IL-2^+/+ and IL-2^–/– mice at 10 wk of age. This level of Foxp3 is even slightly higher than that expressed by the CD25⁺ subset from the IL-2^–/– mice. These data confirmed that the elevated expression of Foxp3 was associated with the in vivo differentiation of CD25^– Th3 cells as well as their suppressive function in TGF-/IL-2^–/– mice. However, as the mice aged, the level of Foxp3 expression at 20 wk in the CD25^– subset did not increase further as would have been expected if there was further accumulation of regulatory CD25^– Th3 cells in the total CD25^– T cell population (Fig. 3b). Nonetheless, the remaining CD25^– cells from 20-wk-old TGF-/IL-2^–/– mice retained their regulatory function in the in vitro suppression assay (Fig. 2b). These results suggest that Foxp3⁺ regulatory cells are continuously generated but do not survive or expand as the animals age due to their requirement for IL-2 as a growth factor.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Foxp3 expression correlates with the suppressive properties of CD4+CD25– cells in TGF-/IL-2–/– mice. Splenocytes of IL-2+/+, IL-2–/–, and TGF-/IL-2–/– mice at 10 wk (a) and 20 wk (b) of age were costained with PE-anti-CD25, PerCP-anti-CD3, and allophycocyanin-anti-CD4. CD25– and CD25+ T subsets of each group were purified by FACS sorting and measured for relative Foxp 3 expression. The data are representative of four experiments with similar results. *, p < 0.001 (TGF-/IL-2–/–25– vs IL-2+/+25– and TGF-/IL-2–/–25– vs IL-2–/–25–, 10 wk); **, p = 0.53 (IL-2+/+25– vs IL-2–/–25– vs TGF-/IL-2–/– 25–, 20 wk).

Lethal autoimmunity is prevented in IL-2-deficient mice by TGF--producing Tg T cells

IL-2^–/– mice develop a lymphoproliferative syndrome with severe hemolytic anemia and die within 20 wk of age (22). If CD25^– Th3 cells in the TGF-/IL-2^–/– mice can efficiently control the hyperactivation of CD4 T cells and thus prevent the multiorgan autoimmunity, this immune regulation should markedly expand the life span of TGF-/IL-2^–/– mice. Indeed, we found lethal autoimmunity was completely prevented in these mice (Fig. 4a). Although 50% of IL-2^–/– mice die between the age of 8–16 wk and have associated severe weight loss and hemolytic anemia, there was no spontaneous death observed in TGF-/IL-2^–/– group (TGF-/IL-2^–/– mice were followed up to 30 wk). Furthermore, there was no weight loss in TGF-/IL-2^–/– mice (Fig. 4b) and there was a partial reversal of hemolytic anemia (Fig. 4c). The existence of anemia in the older mice could also be a reflection of the lack of IL-2 which is needed to maintain Treg populations.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. TGF- transgene expression rescues IL-2-deficient mice from lethal autoimmunity. a, Mortality of IL-2+/+ WT littermate (n = 6), IL-2–/– (n = 25), and TGF-/IL-2–/– (n = 22) mice. Regression line reflects mice that died spontaneously or were euthanized when moribund. b, Three groups of mice were weighed weekly beginning 4 wk after birth and the percentage of the starting weight was calculated. Data are depicted as mean of each group. c, Hematocrit value and hemoglobin concentration of surviving mice that were measured on 10 or 20 wk after birth. Data are shown as mean ± SD of each group. *, p < 0.001 (TGF-/IL-2–/– vs IL-2+/+ and TGF-/IL-2–/– vs IL-2–/–).

View larger version (77K): [in this window] [in a new window]   FIGURE 5. Gross pathology and histology of IL-2–/– and TGF-/IL-2–/– mice. a, Size of a normal IL-2+/+ mouse and its IL-2–/– and TGF-/IL-2–/– littermates at 13 wk of age. b, The spleen and LNs of the IL-2–/– and TGF-/IL-2–/– mouse. c, H&E staining of lung, liver, pancreas, and spleen of IL-2–/– (panels 1–4) and TGF-/IL-2–/– (panels 5–8) littermates at 13 wk of age. Severe inflammatory infiltration and atrophy was seen in parenchyma of lung (panel 1), the portal track and blood vessels of liver (panel 2) and the portal track and the adjacent islet of pancreas (panel 3). Abnormalities of spleen (panel 4) include red and white pulp hyperplasia with loss of marginal zone and accumulation of plasma cells, neutrophils, and reticulocytes in the white pulp. Insets, The enlargements of the periarteriolar lymphoid sheath area (indicated by an arrow). The scale bar represents 50 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Oral administration of Ag induces Ag-specific regulatory Th3 cells that are class II restricted and have the identical TCR as Th1 and Th2 cells; their major characteristic is the predominant secretion of TGF- following TCR ligation (26). The important role of TGF- in down-modulation of T cell-mediated immune responses and control of peripheral tolerance has been clearly established (27, 28), yet the mechanism of action of TGF--producing Th3 cells in vivo is not well-understood. Recently, several lines of in vivo and in vitro evidence have linked TGF- with regulation of Foxp3 expression in T cells. Using a Foxp3-enhanced GFP knockin mouse model, we have shown the de novo generation of Foxp3⁺ Tregs from naive Foxp3^–CD4⁺ non-Tregs when adding TGF- into cell cultures (29). Using T cells that express the intrinsic TGF- transgene upon activation, which have identical properties as Th3 cells, we demonstrated the same differentiation pathway of induced Foxp3⁺ Treg cells in vitro (12). In this study, by introducing the TGF- transgene into IL-2^–/– mouse, we further addressed the differentiation requirements of TGF--derived Th3 cells or iTregs in vivo and their relationship with thymic-derived CD25⁺ Tregs. Unlike thymic-derived CD25⁺ Tregs (23, 25), IL-2 is dispensable for the induction, differentiation, and function of TGF--derived Foxp3⁺ Th3/iTreg cells, indicating that Th3 cells represent a unique regulatory lineage in the periphery.

We observed reversal of lethal autoimmunity and the absence of hyperreactivity of T, B, DCs, and macrophages in the TGF-/IL-2^–/– mice. Foxp3⁺CD25^– Th3 cells that were induced in the periphery of TGF-/IL-2^–/– mice were the major population responsible for the maintenance of self-tolerance. It is theoretically possible that instead of inducing Foxp3⁺ cells de novo from Foxp3^–CD25^– cells, TGF- functions by expanding the few pre-existing Foxp3⁺CD25^+/– cells in IL-2^–/– animals. It has been shown in a recent report that these IL-2^–/– Tregs can develop in the thymus independent of IL-2 and function well if provided exogenous IL-2 from other cells (18). However, expansion of pre-existing IL-2^–/– Tregs seems unlikely given the suppressive nature of TGF- on cell growth. In fact, only IL-2 is critical for the expansion and function of pre-existing Treg cells. As demonstrated by Furtado et al. (25), CD25⁺ T cells from IL-2^–/– mice exhibited regulatory properties upon adoptive transfer into IL-2 ^+/+ mice, in which case only IL-2, but not TGF-, is the variable between host and recipient. Consistent with these observations, after Foxp3⁺ Th3 cells were generated in TGF-/IL-2^–/– mice, IL-2 also seemed to play a vital role in the survival or expansion of these cells. This function of IL-2 is indicated by the failure to accumulate or expand Foxp3⁺ Th3 cells in older TGF-/IL-2^–/– animals which was accompanied by increased hemolytic anemia and ulcerative colitis.

We have also found that overexpression of intrinsic TGF- leads to delayed T cell activation and reduced T cell proliferation (12). Thus, it is theoretically possible that the delay of autoimmunity seen in TGF-/IL-2^–/– mice was related to the T cell hyporesponsiveness in level of TGF- and not by the induction of regulatory cells. Our results demonstrate that T cell hyporesponsiveness contributes partially, but protection cannot be achieved without induction of Foxp3⁺ regulatory cells. When we cross the TGF- transgene into the Foxp3-deficient Scurfy mouse, we did not observe the same amount of protection following overexpression of TGF- in the T cells of Scurfy mice (Y. Carrier and H. L. Weiner, unpublished data). TGF-/Scurfy mice developed the similar level of inflammatory and autoimmune phenotype as the Scurfy mice, indicating that the suppressive effect of TGF- is upstream of Foxp3 expression and dependent on induction of Tregs. The importance of TGF--derived Foxp3 in the induction of immune regulation is also demonstrated by the fact that high-level expression of Foxp3 mRNA in hyperreactive T cells is observed in IL-2^–/– mice (Fig. 3b), which do not have normal Treg function. Thus, the transient expression of Foxp3 does not instruct T cells into Treg differentiation, but is a reflection of T cell activation in the absence of TGF-. Sustained expression of Foxp3 by TGF- is crucial for the Treg differentiation as we have demonstrated in our studies of the myelin oligodendrocyte glycoprotein TCR and TGF- double-Tg model (12). Thus, the absence of hyperlymphoproliferation in TGF-/IL-2^–/– mice appears due to maintenance and induction of Foxp3 expression by TGF- in the periphery.

In summary, although pre-existing thymic-derived Foxp3⁺ cells in the IL-2^–/– mouse may have limited function, we believe the marked increase in self-tolerance we observed is due to the induction of a separate lineage in the periphery, consisting of TGF--derived Foxp3⁺ Th3 cells. The induction and differentiation of these Th3 cells do not require IL-2 and IL-2 is not required for their function, which distinguishes these TGF--derived regulatory cells from thymic-derived Tregs. As we have previously shown using the myelin oligodendrocyte glycoprotein TCR x TGF- double-Tg model, Th3 cells respond to Ag stimulation and can differentiate from the same precursor as pathogenic T cells (12), which is also different from thymic-derived Tregs. Thus, as a separate lineage induced in the periphery, TGF--derived Foxp3⁺ Th3 cells contribute to the peripheral Foxp3⁺ Treg pool that includes nonthymic-derived CD25⁺ Tregs, the induction of which can serve to maintain or re-establish peripheral tolerance.

	Acknowledgment

 
We thank D. Kozoriz for cell sorting.

	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 National Multiple Sclerosis Society Fellowship Grant FG1479A1/1 (to Y.C.) and by National Institutes of Health Grants AI435801 and NS38037 (to H.L.W.).

² Address correspondence and reprint requests to Dr. Howard L. Weiner, Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115. E-mail address: hweiner{at}rics.bwh.harvard.edu

³ Abbreviations used in this paper: Treg, regulatory T cell; Tg, transgenic; iTreg, induced Treg; WT, wild type; LN, lymph node; DC, dendritic cell.

Received for publication July 20, 2006. Accepted for publication October 18, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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